Split (1)

train · ~9.11M rows (showing the first 93.5k)

identifier stringlengths 11 32	pdf_url stringlengths 17 4.62k ⌀	lang stringclasses 120 values	error stringclasses 1 value	title stringlengths 2 500 ⌀	source_name stringlengths 1 435 ⌀	publication_year float64 1.9k 2.02k	license stringclasses 3 values	word_count int64 0 1.64M	text stringlengths 1 9.75M
https://openalex.org/W2328107542	https://zenodo.org/records/1601099/files/article.pdf	English	null	Excursion to Godstone	Proceedings of the Geologists' Association	1,917	public-domain	465	Ordnance Map, New Series, r-inch, Sheet 286. Geological Survey Map, Sheet 6. 1900. \ VHITAKER, W.-" Excursion to Caterham, Godston e, etc." Proc, Geot. A ssoc., vol, xvi, p. 510. JUNE 17TH, 1916. REPORT BY W. WHITAKER, F.R.S., F.G.S., Director of the E xcursion. THIS Excursion was practically the same as the first part of that of June rSth, 1900. The party reached Caterharn by the train due at 1.41 p.rn., and walked southward to the escarpment of the North Downs at Upwood, where there was plentiful surface-evidence of the small outli er of Blackh eath pebble-beds that crosses the summit. p Passing down to the outside of the wood there was a good view over the outcrop of the beds below the Chalk, down to the Wealden Beds. Descending the Chalk-slope we came to shafts and openings to the und erground workings in the Upper Green- sand, for firestone and hearthstone, and at one of the shafts there was some of what has been called Scarp Drift, a wash of material down the face of the escarpment deposited at or near the foot. H ere there were good sections of the Upper Greensand, one a fresh shallow pit in the stone. The workings on the western side of the high road seem to have revived, and there was a good stock of dre ssed stone to be seen. Crossing the high road and going along the beautiful lane by Quarry Farm, we came to the stone-quarry, a little north-east of the farm, and, having bee n provided with candl es, were taken along the und erground workings, which consist of headin gs carried northward down the dip of the beds, so that they reach deep down under the hill, which rises in that direction. Then other quarries, about a quarter of a mile eastward, were visited, and the entra nces of these showed very good sections of the Scarp Drift, parts of which contained a great num ber of land- shells, notably Cy clostoma eiegans, a species common in Chalk- tracts. A few specimens of the large land-snail, H elix p01llatia, were also got. A note on the mollusca is append ed. g pp The walk was then continued over the outcrops of the Upper Greensand and of the Gault to that of the Folkestone Beds, by Godstone Church, whence a footpath led to the village, and to tea. The return-j ourney was made along the high road to Caterham through the gap in the hills, a passing glance being given to the sand-pit at the northern end of Godstone. Miss Grace Bigby acted as Excursion Secretary. g y p 1900. \ VHITAKER, W.-" Excursion to Caterham, Godston e, etc." Proc, Geot. A ssoc., vol, xvi, p. 510. p Geological Survey Map, Sheet 6.
https://openalex.org/W4380722144	https://zenodo.org/records/8039677/files/1.6.pdf	Quechua	null	MEHNAT BOZORIDA MARKETING TADQIQOTLARINI TASHKIL QILISH VA RIVOJLANTIRISH YO`LLARI	Zenodo (CERN European Organization for Nuclear Research)	2,023	cc-by	2,532	YANGILANAYOTGAN OʻZBEKISTON TARAQQIYOTIDA IQTISODIY FANLARNI OʻQITISHNING DOLZARB MASALALARI RESPUBLIKA ILMIY-AMALIY KONFERENSIYASI 16-iyun, 2023- yil RESPUBLIKA ILMIY-AMALIY KONFERENSIYASI 16-iyun, 2023- yil uchun etarli bo`lishi mumkin. Ta`lim xizmatlari bozorida boshqa maqsadli auditoriya - ta`lim xizmatlarini xaridorlarning xatti-harakatlarini tahlil qilish kerak. Xaridorlarni o`rganish tadqiqotning eng muhim turlaridan biridir, chunki aynan ular iste`molchining tanloviga ta`sir qilish va ta`lim xizmatini sotib olish orqali ta`lim muassasasiga asosiy foyda keltiradi. Shuning uchun, uning hajmi va faoliyatining o`ziga xos xususiyatlaridan qat’i nazar, har qanday muvaffaqiyatli muassasaning diqqat markazida bo`lgan xaridordir. Texnikumni rivojlantirishning to`g`ri rejasini ishlab chiqish uchun ta`lim xizmatlarini xaridorlarni segmentlash va joylashishni aniqlash uchun eng jozibador segmentlarni ajratib ko`rsatish kerak. Xarid qilish to`g`risida qaror qabul qilish jarayonining bosqichlarida turli omillarning ta`sir darajasini baholash kerak. Narxni va xaridorlarning boshqa taxminlarini tahlil qilish kerak. uchun etarli bo`lishi mumkin. Ta`lim xizmatlari bozorida boshqa maqsadli auditoriya - ta`lim xizmatlarini xaridorlarning xatti-harakatlarini tahlil qilish kerak. Xaridorlarni o`rganish tadqiqotning eng muhim turlaridan biridir, chunki aynan ular iste`molchining tanloviga ta`sir qilish va ta`lim xizmatini sotib olish orqali ta`lim muassasasiga asosiy foyda keltiradi. Shuning uchun, uning hajmi va faoliyatining o`ziga xos xususiyatlaridan qat’i nazar, har qanday muvaffaqiyatli muassasaning diqqat markazida bo`lgan xaridordir. Texnikumni rivojlantirishning to`g`ri rejasini ishlab chiqish uchun ta`lim xizmatlarini xaridorlarni segmentlash va joylashishni aniqlash uchun eng jozibador segmentlarni ajratib ko`rsatish kerak. Xarid qilish to`g`risida qaror qabul qilish jarayonining bosqichlarida turli omillarning ta`sir darajasini baholash kerak. Narxni va xaridorlarning boshqa taxminlarini tahlil qilish kerak. 4) Ma`lumotlarni tahlil qilish asosida ta`lim xizmatlari iste`molchisining batafsil portretini tuzish, ta`lim xizmatlari bozorida iste`molchilarning xatti-harakatlarining turlari va omillarini ajratib ko`rsatish kerak. Xarid qilish xulq-atvorini tahlil qilish natijasi xaridorlarni segmentatsiyalash va bozorda sotib olish xatti-harakatlari omillarini baholash bo`lishi kerak. 5) Tadqiqotning yakuniy bosqichi – ta`lim muassasasini rivojlantirishning strategik rejasini tuzish bo`yicha tavsiyalar ishlab chiqish. Shuningdek, ushbu bosqichda olib borilgan tadqiqotlarni, duch kelgan qiyinchiliklar va xatolarni tahlil qilish va keyingi marketing tadqiqotlari uchun tavsiyalar ishlab chiqish kerak. So`nggi yillar ichida Samaarqand viloyatidagi ta`lim xizmatlari bozorining holatini tahlil qilish ushbu bozorni rivojlantirishning asosiy tendentsiyalarini aniqlashga imkon berdi: Viloyatidagi ta`lim xizmatlari bozori boshlang`ich, o`rta va professional ta`limning bir qator ta`lim muassasalari bilan ifodalanadi. Barcha muassasalarning faoliyati ma’lum darajada o`zaro bog‘langan: Boshlang`ich kasb-hunar ta`limi iste`molchilari kelajakda o`rta va o`rta maxsus va professional ta`limi iste`molchilariga aylanishi mumkin. MEHNAT BOZORIDA MARKETING TADQIQOTLARINI TASHKIL QILISH VA RIVOJLANTIRISH YO`LLARI 1Musayeva Shoira Azimovna, 2Usmonova Dilfuza Ilxomovna 1Samarqand iqtisodiyot va servis institute professori 2Samarqand iqtisodiyot va servis institute dotsenti https://doi.org/10.5281/zenodo.8039677 Annotatsiya. Mazkur maqolada ta`lim xizmatlarini rivojlantirishning ustuvor yo`nalishlari, mamlakatimizda ta`lim xizmatlarining etakchi sub`ekti bo`lgan ta`lim muassasalarining hozirgi holati va ulardagi mavjud muammolar, hozirgi davrda ta`limning mazmuni va uni tashkil ko’rib chiqilgan. Kalit so’zlar: Mehnat bozori, ta`lim xizmatlari , marketing vositasi , tadqiqoti kontseptsiyasi , maqsad va vazifa. Ta`lim muassasasida boshqaruv qarorlarini qabul qilishning eng muhim marketing vositasi ta`lim xizmatlari bozorini marketing tadqiqotlari hisoblanadi. Ta`lim muassasasini rivojlantirishning strategik rejasini ishlab chiqishning tahliliy bosqichida ma`lumotni birlashtirish uchun ta`lim xizmatlari bozorini o`rganishning mavjud bir nechta usullarini bozorning ikki sub`ekti - iste`molchilarning xatti-harakatlari va qarorlariga ta`sir etuvchi omillarni tahlil qilish bilan to`ldirish kerak. Ta`lim xizmatlari bozorini o`rganish bo`yicha taklif etilayotgan metodologiyamiz bir necha bosqichlarni o`z ichiga oladi. 1) Tadqiqotning birinchi bosqichida marketing tadqiqoti kontseptsiyasi ishlab chiqiladi, bu tadqiqotning asosiy g‘oyasini aks ettiruvchi hujjatdir. Kontseptsiya tadqiqotning dolzarbligini asoslashi, maqsad va vazifalarini, tadqiqot ob’ekti va predmetini belgilashi, vaqtini ko`rsatishi kerak. Usullarni tanlash va asoslash, tadqiqotni axborot bilan ta’minlash muhim ahamiyatga ega. Tadqiqot konsepsiyasi asosida marketing tadqiqotlari dasturi ishlab chiqilmoqda. 2) Ikkinchi bosqichda, tadqiqot kontseptsiyasiga ko‘ra, Ta`lim xizmatlari bozorini tahlil qilish kerak: bozor hajmini, uning sig‘imini, "korporativ" tuzilmasini, raqobat darajasi va jiddiyligini baholash va asosiy tendentsiyalarni aniqlash. Ta`lim xizmatlari bozorini har tomonlama o`rganish vazifasi bozor, uning xususiyatlari va rivojlanish tendentsiyalari haqida to`liq ma`lumot olishdan iborat. Tadqiqotning muhim bosqichi ekspert so`rovi bo`lishi mumkin, bu nafaqat sub`ektiv, balki ob`ektiv tomondan ta`lim xizmatlari bozoridagi vaziyatga, uning rivojlanish istiqbollariga, iste`molchi va xaridlarga ta`sir qiluvchi omillarni tahlil qilish imkonini beradi. Ekspert sifatida muassasa o`quv yurtlari rahbarlari, viloyatning Ta`lim, yoshlar siyosati sohasi xodimlari va boshqalar qatnashishi mumkin. 3) Tadqiqotning uchinchi bosqichida ta`lim xizmatlari iste`molchilarining xatti- harakatlarini tahlil qilish kerak: hayotiy maqsadlar, qiziqishlar, baholashlar, istaklar, xatti- harakatlar motivlari, ularning ichki qadriyatlar tizimi bilan bog`liq bo`lgan afzalliklar va intilishlar. O`z iste`molchilarini, ularning xohish-istaklarini, imtiyozlarini o`rganib, Texnikumlar ularga aynan o`ziga kerak bo`lgan narsani taklif qilishi va buni raqobatchilardan yaxshiroq qilishi mumkin. Iste`molchini o`rganishda asosiy vazifa - ularning xulq-atvoriga ta`sir qiluvchi omillarni aniqlash va sotib olish xulq-atvori modellarini tahlil qilish. Aksariyat bozorlarda kamroq qimmatli xaridlar amalga oshiriladi yoki iste`molchi va xaridor bir shaxs sifatida harakat qila oladi, bu ma`lumot boshqaruv qarorlarini qabul qilish YANGILANAYOTGAN OʻZBEKISTON TARAQQIYOTIDA IQTISODIY FANLARNI OʻQITISHNING DOLZARB MASALALARI RESPUBLIKA ILMIY-AMALIY KONFERENSIYASI 16-iyun, 2023- yil 1 jad Ta`lim muassasalari haqida qisqacha ma`lumot (2022 yil yanvar holatiga) j Ta`lim muassasalari haqida qisqacha ma`lumot (2022 yil yanvar holatiga) № Ta`lim muassasalari TM soni TM quvvati Mavjud o`quvchi soni (2020/2021o`quv yili) To‘ldirilish koeffesenti 1 Kasb-hunar maktablari 37 24190 17479 0,7 2 Texnikumlar 23 20220 15688 0,8 3 Kollejlar 14 11605 8664 0,8 Jami 74 56015 4181 0,8 Ta`lim muassasalari haqida qisqacha ma`lumot (2022 yil yanvar holatiga) Samarqand viloyatida o`rta maxsus, professional ta`lim xizmatlariga bo`lgan potentsial talab 41831 kishini tashkil etadi, ammo bu ko`rsatkich doimiy ravishda pasayib bormoqda. Bu bir tomondan oliy ta`limga qamrovning ortib borayotganligi, ikkinchi tomondan o`rta bo`g`inda Ta`limning pullik bo`lganligi bilan izohlanadi. Migratsiya darajasi vaziyatni sezilarli darajada yaxshilay olmaydi, chunki emigratsiya darajasi ancha yuqori. Talabning barqaror pasayishi fonida bu bozorning yaqqol nomutanosibligi taklifning ko`payishi hisoblanadi. Statistik ma`lumotlarga ko`ra, Samarqand viloyatining 2022 yil yanvar holariga ta`lim xizmatlari bozorida taklif 56 015 kishini tashkil etdi. Bozordagi taklifga ekvivalent qiymat - bu viloyatidagi Ta`lim xizmatlari bozorining sig`imi. 28 YANGILANAYOTGAN OʻZBEKISTON TARAQQIYOTIDA IQTISODIY FANLARNI OʻQITISHNING DOLZARB MASALALARI RESPUBLIKA ILMIY-AMALIY KONFERENSIYASI 16-iyun, 2023- yil Q RESPUBLIKA ILMIY-AMALIY KONFERENSIYASI 16-iyun, 2023- yil Bozor imkoniyatlarining qisqarishi qattiq raqobat muhitiga olib keladi, bu ayniqsa kasb- hunar Ta`limi sohasida yaqqol namoyon bo`ladi. Bu ham nodavlat universitetlar sonining o`sishi bilan bog`liq. Viloyatining raqobat muhitini tahlil qilish ikkita asosiy ko`rsatkich bo`yicha baholash imkonini berdi: texnikumlarning moddiy-texnik bazasi (o`quv jarayoni uchun jihozlangan o`z hududining mavjudligi) va talabalar soni. Texnikumlarrning moddiy-texnik bazasi nuqtai nazaridan Ta`lim xizmatlari bozori o`rtacha konsentratsiyali deb tavsiflanadi. Viloyatdagi 74 ta Professional ta`lim muassasalari 1998 yildan bugungi kunga qadar Respublika va mahalliy budjet hisobidan 26 milliard 822 mln so‘m, shundan, xorijiy mamlakatlar kreditlari hisobidan 15 milliard 866 mln so‘m, jami 42 milliard 708 mln so‘mlik o`quv-laboratoriya, kompyuter texnikasi, avtomobil va traktor texnikasi, mini sex va ustaxona jixozlari bilan ta`minlangan. Ushbu jixozlardan ta`lim jarayonida foydalanilmoqda. Viloyat bozori sub’ektlarining ta`lim xizmatlari portfelini tahlil qilish shuni ko`rsatdiki, texnikumlarda talab qilinadigan mutaxassisliklar sog`liqni saqlash (67%), muhandislik ishi (11.7%), biznes va boshqaruv (3.7%), qishloq, o‘rmon va baliq xo‘jaligi (2.9), arxetektura va qurilish (2.7%), veterenariya (2.7 %), kompyuter texnalogiyalari va informatika (2.6%), xizmat ko`rsatish va transport (3.3%), huquq (1.2%), atrof muhit muhofazasi va qishloq xo‘jalik texnikasi (0.8%), ishlab chiqarish va aloqa, axborotlashtirish (1.2%). Oʻrta boʻgʻin mutaxassislarini tayyorlash tarkibida eng katta ulushni tibbiyot va muhandislik ishi mutaxassisliklar (xamshiralik ishi, feldsher-akusherlik ishi, farmatseftika, suv va kanalizatsiya tizimlarini montaj qilish, gaz tarmoqlarini montaj qilish, ko‘chmas mulkni ro`yxatga olish mutaxassisliklar) tashkil etadi. YANGILANAYOTGAN OʻZBEKISTON TARAQQIYOTIDA IQTISODIY FANLARNI OʻQITISHNING DOLZARB MASALALARI RESPUBLIKA ILMIY-AMALIY KONFERENSIYASI 16-iyun, 2023- yil Demak, Ta`lim, sog‘liqni saqlash, madaniyat va san’at, iqtisod va menejment yo`nalishlarida tahsil olayotgan talabalar orasida xotin-qizlar 74-80 foizni tashkil etdi. Avtotransport vositalari, geologiya, foydali qazilmalarni qidirish va o`zlashtirish, energetika, energetika va elektrotexnika, metallurgiya, mashinasozlik va materiallarni qayta ishlash, avtomatlashtirish kabi mutaxassisliklar bo`yicha oliy va o`rta muhandislik-texnik ta`lim olayotgan talabalar orasida erkaklar (79-92%) ustunlik qiladi. Mehnat bozorida Boshlang`ich va oliy kasb-hunar ma`lumotiga ega bitiruvchilarga o`rta kasb-hunar ta`limiga ega bo`lganlarga nisbatan talab katta. M h t b id k ` t l b ili di t i likl i i l h ki YANGILANAYOTGAN OʻZBEKISTON TARAQQIYOTIDA IQTISODIY FANLARNI OʻQITISHNING DOLZARB MASALALARI RESPUBLIKA ILMIY-AMALIY KONFERENSIYASI 16-iyun, 2023- yil 20 Trans port– 820 000 215 88 127 119 63 56 96 25 71 21 Atrof- muhit muhof azasi- 830 000 24 15 9 14 11 3 10 4 6 22 Hayot iy faoliy at havfsi zligi - 840 000 0 0 0 0 0 0 0 0 0 YANGILANAYOTGAN OʻZBEKISTON TARAQQIYOTIDA IQTISODIY FANLARNI OʻQITISHNING DOLZARB MASALALARI RESPUBLIKA ILMIY-AMALIY KONFERENSIYASI 16 iyun 2023 yil Mutaxassisliklarni tanlashda sezilarli gender farqi mavjud bo`lib, bu ayniqsa o`rta va oliy o`quv yurtlarida kasb tanlashda seziladi. Demak, Ta`lim, sog‘liqni saqlash, madaniyat va san’at, iqtisod va menejment yo`nalishlarida tahsil olayotgan talabalar orasida xotin-qizlar 74-80 foizni tashkil etdi. Avtotransport vositalari, geologiya, foydali qazilmalarni qidirish va o`zlashtirish, energetika, energetika va elektrotexnika, metallurgiya, mashinasozlik va materiallarni qayta ishlash, avtomatlashtirish kabi mutaxassisliklar bo`yicha oliy va o`rta muhandislik-texnik ta`lim olayotgan talabalar orasida erkaklar (79-92%) ustunlik qiladi. Mehnat bozorida Boshlang`ich va oliy kasb-hunar ma`lumotiga ega bitiruvchilarga o`rta kasb-hunar ta`limiga ega bo`lganlarga nisbatan talab katta. Mehnat bozorida eng ko`p talab qilinadigan mutaxassisliklarni aniqlash mumkin. Boshlang`ich kasb-hunar ta`limida - konchilik, ishlab chiqarish, qishloq xo‘jaligi, qurilish va transport va aloqa sohalarida ishlash uchun ishchilar mutaxassisliklari. O`rta kasb-hunar ta`limida - avtomatlashtirish va boshqarish, madaniyat va san’at, ta`lim va pedagogika, iqtisodiyot va boshqaruv, qurilish va arxitektura, transport vositalari. Oliy kasbiy taʼlimda mehnat bozorida eng koʻp talab qilinadigan mutaxassisliklar metallurgiya, mashinasozlik, materiallarni qayta ishlash, transport vositalari, informatika va kompyuter texnologiyalari, asbobsozlik va optika muhandisligi, avtomatlashtirish va boshqarish, qurilish va arxitektura, taʼlim va pedagogika, madaniyat va san`at, energetika, energetika va elektrotexnika. O`zbekiston respublikasi ta`lim tizimining o`ziga xos xususiyati davlat mulki xissasining qisqarib borayotganligidadir. Ta`lim asosini tashkil etuvchi bilimning o`ziga xos tarafi uning iste`mol qilinishi bilan yo`q bo`lib ketmasligi, balki, insonda gavdalanishi va oshib borishidadir. YANGILANAYOTGAN OʻZBEKISTON TARAQQIYOTIDA IQTISODIY FANLARNI OʻQITISHNING DOLZARB MASALALARI RESPUBLIKA ILMIY-AMALIY KONFERENSIYASI 16-iyun, 2023- yil Samarqand viloyatidagi texnikumlarda kunduzgi ta`lim shaklida ta`lim olayotgan talabalarning mutaxasislik kesimidagi soni to`g`risidagi 29 j MA`LUMOT T/r Ta`li m sohala ri soni Jami o`quvchila r soni shundan: Jami o`quvchila r sonidan davlat granti to`lov kontrak t 1-bosqichda 2-bosqichda Jami gra nt ko ntr akt Jami gra nt ko ntr akt Viloy at bo`yic ha jami: 14599 2325 12274 6773 13 06 53 95 7714 99 2 67 22 1 Pedag ogika 1 1 0 1 1 0 0 0 0 29 YANGILANAYOTGAN OʻZBEKISTON TARAQQIYOTIDA IQTISODIY FANLARNI OʻQITISHNING DOLZARB MASALALARI RESPUBLIKA ILMIY-AMALIY KONFERENSIYASI 16-iyun, 2023- yil YANGILANAYOTGAN OʻZBEKISTON TARAQQIYOTIDA IQTISODIY FANLARNI OʻQITISHNING DOLZARB MASALALARI RESPUBLIKA ILMIY-AMALIY KONFERENSIYASI 16-iyun, 2023- yil – 110 000 2 San’at – 210 000 0 0 0 0 0 0 0 0 0 3 Guma nitar fanlar -220 000 0 0 0 0 0 0 0 0 0 4 Jurnal istika va ijtimo iy axbor ot - 310 000 0 0 0 0 0 0 0 0 0 5 Bizne s va boshq aruv – 320 000 552 142 410 217 85 13 2 335 57 27 8 6 Huqu q – 330 000 184 27 157 72 0 0 0 0 0 7 Tabiiy fanlar - 410 000 0 0 0 0 0 0 0 0 0 8 Muha ndisli k ishi – 510 000 1720 286 1434 938 16 2 77 6 782 12 4 65 8 9 Komp yuter texnal ogiyal ari va 384 188 196 151 10 7 44 233 81 15 2 30 30 YANGILANAYOTGAN OʻZBEKISTON TARAQQIYOTIDA IQTISODIY FANLARNI OʻQITISHNING DOLZARB MASALALARI RESPUBLIKA ILMIY-AMALIY KONFERENSIYASI 16-iyun 2023- yil YANGILANAYOTGAN O ZBEKISTON TARAQQIYOTIDA IQTISODIY FANLARNI OʻQITISHNING DOLZARB MASALALARI RESPUBLIKA ILMIY-AMALIY KONFERENSIYASI 16-iyun, 2023- yil 31 infor matik a - 520 000 10 Ishlab chiqar ish va qayta ishlas h tarmo qlari- 530 000 133 78 55 66 39 27 67 39 28 11 Arxet ektura va qurilis h- - 540 000 395 131 264 178 85 93 217 46 17 1 12 Aloqa va axbor otlash tirish, teleko mmun ikatsi ya texnal ogiyal ari - 550 000 41 9 32 17 5 12 24 4 20 13 Qishl oq, o‘rmo n va baliq xo‘jali gi – 610 431 274 157 143 11 3 30 288 16 1 12 7 11 YANGILANAYOTGAN OʻZBEKISTON TARAQQIYOTIDA IQTISODIY FANLARNI OʻQITISHNING DOLZARB MASALALARI RESPUBLIKA ILMIY-AMALIY KONFERENSIYASI 16-iyun, 2023- yil OʻQITISHNING DOLZARB MASALALARI RESPUBLIKA ILMIY-AMALIY KONFERENSIYASI 16-iyun, 2023- yil 32 000 14 Qishq oq xo‘jali gi texnik asi - 620 000 91 65 26 29 23 6 62 42 20 15 Veter enariy a- 630 000 387 217 170 135 13 5 0 252 82 17 0 16 Qishl oq xo‘jali gi irregat siyasi va melior atsiya si - 640 000 0 0 0 0 0 0 0 0 0 17 Sog‘li qni saqlas h– 710 000 9777 609 9168 4600 44 1 41 59 5177 16 8 50 09 18 Ijtimo iy ta`min ot– 720 000 0 0 0 0 0 0 0 0 0 19 Xizm at ko`rsa tish – 810 000 264 195 69 93 36 57 171 15 9 12 32 32 YANGILANAYOTGAN OʻZBEKISTON TARAQQIYOTIDA IQTISODIY FANLARNI OʻQITISHNING DOLZARB MASALALARI RESPUBLIKA ILMIY-AMALIY KONFERENSIYASI 16-iyun, 2023- yil 20 Trans port– 820 000 215 88 127 119 63 56 96 25 71 21 Atrof- muhit muhof azasi- 830 000 24 15 9 14 11 3 10 4 6 22 Hayot iy faoliy at havfsi zligi - 840 000 0 0 0 0 0 0 0 0 0 Mutaxassisliklarni tanlashda sezilarli gender farqi mavjud bo`lib, bu ayniqsa o`rta va oliy o`quv yurtlarida kasb tanlashda seziladi. YANGILANAYOTGAN OʻZBEKISTON TARAQQIYOTIDA IQTISODIY FANLARNI OʻQITISHNING DOLZARB MASALALARI RESPUBLIKA ILMIY-AMALIY KONFERENSIYASI 16-iyun, 2023- yil Ta`lim xizmatlarining o`ziga xos xususiyatlari quyidagilardan iborat:  Ta`lim xizmatlarining o`ziga xos xususiyatlari quyidagilardan iborat:  ularning xar xil ne`mat turlariga tegishli ekanligi; ne`mat sifatida cheklanmaganligi; Q RESPUBLIKA ILMIY-AMALIY KONFERENSIYASI 16-iyun, 2023- yil iste`mol qiymatiga egaligi, ta`limga bo`lgan extiyojlarni qondiruvchi o`ziga xos tovar ekanligi (moddiy tavsifga ega emasligi, saqlash va yo`q qilib tashlashning mumkin emasligi);  ta`lim xizmatlarining individual tavsifga ega ekanligi;  ta`lim xizmatlarining individual tavsifga ega ekanligi;  ta`lim xizmatlarini ko`rsatish va iste`mol qilish jarayonining bir vaqtning o`zida ro`y berishi;  iste`molchilarning ta`lim xizmatlarining ko`rsatilishi jarayonida bevosita ishtirok etishi;  ta`limga sarflanadigan xarajatlardan oladigan samarani miqdor jixatidan baholashning qiyinligi;  ta`lim xizmatlariga talabning yuqori darajada egiluvchanligi. ta`lim xizmatlariga talabning yuqori darajada egiluvchanligi. Shu bois, ta`lim xizmatlarini rivojlantirishning ustuvor yo`nalishlarini belgilashda, birinchidan, mamlakatimizda ta`lim xizmatlarining etakchi sub`ekti bo`lgan ta`lim muassasalarining hozirgi holati va ulardagi mavjud muammolar, ikkinchidan, esa hozirgi davrda ta`limning mazmuni va uni tashkil etishdagi o`zgarishlarni e`tiborga olish zarur. Chunki bu o`zgarishlar fan-texnika taraqqiyotining ajralmas qismi bo`lib hisoblanadi. REFERENCES 1. O`zbekiston Respublikasi Qonunlari 2. O`zbekston Respublikasi Konstitutsiyasi. – T.: “O`zbekiston”, 2023y 3. O`zbekston Respublikasi «Ta`lim to`g‘risidagi» Qonuni 23.09.2020 y 4. Kadrlar tayyorlash milliy dasturi 1997 yil 29 avgust 5. O`zbekston Respublikasi Prezidentining Farmonlari va qarorlari 6. Prezidentimizning 2019 yil 6 sentyabrda “Professional ta`lim tizimini yanada takomillashtirishga doir qo`shimcha chora-tadbirlar to`g‘risida”gi 5812 sonli Farmoni. 6. Prezidentimizning 2019 yil 6 sentyabrda “Professional ta`lim tizimini yanada takomillashtirishga doir qo`shimcha chora-tadbirlar to`g‘risida”gi 5812 sonli Farmoni. 7. O`zbekiston Respublikasi Prezidentining 2020-yil 31-dekabrdagi “Malakalarni baholash tizimini tubdan takomillashtirish va mehnat bozorini malakali kadrlar bilan ta’minlash chora-tadbirlari to`g`risida”gi PQ-4939-son qarori. 7. O`zbekiston Respublikasi Prezidentining 2020-yil 31-dekabrdagi “Malakalarni baholash tizimini tubdan takomillashtirish va mehnat bozorini malakali kadrlar bilan ta’minlash chora-tadbirlari to`g`risida”gi PQ-4939-son qarori. 8. O ‘zbekiston Respublikasi Prezidentining “YOshlarni ma’naviy-axloqiy va jismoniy barkamol etib tarbiyalash, ularga Ta`lim-tarbiya berish tizimini sifat jihatidan yangi bosqichga ko‘tarish chora-tadbirlari to`g`risida” 15/08/2018 - 18:02 11699 qarori.. 9. Musaeva Sh.A. Marketing tadqiqotlari. Darslik “STAR-SEL” MChJ nashriyot va ijodiy bo‘limi. Samarqand-2023 9. Musaeva Sh.A. Marketing tadqiqotlari. Darslik “STAR-SEL” MChJ nashriyot va ijodiy bo‘limi. Samarqand-2023 10. Musaeva Sh.A. Integrallashtirilgan marketing kommunikatsiyasiO‘quv qo‘llanma “Mahorat” nashriyoti, Samarqand – 2022 11. Musaeva Sh.A., Usmonova D.I. Innovatsion marketing “TURON EDITION” 2021 yil uchun o‘quv qo‘llanma.Kalyujnovoy, A.YA. YAkobsona. – M.: «Omega-L», 2009. – 476 s. 34
https://openalex.org/W3212804023	https://europepmc.org/articles/pmc8564389?pdf=render	English	null	Predictive Model of Dysphagia and Brain Lesion-Symptom Mapping in Acute Ischemic Stroke	Frontiers in aging neuroscience	2,021	cc-by	9,189	Predictive Model of Dysphagia and Brain Lesion-Symptom Mapping in Acute Ischemic Stroke Lulu Zhang1†, Xiang Tang1†, Can Wang1, Dongxue Ding1, Juehua Zhu1, Yun Zhou1, Shanshan Diao1, Yan Kong1, Xiuying Cai1, Cuiping Li2, Ye Yao3,4,5* and Qi Fang1* Lulu Zhang1†, Xiang Tang1†, Can Wang1, Dongxue Ding1, Juehua Zhu1, Yun Zhou1, Shanshan Diao1, Yan Kong1, Xiuying Cai1, Cuiping Li2, Ye Yao3,4,5* and Qi Fang1* 1 Department of Neurology, First Afﬁliated Hospital of Soochow University, Suzhou, China, 2 Shanghai Zhiyu Software Technology Co., Ltd., Shanghai, China, 3 Department of Biostatistics, School of Public Health, Fudan University, Shanghai, China, 4 National Clinical Research Center for Aging and Medicine, Huashan Hospital, Fudan University, Shanghai, China, 5 Key Laboratory of Public Health Safety of Ministry of Education, Fudan University, Shanghai, China Background and purpose: Early recognition and management of post-stroke dysphagia (PSD) based on MRI may reduce the incidence of complications. Combining clinical symptoms with applications of MRI, we aimed to identify the risk factors of PSD, develop a prediction scale with high accuracy and map key dysphagia brain areas. Keywords: post-stroke dysphagia, MRI, water-swallowing test, volume-viscosity swallow test, voxel-based lesion-symptom mapping Methods: A total of 275 acute ischemic stroke patients were enrolled in this study, and 113 (41.1%) patients were diagnosed with PSD. All patients underwent the water-swallowing test (WST) and volume-viscosity swallow test (V-VST) within ﬁrst 24 h following admission to assess swallowing. Vascular factors were evaluated and MRI brain scans were obtained within 3 days after symptom onset for each participant admitted to the hospital. T-test, chi-squared test and Fisher’s exact test were used to investigate the associations of various patient characteristics with dysphagia, and multivariable logistic regression models were used to construct a prediction scale. Scale accuracy was assessed using receiver operating characteristic (ROC) analysis. We extracted white matter hyperintensities for each patient as potential brain lesions. Voxel-based lesion-symptom mapping (VLSM) was used to identify key brain areas for dysphagia. Edited by: Aurel Popa-Wagner, University of Medicine and Pharmacy of Craiova, Romania Reviewed by: Paul Muhle, Universitätsklinikum Münster, Germany Jiyang Jiang, University of New South Wales, Australia Correspondence: Ye Yao yyao@fudan.edu.cn Qi Fang fangqi@suda.edu.cn †These authors have contributed equally to this work and share ﬁrst authorship Correspondence: Ye Yao yyao@fudan.edu.cn Qi Fang fangqi@suda.edu.cn †These authors have contributed equally to this work and share ﬁrst authorship Results: Risk factors related with PSD were older age, history of atrial ﬁbrillation, higher fasting blood glucose, NIH stroke scale, TOAST classiﬁcation, progressive stroke, middle cerebral artery lesion and anterior cerebral artery lesion. Three variables with most signiﬁcant associations, including NIH stroke scale, TOAST classiﬁcation and progressive stroke, combined with age and gender, were used to construct a dysphagia prediction scale with high accuracy (AUC = 0.86). VLSM identiﬁed left inferior parietal gyrus as a key brain region for PSD. Received: 04 August 2021 Accepted: 29 September 2021 Published: 20 October 2021 ORIGINAL RESEARCH published: 20 October 2021 doi: 10.3389/fnagi.2021.753364 Abbreviations: PSD, post-stroke dysphagia; VLSM, voxel-based lesion-symptom mapping; IPG, inferior parietal gyrus; NIHSS, National Institutes of Health Stroke Scale; WST, water-swallowing test; V-VST, volume-viscosity swallow test. Study Design and Participants Swallowing is a complex and bilateral neuromuscular mechanism that requires multiple brain regions to control the involved muscles and structures (Leopold and Daniels, 2010). Post-stroke dysphagia (PSD) is a common and disabling condition that occurs in up to 70% of patients, and it is associated with pneumonia, malnutrition, prolonged hospital stay, increased mortality and poor long-term outcome. Mild and moderate dysphagia often resolve within the ﬁrst week, but almost one-third of patients develop post-stroke pneumonia that requires treatment (Hinchey et al., 2005). Study participants were from acute ischemic stroke (AIS) patients admitted to our stroke unit between October 2017 and May 2018. Inclusion criteria were based on the diagnosis of AIS as conﬁrmed on diﬀusion-weighted MRI (DWI). Patients with the following conditions were excluded from the study: (1) preexisting dysphagia; (2) concomitant intracerebral hemorrhage; (3) brain tumors; (4) severe hepatic and renal dysfunction; (5) end-stage severe disease; and (6) concomitant diseases likely to cause dysphagia, including dementia. All subjects were divided into two groups according to the following swallowing assessment: (1) patients with PSD; and (2) patients without PSD. Therefore, early recognition and management may reduce the incidence of complications, but these goals remain challenging in clinical practice. PSD remains a neglected area of research despite its high impact on prognosis. Several studies identiﬁed the prevalence of PSD and risk factors to predict dysphagia. Elderly patients with stroke have a greater chance to present swallowing dysfunction because of a reduced cough reﬂex, alterations in swallowing, and breathing coordination (Marik and Kaplan, 2003). The National Institute of Health Stroke Scale (NIHSS) score may be used as an adjunct to predict PSD with moderate sensitivity and speciﬁcity (Labeit et al., 2018). PSD was assessed using the water-swallowing test (WST) and volume-viscosity swallow test (V-VST). WST was performed using 30 ml of water while sitting at a 90◦angle (Osawa et al., 2013). V-VST is a clinical assessment that uses boluses of diﬀerent volumes (5, 10, and 20 ml) and viscosities (thin liquid, nectar-like, and spoon thick) in combination with a pulse-oximeter to evaluate the eﬃcacy and safety of swallowing with minimum risk for the patient. Signs and symptoms of impaired eﬃcacy of swallowing included oral residue, eﬃciency of labial seal, fractional swallow and pharyngeal residue. Signs of impaired safety of swallowing included changes in voice quality (including wet voice), coughing and decreased oxygen saturation (SpO2) ≥3% from basal level (Rofes et al., 2018). INTRODUCTION stroke healthcare settings. The present study took advantage of comprehensive clinical data from early clinical swallowing examinations and voxel-based image analysis approaches. The aims of this study were (1) to establish risk factors and construct a predictive model of dysphagia and (2) map lesion-symptoms for PSD to predict PSD in AIS and help healthcare settings. Non-invasive magnetic resonance imaging (MRI) provides high sensitivity and speciﬁcity for ischemic stroke. The latest developments of various MRI imaging techniques provide a comprehensive understanding of brain function. It was widely recognized that MRI play critical role in the diagnosis of stroke, particularly in the hyperacute stage. The applications of MRI make sense to identify AIS patients rapidly and facilitate clinical decision making to convert severe stroke to minor stroke. Citation: Conclusion: Risk factors of PSD were identiﬁed and a predictive model of dysphagia was constructed intelligently and automatically. The left inferior parietal gyrus was identiﬁed as a key brain area for dysphagia, which provides a new symptom-based treatment target for early rehabilitation in the future. Zhang L, Tang X, Wang C, Ding D, Zhu J, Zhou Y, Diao S, Kong Y, Cai X, Li C, Yao Y and Fang Q (2021) Predictive Model of Dysphagia and Brain Lesion-Symptom Mapping in Acute Ischemic Stroke. Front. Aging Neurosci. 13:753364. doi: 10.3389/fnagi.2021.753364 October 2021 \| Volume 13 \| Article 753364 1 Frontiers in Aging Neuroscience \| www.frontiersin.org Predictive Model and Lesion-Symptom Mapping Zhang et al. Study Design and Participants Validation studies showed excellent psychometric properties for V-VST (sensitivity 0.94 and speciﬁcity 0.88) in detecting PSD (Vilardell et al., 2017). V-VST was performed within 24 h after admission before oral feeding, with the exception of patients with low levels of consciousness. Patients who presented with any signs of impaired eﬃcacy and/or safe swallowing were considered as having PSD. A trained neurologist who was blinded to the patient’s clinical results examined all patients. Previous research showed associations between distinct lesion locations and PSD. The brain stem (especially the medulla and pons) is the central control of swallowing, but cortical and subcortical regions play integral roles in mediating swallowing (Wilmskoetter et al., 2019). An independent correlation between leukoaraiosis severity and PSD was previously reported, which emphasizes the important role of subcortical white matter in swallowing. Disruption of cortical–subcortical white matter connections resulted in dysphagia by lowering the threshold of the input to the swallowing center (Toscano et al., 2015). Previous reports stated that brain lesions in the right hemisphere caused pharyngeal dysphagia, and lesions in the left hemisphere caused dysfunction during the oral phase of swallowing (Ickenstein et al., 2005). However, there are no deﬁnite conclusions about whether one hemisphere is more involved than the brain stem and whether one hemisphere was dominant. The ethics committee of our hospital approved the study protocol (No. 2021041). Informed consent was obtained from all patients or legal guardians if the patient’s communication and/or understanding were impaired. All of these ﬁndings are inconsistent due to the comparatively small sample sizes, diﬀerent dysphagia screening methods, various inclusion criteria and inaccurate classiﬁcation of brain regions. Most previous research lacked comprehensive clinical and imaging studies. Therefore, it is urgent to ﬁnd a clear picture of PSD with risk factors and clinical outcomes to improve Statistical Tests Continuous variables are presented as the means ± standard deviation (SD) or minimum-maximum and were analyzed using Student’s t-test and one-way ANOVA. Categorical data were examined using the chi-squared test or Fisher’s exact test. A multivariate logistic regression was used to construct a PSD prediction scale, and its accuracy was assessed using receiver operating characteristic (ROC) analysis with p-values extracted by permutation tests. Voxel-based lesion-symptom mapping (VLSM) (Bates et al., 2003) was used to analyze the relationship between tissue lesions (white matter hyperintensities) and PSD on a voxel-by-voxel basis to help identify key brain areas of PSD. For each voxel, patients were divided into two groups according to whether they did or did not have a lesion aﬀecting that voxel. PSD or not were then compared for these two groups, yielding a logistic regression for each voxel. Moreover, we added covariates (e.g., age, gender, and lesion size) in the regression analysis to remove their eﬀects. In addition, we applied cluster analysis to do multiple comparison corrections. Statistical analyses were performed using MATLAB software, version 2019b (MathWorks Inc.). A statistical threshold corrected at cluster- level for familywise error correction at p = 0.05 was set to reduce type-1 errors in the voxel-level analysis. The image preprocessing includes registration for aligning all images into the same coordinate system, imaging normalization and white matter hyperactivity extraction. Although the images were acquired during the same session, a certain amount of subject motion and movement is unavoidable between the sequences, which leads to image misalignment. Basically, we extracted white matter hyperintensities from T2 images and stroke lesions from DWI images in the similar way with a stricter parameter, 0.5. In addition, T1 images ware applied to help image preprocessing including normalizations. For each subject, the T2 images were aligned with the T1 images using the 3D rigid body image registration algorithm proposed by Ashburner et al. (2000). We used the non-rigid normalization toolbox and transformed the T1 images into a standard template of MNI (Montreal Neurological Institute) space (Smith et al., 2018). T2 and DWI images were also transformed into standard space based on the deformation ﬁeld of the corresponding T1 images. We used the Lesion Segmentation Toolbox (Schmidt et al., 2012) to identify white matter hyperintensities (lesion localized) and stroke lesions automatically of white matter regions and white matter anatomy of gray matter regions for each subject. RESULTS A total of 275 patients fulﬁlled the inclusion criteria and were included in the study (Supplementary Figure 1 summarized details of study recruitment). A total of 113 (41.1%) of the patients had PSD: 44 patients were assessed as V grade in the WST and were directly given nasal feeding. Other 69 patients were assessed using the V-VST. A total of 14.5% (n = 10) had clinical signs of impaired safety, 18.8% (n = 13) had impaired eﬃcacy, and 66.7% (n = 46) had both swallowing impairments. The demographic and clinical characteristics of the study population and the diﬀerences between PSD and non- PSD patients are shown in Table 1. The groups signiﬁcantly diﬀered in age, and PSD patients were older than the non- PSD patients (67.92 ± 12.22 vs. 63.38 ± 13.19 years, t = 2.89, P < 0.01). Patients with history of AF (29.9% vs. 9.4%, χ2 = 11.05, P < 0.01) and higher FBG (6.59 ± 2.84 vs. 5.54 ± 1.74 µmol/l, t = 3.78, P < 0.01) were more likely to suﬀer from dysphagia. PSD patients showed higher scores in NIHSS (10.50 ± 6.95 vs. 3.38 ± 3.30, t > 11.34, P < 0.01) and often were characterized by concomitant stenosis or occlusion of the MCA (56.9% vs. 21.8%, χ2 = 11.85, P < 0.01) and ACA (3.7% vs. 1.2%, Z = 7.05, P < 0.01). Patients with progressive stroke had a higher chance of exhibiting PSD (31.4% vs. 3.2%, χ2 = 28.03, P < 0.01). We did a ROI analysis of stroke lesions in PSD and identiﬁed temporal lobe (815.40 ± 1305.77 vs. 548.93 ± 723.34, t = 2.02, P < 0.05), hippocampus (318.61 ± 331.74 vs. 236.38 ± 273.14, t = 2.06, P < 0.05), basal nucleus (717.36 ± 701.56 vs. 528.68 ± 495.54, Imaging Procedures All patients were scanned in a 3T MR scanner (MAGNETOM Skyra; Siemens Healthineers, Erlangen, Germany). A 20-channel brain array coil was used for signal reception. The images obtained included transverse T1-weighted turbo spin-echo (TSE) images [repetition time (ms)/echo time (ms), 700/14; section thickness, 3 mm; intersection gap, 0.5 mm; ﬁeld of view, 25 cm; matrix, 384 × 336] and transverse T2-weighted TSE images [repetition time (ms)/eﬀective echo time (ms) 6,000/124; section October 2021 \| Volume 13 \| Article 753364 Frontiers in Aging Neuroscience \| www.frontiersin.org 2 Predictive Model and Lesion-Symptom Mapping Zhang et al. progressively worsened after regular treatment for cerebral infarction, and the NIHSS score increased no less than 2 points (Zhang et al., 2017). thickness, 3 mm; intersection gap, 0.3 mm; ﬁeld of view, 25 cm; matrix, 384 × 336]. DWI was obtained to calculate an apparent diﬀusion coeﬃcient using a 2D echo planar imaging sequence with multiple b-value acquisitions (0, 100, 800, 1,000, and 1,500 s/mm2), with the diﬀusion-sensitizing gradients applied along the X, Y, and Z axes. MRI brain scans were obtained within 3 days after symptom onset for each participant admitted to the hospital. Lesions were localized according to hemisphere (right, left, or bilateral). thickness, 3 mm; intersection gap, 0.3 mm; ﬁeld of view, 25 cm; matrix, 384 × 336]. DWI was obtained to calculate an apparent diﬀusion coeﬃcient using a 2D echo planar imaging sequence with multiple b-value acquisitions (0, 100, 800, 1,000, and 1,500 s/mm2), with the diﬀusion-sensitizing gradients applied along the X, Y, and Z axes. MRI brain scans were obtained within 3 days after symptom onset for each participant admitted to the hospital. Lesions were localized according to hemisphere (right, left, or bilateral). Statistical Tests We subtracted stroke lesions found by DWI images from white matter hyperintensities extracted by T2 images, which were considered potential lesions (Debette and Markus, 2010). Two trained neurologists who were blinded examined all of the preprocessing results to assure the imaging quality. Frontiers in Aging Neuroscience \| www.frontiersin.org Vascular Risk Factors and Other Co-variates We collected the following variables: age, gender, medical history (including hypertension, diabetes mellitus, smoking, history of stroke, atrial ﬁbrillation, and other heart diseases), clinical data on admission (including relevant laboratory indicators, stroke severity as measured by the NIHSS, and thrombolytic treatment), location of stroke (left, right, bilateral hemispheres or posterior circulation) and aﬀected vessels [anterior cerebral artery (ACA), middle cerebral artery (MCA), internal carotid artery (ICA) and posterior cerebral artery (PCA)]. The etiology of stroke was determined according to TOAST, which refers to ﬁve classiﬁcations: (1) large-artery atherosclerosis (LAA), (2) cardioembolism (CE), (3) small- vessel occlusion (SVO), (4) stroke of other determined etiology (SOE), and (5) stroke of undetermined etiology (SUE). The treating team made the diagnoses of progressive stroke. The following diagnostic criteria were used for progressive stroke: (a) the disease course extended from 6 h to 7 days; (b) the primary nervous symptoms and signs of the cerebral infarction October 2021 \| Volume 13 \| Article 753364 Frontiers in Aging Neuroscience \| www.frontiersin.org 3 Predictive Model and Lesion-Symptom Mapping Zhang et al. t = 2.42, P < 0.01) and internal capsule (303.10 ± 584.71 vs. 118.25 ± 308.46, t = 3.18, P < 0.001) in left hemisphere to be associated with PSD as shown in Table 1. Post-stroke pneumonia was not associated with a higher chance of having PSD. A strong association was found between dysphagia and stroke using TOAST classiﬁcations (χ2 = 39.51, P < 0.01). We investigated the associations of dysphagia and stroke using the TOAST classiﬁcation as shown in Figure 1. Patients with LAA and CE suﬀered dysphagia more often (LAA: OR = 2.81, P < 0.01; CE: OR = 2.54, P < 0.05), and patients with SVO suﬀered less to be associated with PSD as shown in Table 1. Post-stroke pneumonia was not associated with a higher chance of having PSD. A strong association was found between dysphagia and TABLE 1 \| Demographic and clinical data of patients with dysphagia and controls. Vascular Risk Factors and Other Co-variates Demographic and clinical data Dysphagia Controls t/Z/χ 2 p (n = 113) (n = 162) Age (years) 67.92 ± 12.22, 31.00–93.00 63.38 ± 13.19, 22.00–85.00 t = 2.89 2.10 × 10−3* Gender male/female 75/38 107/55 χ2 = 0.00 0.96 Systolic blood pressure (mmHg) 145.37 ± 21.33, 103.00–201.00 142.77 ± 21.01, 104.00–207.00 t = 1.00 0.32 Diastolic blood pressure (mmHg) 81.30 ± 13.12, 40.00–118.00 80.62 ± 12.89, 7.00–117.00 t = 0.43 0.67 History of hypertension yes/no 90/23 111/51 χ2 = 4.19 0.04 History of diabetes yes/no 26/87 31/131 χ2 = 0.61 0.44 Smoking yes/quit/no 30/4/79 39/4/119 Z = 0.32 0.75 History of AF† yes/no 26/87 14/148 χ2 = 11.05 8.85 × 10−4* Other heart diseases yes/no 6/107 5/157 Z = 0.90 0.37 Previous stroke yes/no 23/90 21/141 χ2 = 2.71 0.10 Triglyceride (mmol/L) 1.27 ± 0.53, 0.51–3.83 1.47 ± 0.95, 0.38–9.11 t = −1.97 0.05 Total cholesterol (mmol/L) 4.26 ± 0.99, 2.21–6.93 4.19 ± 1.09, 0.65–8.21 t = 0.55 0.58 LDLC (mmol/L) 2.43 ± 0.81, 0.69–4.88 2.38 ± 0.83, 0.65–5.15 t = 0.56 0.57 Creatinine (µmol/L) 70.19 ± 20.08, 38.50–191.70 69.37 ± 17.12, 32.50–121.00 t = 0.36 0.72 Uric acid (µmol/L) 285.82 ± 95.45, 9.30–553.50 297.65 ± 88.86, 92.50–574.20 t = −1.05 0.29 Fasting blood glucose (µmol/L) 6.59 ± 2.84, 3.43–19.03 5.54 ± 1.74, 3.34–12.67 t = 3.78 1.00 × 10−4* Homocysteine‡ (µmol/L) 12.90 ± 10.74, 2.40–97.00 11.43 ± 7.82, 3.10–56.20 t = 1.17 0.12 Hemoglobin A1c‡ (%) 6.93 ± 1.88, 3.70–12.30 6.70 ± 1.88, 4.90–14.80 t = 0.80 0.21 ECG† (AF) yes/no 30/83 19/143 χ2 = 9.98 1.58 × 10−3* NIH stroke scale 10.50 ± 6.95, 0.00–36.00 3.38 ± 3.30, 0.00–19.00 t = 11.34 5.50 × 10−25 NIH stroke scale (After discharge) 8.58 ± 5.98, 0.00–35.00 2.22 ± 2.55, 0.00–16.00 t = 12.06 1.93 × 10−27 TOAST classiﬁcation† 56/31/20/2/4 42/21/79/10/10 χ2 = 39.51 5.46 × 10−8 Progressive stroke yes/no 27/86 5/157 χ2 = 28.03 1.20 × 10−7 Lesioned hemi left/right/both 43/52/18 84/55/23 χ2 = 5.37 0.07 Brain stem lesioned yes/no 25/88 25/137 χ2 = 2.00 0.16 MCA† Affected yes/no 41/72 29/133 χ2 = 11.85 5.75 × 10−4* PCA† Affected yes/no 5/108 16/146 χ2 = 2.81 0.09 ICA† Affected yes/no 21/92 16/146 χ2 = 4.33 0.04 ACA† Affected yes/no 4/109 2/160 Z = 7.05 1.76 × 10−12** BA† Affected yes/no 6/107 4/158 Z = 0.00 1.00 VA† Affected yes/no 5/108 3/159 Z = 0.00 1.00 Thrombolytic yes/no 19/94 23/139 χ2 = 0.35 0.55 Epilepsy yes/no 3/110 4/158 Z = 0.00 1.00 Lesion size of temporal lobe (left) (mm∧3) 815.40 ± 1305.77 548.93 ± 723.34 t = 2.02 0.02 (Continued) TABLE 1 \| Demographic and clinical data of patients with dysphagia and controls. Vascular Risk Factors and Other Co-variates The incidence rate of total population is 41.1%. Large-artery atherosclerosis and cardioembolism hold odd ratios signiﬁcantly larger than 1 with dysphagia (OR = 2.81, p = 5.68 × 10−5 and OR = 2.54, p = 2.57 × 10−3), while small-vessel occlusion holds an odd ratio signiﬁcantly lower than 1 (OR = 0.23, p = 1.29 × 10−7). FIGURE 1 \| Dysphagia and stroke associations by TOAST classiﬁcation. (A) Composition ratios of TOAST classiﬁcation (blue: large-artery atherosclerosis, orange: cardioembolism, green: small-vessel occlusion, violet: stroke of other determined etiology and red: stroke of undetermined etiology) in patients with dysphagia and controls, which indicates a signiﬁcantly strong association between dysphagia and stroke by TOAST classiﬁcations (Chi-squared test, χ2 = 39.51, p = 5.46 × 10−8). (B) Incidence rate of dysphagia in different TOAST classiﬁcations (patients with dysphagia in green and controls in blue). The incidence rate of total population is 41.1%. Large-artery atherosclerosis and cardioembolism hold odd ratios signiﬁcantly larger than 1 with dysphagia (OR = 2.81, p = 5.68 × 10−5 and OR = 2.54, p = 2.57 × 10−3), while small-vessel occlusion holds an odd ratio signiﬁcantly lower than 1 (OR = 0.23, p = 1.29 × 10−7). often (OR = 0.23, P < 0.01). Patients of other determined and UD did not show signiﬁcant diﬀerences. doing multivariate analysis. The three variables with the most signiﬁcant associations, NIH stroke scale, TOAST classiﬁcation and progressive stroke, were combined with age and gender and used to construct a dysphagia prediction scale using a multivariate logistic model. As shown in Table 2, NIH stroke scale (OR = 1.27, t = 6.30, P < 0.01) and progressive stroke (OR = 5.65, t = 2.97, P < 0.01) showed the most signiﬁcant associations with PSD in multivariate statistical analysis, and the Vascular Risk Factors and Other Co-variates (Continued) October 2021 \| Volume 13 \| Article 753364 Frontiers in Aging Neuroscience \| www.frontiersin.org 4 Predictive Model and Lesion-Symptom Mapping Zhang et al. TABLE 1 \| (Continued) Demographic and clinical data Dysphagia Controls t/Z/χ 2 p (n = 113) (n = 162) Lesion size of hippocampus (left) (mm∧3) 318.61 ± 331.74 236.38 ± 273.14 t = 2.06 0.02 Lesion size of basal nucleus (left) (mm∧3) 717.36 ± 701.56 528.68 ± 495.54 t = 2.42 8.20 × 10−3 Lesion size of internal capsule (left) (mm∧3) 303.10 ± 584.71 118.25 ± 308.46 t = 3.18 8.00 × 10−4* Continuous data are shown as mean ± SD, minimum and maximum values in patients with dysphagia and controls with statistical signiﬁcance based on two sample t-test. Categorical data differences in patients and controls are represented with statistical signiﬁcance based on chi-squared test (χ2 and p) or Fisher exact test (Z and p). p < 0.005, p < 1.00 × 10−6. †AF, refers to atrial ﬁbrillation; LDLC, refers to low density lipoprotein cholesterol; ECG, refers to Electrocardiogram; MCA, refers to middle cerebral artery; PCA, refers to posterior cerebral artery; ICA, refers to internal carotid artery; ACA, refers to anterior cerebral artery; BA, refers to basilar artery and VA, refers to vertebral artery. TOAST refers to ﬁve classiﬁcations: (1) large-artery atherosclerosis, (2) cardioembolism, (3) small-vessel occlusion, (4) stroke of other determined etiology, and (5) stroke of undetermined etiology. ‡87 patients with dysphagia and 131 controls took part in Homocysteine tests, while 66 patients with dysphagia and 100 controls attended Hemoglobin A1c tests. FIGURE 1 \| Dysphagia and stroke associations by TOAST classiﬁcation. (A) Composition ratios of TOAST classiﬁcation (blue: large-artery atherosclerosis, orange: di b li ll l l i i l k f h d i d i l d d k f d i d i l ) i i i h d h i d FIGURE 1 \| Dysphagia and stroke associations by TOAST classiﬁcation. (A) Composition ratios of TOAST classiﬁcation (blue: large-artery atherosclerosis, orange: cardioembolism, green: small-vessel occlusion, violet: stroke of other determined etiology and red: stroke of undetermined etiology) in patients with dysphagia and controls, which indicates a signiﬁcantly strong association between dysphagia and stroke by TOAST classiﬁcations (Chi-squared test, χ2 = 39.51, p = 5.46 × 10−8). (B) Incidence rate of dysphagia in different TOAST classiﬁcations (patients with dysphagia in green and controls in blue). Post-stroke Dysphagia Risk Factors and Prediction Scale In order to avoid the inﬂuence of multiple comparison, we raised the threshold to (p < 1.00 × 10−6 with ∗∗in Table 1) when October 2021 \| Volume 13 \| Article 753364 Frontiers in Aging Neuroscience \| www.frontiersin.org 5 Predictive Model and Lesion-Symptom Mapping Zhang et al. TABLE 2 \| Multivariable logistic regression model for predicting patients with dysphagia. Variables Odds ratio 95% CI t P-value Age 1.03 1.00, 1.06 1.72 0.09 Gender 1.78 0.87, 3.66 1.59 0.11 NIH stroke scale 1.27 1.18, 1.38 6.30 3.04 × 10−10 Large-artery atherosclerosis 1.36 0.39, 4.79 0.48 0.63 Cardioembolism 0.88 0.20, 3.90 −0.17 0.86 Small-vessel occlusion 0.59 0.15, 2.23 −0.79 0.43 Progressive stroke 5.65 1.80, 17.76 2.97 0.0029* p < 0.005, p < 1.00 × 10−6. TABLE 2 \| Multivariable logistic regression model for predicting patients with dysphagia. Once the predictive variables were determined and the accuracy was conﬁrmed by the ROC analysis, an online calculator tool was constructed to help neurologists to acquire PSD risks of AIS patients intelligently and automatically (with risk scores and classiﬁcations included, http://180.167.250.222:10080/ Online-Dysphagia-Risk-Calculator-Tool-for-AISPatients.html, Supplementary Figure 2). DISCUSSION Combining clinical symptoms with applications of MRI (T1, T2, and DWI sequences) and VLSM analysis methods, our study demonstrated two major ﬁndings in acute ischemic stroke patients: (1) potential clinical risk factors of the onset of dysphagia following acute ischemic stroke, including older age, history of atrial ﬁbrillation, higher fasting blood glucose, NIH stroke scale, TOAST classiﬁcation, progressive stroke, middle cerebral artery lesion and anterior cerebral artery lesion with a PSD prediction scale; and (2) lesions in diﬀerent anatomical locations in patients with AIS showed speciﬁc and functional connections between PSD and left inferior parietal gyrus using VLSM. FIGURE 2 \| ROC curves generated for stroke with dysphagia. Receiver operating characteristic (ROC) curve is generated for NIH stroke scale (NIHSS), TOAST classiﬁcation and progressive stroke (age and gender effects included) with dysphagia (red line, AUC = 0.86, permutation p < 1 × 10−5) based on multiple logistic regression. In addition, ROC curves are generated separately for NIHSS (orange, AUC = 0.84), TOAST classiﬁcation (blue, AUC = 0.73) and progressive stroke (green, AUC = 0.68) with age and gender effects included. MRI has been proven to be high sensitivity and speciﬁcity for ischemic stroke, particularly in the hyperacute stage. Diﬀerent MRI methods have been worldwide applied in the assessment of AIS patients in clinical practice (Asaithambi et al., 2020). Combining clinical symptoms with applications of MRI, we established a predictive model of dysphagia in AIS patients intelligently and automatically. With VLSM, the left inferior parietal gyrus was identiﬁed as a key brain area for dysphagia. After the automatic operation of the conventional MRI sequences mentioned above and VLSM analysis methods, the eﬃciency of MRI applications was improved. The role of MRI has broadened and progressed from making diagnoses to providing important information for clinical decision making and AIS patients management. An increasing body of evidence suggest that the physiology revealed by MRI can be an advance over time in clinical decisions in the management of AIS patients, which ﬁnally can improve the prognosis of patients (Campbell and Parsons, 2018). FIGURE 2 \| ROC curves generated for stroke with dysphagia. Receiver operating characteristic (ROC) curve is generated for NIH stroke scale (NIHSS), TOAST classiﬁcation and progressive stroke (age and gender effects included) with dysphagia (red line, AUC = 0.86, permutation p < 1 × 10−5) based on multiple logistic regression. Voxel-Based Lesion-Symptom Mapping of Post-stroke Dysphagia The VLSM was used to identify lesions (white matter hyperintensities) that were strongly connected with PSD in 275 patients with AIS. We found a list of voxels with potential relationships. To eliminate noise eﬀects, we performed a cluster analysis and found that the largest cluster (greater than 100 mm3) fell on the left IPG with the center at MNI coordinates: X = −38, Y = −39, Z = 51, t = 3.59, P < 0.0001). Figure 3 showed colorized depictions of VLSM logistic regression analysis results evaluating lesion and PSD relationships on a voxel-by-voxel basis. The left IPG had a speciﬁc and functional connection with PSD in the VLSM analysis. p < 0.005, **p < 1.00 × 10−6. other variables, including age, gender and TOAST classiﬁcations, showed no association. A ROC analysis was performed to examine the accuracy of the dysphagia prediction scale. As shown in Figure 2, NIH stroke scale, TOAST classiﬁcation and progressive stroke together showed a signiﬁcantly high AUC (area under the ROC curve) of 0.86 with a permutation P < 1 × 10−5, which indicated higher accuracy of the dysphagia prediction scale. The AUC of predictions based on NIH stroke scale (orange curve, AUC = 0.84), TOAST classiﬁcation (blue curve, AUC = 0.73) and progressive stroke (green curve, AUC = 0.68) separately are also represented. Moreover, in the samples with images, as shown in Supplementary Figure 3, NIH stroke scale, TOAST classiﬁcation and progressive stroke together showed AUC of 0.84 with a permutation P < 1 × 10−5. NIH stroke scale, TOAST classiﬁcation, progressive stroke and regional lesion location together showed a signiﬁcantly higher AUC of 0.85. Age and gender eﬀects were included in the multivariate logistic model to construct prediction scales. DISCUSSION In addition, ROC curves are generated separately for NIHSS (orange, AUC = 0.84), TOAST classiﬁcation (blue, AUC = 0.73) and progressive stroke (green, AUC = 0.68) with age and gender effects included. FIGURE 2 \| ROC curves generated for stroke with dysphagia. Receiver operating characteristic (ROC) curve is generated for NIH stroke scale (NIHSS), TOAST classiﬁcation and progressive stroke (age and gender effects included) with dysphagia (red line, AUC = 0.86, permutation p < 1 × 10−5) based on multiple logistic regression. In addition, ROC curves are generated separately for NIHSS (orange, AUC = 0.84), TOAST classiﬁcation (blue, AUC = 0.73) and progressive stroke (green, AUC = 0.68) with age and gender effects included. October 2021 \| Volume 13 \| Article 753364 Frontiers in Aging Neuroscience \| www.frontiersin.org 6 Zhang et al. Predictive Model and Lesion-Symptom Mapping FIGURE 3 \| Voxel-based lesion-symptom mapping for dysphagia symptoms. Representative slices from VLSM maps computed for dysphagia symptoms of 275 patients with stroke. These maps are colorized depictions of logistic regression analysis results evaluating patient performance on a voxel-by-voxel basis. The warmer the color is, patients with lesions on the corresponding voxel hold higher chance to develop dysphagia. Age, gender, NIH stroke scale, TOAST classiﬁcation and progressive stroke effects have been removed. FIGURE 3 \| Voxel-based lesion-symptom mapping for dysphagia symptoms. Representative slices from VLSM maps computed for dysphagia symptoms of 275 patients with stroke. These maps are colorized depictions of logistic regression analysis results evaluating patient performance on a voxel-by-voxel basis. The warmer the color is, patients with lesions on the corresponding voxel hold higher chance to develop dysphagia. Age, gender, NIH stroke scale, TOAST classiﬁcation and progressive stroke effects have been removed. PSD is prevalent in hospitalized patients, and it is associated with increased mortality and comorbidities. The reported incidence ranges substantially across studies from 50 to 80% (Woodhouse et al., 2018), depending on the diﬀerent evaluation methods. The prevalence of PSD (41.1%) in our study is consistent with other studies in which similar bedside clinical assessments were used. a consequence, it is assumed that cortical lesions in elderly patients are more likely to lead to PSD due to an already reduced compensatory reserve. With regards to gender, the impact on PSD has not yet been systematically explored. One retrospective study identiﬁed that female sex was associated with prolonged dysphagia and increased death (Smithard et al., 2007). Frontiers in Aging Neuroscience \| www.frontiersin.org DISCUSSION Transcranial magnetic stimulation (TMS) is a tool to alter brain activity in CNS disease and has been used successfully to study swallowing. It was reported that both ipsilesional high frequency and contralesional low frequency TMS could improvement in the symptoms and signs of dysphagia in the acute and chronic stroke (Michou and Hamdy, 2009). Lin et al. (2018) indicated that vagus nerve modulation using TMS in patients with stroke involving the brainstem can facilitate the recovery of swallowing function. In more recent literature, TMS was approved to have a positive value on the treatment of dysphagia after stroke by several authors (Verin and Leroi, 2009; Khedr and Abo-Elfetoh, 2010). Therefore, the IPG may be a new symptom-based treatment target of PSD. Therefore, the IPG may be a new symptom-based treatment target of PSD. A strong dominance of left hemispheric lesions was observed in our study, and this result conﬁrmed previous research that unilateral hemispheric lesions produce PSD (Daniels et al., 1996). However, there was no uniﬁed conclusion about the dominant role of the left and right cerebral hemispheres. Some functional imaging showed time-dependent shifts of neural activation from the left to right sensorimotor cortex during swallowing (Teismann et al., 2009). Recent evidence suggested that swallowing was represented bilaterally, but asymmetrically, with interindividual and characteristic diﬀerences (Saito et al., 2016). Previous studies attempted to identify the association between the location of brain lesions and PSD based on functional neuroimaging. The nucleus ambiguus in the rostral and ventrolateral medulla, the nucleus tractus solitarius and the reticular formation contain the central pattern generator for swallowing (Daniels et al., 2017). Cortical regions associated with primary and secondary sensorimotor areas were identiﬁed as important in swallowing in neuroimaging studies. Previous studies showed several regions related to swallowing, such as supramarginal gyrus, the precentral gyrus, post-central gyrus, insula, and cingulate cortex (Rolls, 2016). Multiple subcortical and white matter regions were also involved in abnormal swallowing (Malandraki et al., 2009). Gonzalez-Fernandez et al. (2008) found a positive association between dysphagia and acute stroke involving the internal capsule after adjusting for stroke severity (NIHSS) and stroke volume. Daniels and Foundas (1999) identiﬁed that periventricular white matter was highly associated with dysphagia. These regions may provide interconnections between cortical swallowing centers and the central pattern generator. DISCUSSION Our study conﬁrmed that an increased NIH stroke scale and progressive stroke were associated with PSD in patients with AIS. The PSD risk increased 1.27 times with every one NIHSS score increase, and patients with progressive stroke had a 5.65-times higher PSD risk. Higher NIHSS stroke scale and progressive stroke should be considered two main symptom targets during healthcare settings for PSD. due to a lot of reasons. First, the comparatively small sample sizes had limited our ability to predict which patients were more likely to develop swallowing dysfunction. Moreover, dysphagia is time-dependent and conclusions about the association between stroke locations and dysphagia required early assessment of both stroke and swallowing. Not only that, but studies were often conﬁned to a low number of roughly classiﬁed brain regions of interest (Suntrup et al., 2015). The limitations of functional imaging may also account for this lack of consensus. Functional neuroimaging requires a long time, and patients inevitably move, which compromises the quality of imaging data. There are also diﬀerent results from diﬀerent neuroimaging methods. Imaging ﬁndings change over time due to treatment, disease duration and severity (Fox, 2018). The present study showed that lesions in diﬀerent anatomical locations presented strong connections between the left IPG and PSD using VLSM. To the best of our knowledge, the IPG is widely accepted as associated with the egocentric, perceptual, exploratory extrapersonal, and personal, bodily components of neglect (Vallar and Calzolari, 2018). The roles of the IPG in conceptual processing and context-driven word production were also observed previously (Piai et al., 2018). The most common site of involvement in this study was the IPG. Swallowing is a complicated and multisensory process that depends on several sensory modalities. Therefore, it was not surprising that the IPG, as the secondary somatosensory cortex, may contribute to sensorimotor integration during swallowing. Our ﬁndings suggest the importance of intact sensory aﬀerents for the motor output network of swallowing (Galovic et al., 2013). This new approach using network mapping identiﬁed previously unappreciated connections in patients with PSD and provide a new symptom-based treatment target. The current treatments for PSD are diet improvement, and compensatory and rehabilitation methods. However, these rehabilitation methods are primarily experience-based for dysfunction of a typical swallowing organ, and no evidence shows that these methods may be used as a routine clinical treatment for dysphagia after stroke (Mitchell, 2016). DISCUSSION Considerable research found that stroke severity, as measured by NIHSS score, was an independent risk factor for PSD adjusting for other variables (Labeit et al., 2018; Toscano et al., 2019). Two studies evaluated the correlation of NIHSS score and dysphagia severity. And one article found that a cutoﬀvalue of 10 points carried the best sensitivity for predicting PSD (Jeyaseelan et al., 2015). Another article set the cutoﬀvalue at 12 points (Okubo et al., 2012). However, no research focused on the relationship between stroke subtype based on the TOAST criteria and PSD. The only related article found no signiﬁcant diﬀerence between TOAST classiﬁcation and the requirement for percutaneous endoscopic gastrostomy tube insertion in patients after stroke (Li et al., 2014). Our study provided evidence that patients with LAA and CE were more likely to suﬀer from PSD. In general, patients with LAA and CE had a higher NIHSS score than patients with other subtypes (Adams et al., 1999). Therefore, one Previous studies identiﬁed various clinical variables, such as, age, dysarthria, lesion volume and stroke severity. However, these ﬁndings were inconsistent. Our study identiﬁed certain demographic and clinical factors that inﬂuenced PSD to help identify patients with a greater risk of PSD at an early stage. Age and stroke severity are consistently considered predictors for PSD. In addition to that, a predictive model of PSD was constructed intelligently and automatically. Older age is consistently considered as a risk factor for dysphagia after stroke. Older patients are particularly vulnerable to PSD because of multiple age-related changes. On the one hand, elderly patients cannot compensate for sarcopenia (Wirth et al., 2016). On the other hand, a broader cortical activation in both hemispheres was identiﬁed measured by MEG during swallowing in elderly subjects compared to the young (Teismann et al., 2010). As October 2021 \| Volume 13 \| Article 753364 7 Predictive Model and Lesion-Symptom Mapping Zhang et al. potential explanation is that patients with LAA and CE tend to have severe neurological deﬁcits (Tan et al., 2018). There was little literature on the relationship between progressive stroke and PSD. The mechanism of progressive stroke was not clear. The hypotheses involved included propagation of thrombus, development of brain edema, production of inﬂammatory cytokines and metabolic factors. Besides, progressive stroke was reported to be positively correlated with large vessel disease or branch atheromatous disease (Saia and Pantoni, 2009). Frontiers in Aging Neuroscience \| www.frontiersin.org DISCUSSION There is no doubt that brain stem is the central control of swallowing and plays an absolutely important role in PSD as evidenced in by the high incidence of PSD in cases with lateral medullary stroke. However, in our study, only 50/275 (18.2%) of the patients were with brain stem lesions. The absence of signiﬁcant eﬀects in brain stem lesions in our study may result from small patient collectives. Our study had some limitations. First, we must consider that PSD in our study was not conﬁrmed via instrumental testing, such as videoﬂuoroscopy. Instead, swallowing was assessed clinically using bedside evaluations (WST and VVST), which may lack the ability to evaluate silent aspiration. Furthermore, instrumental validation could have allowed for a more detailed There was no consistent conclusion about the relationship between brain lesions and the occurrence of PSD. A number of studies in the past have strived to identify lesion patterns that predict dysphagia (Galovic et al., 2013; Suntrup-Krueger et al., 2017). However, all of these ﬁndings were inconsistent October 2021 \| Volume 13 \| Article 753364 Frontiers in Aging Neuroscience \| www.frontiersin.org 8 Predictive Model and Lesion-Symptom Mapping Zhang et al. DATA AVAILABILITY STATEMENT The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fnagi. 2021.753364/full#supplementary-material The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fnagi. 2021.753364/full#supplementary-material The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. Supplementary Figure 1 \| Details of study recruitment. AIS, acute ischemic stroke; TIA, transient ischemic attack. CONCLUSION In conclusion, the present study showed that dysphagia was a frequent symptom in patients with AIS. Possible predictors of PSD were identiﬁed and included older age, history of atrial ﬁbrillation, higher fasting blood glucose, NIH stroke scale, TOAST classiﬁcation, progressive stroke, middle cerebral artery lesion and anterior cerebral artery lesion, which were used to construct a dysphagia prediction scale with high accuracy. An online predictive model of dysphagia was constructed to help neurologists to acquire PSD risks of AIS patients intelligently and automatically. The left inferior parietal gyrus was a key brain region of PSD associated with dysphagia after AIS and may be a new symptom-based treatment target. ETHICS STATEMENT Supplementary Figure 2 \| Online dysphagia risk calculator tool for patients with acute ischemic stroke. The tool is available at http://180.167.250.222: 10080/Online-Dysphagia-Risk-Calculator-Tool-for-AISPatients.html. The studies involving human participants were reviewed and approved by the Ethics Committee of the First Aﬃliated Hospital of Soochow University approved the study protocol (No. 2021041). The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identiﬁable images or data included in this article. Supplementary Figure 3 \| ROC curves generated for stroke with dysphagia. Receiver operating characteristic (ROC) curve is generated for NIH stroke scale (NIHSS), TOAST classiﬁcation, progressive stroke and regional lesion location (age and gender effects included) with dysphagia (red line, AUC = 0.85, permutation p < 1 × 10−5) based on multiple logistic regression. ROC curves are generated for NIHSS, TOAST classiﬁcation and progressive stroke (blue, AUC = 0.84) with age and gender effects included. Supplementary Figure 3 \| ROC curves generated for stroke with dysphagia. Receiver operating characteristic (ROC) curve is generated for NIH stroke scale (NIHSS), TOAST classiﬁcation, progressive stroke and regional lesion location (age and gender effects included) with dysphagia (red line, AUC = 0.85, permutation p < 1 × 10−5) based on multiple logistic regression. ROC curves are generated for NIHSS, TOAST classiﬁcation and progressive stroke (blue, AUC = 0.84) with age and gender effects included. ACKNOWLEDGMENTS We thank the participants and investigators who took part in the study. AUTHOR CONTRIBUTIONS score to evaluate severity of PSD. However, instrumental assessment of swallowing function existed limitation in clinical practice, especially to AIS patients. The bedside evaluations we performed were not only quite simple and easy, but also had good sensitivity and speciﬁcity, which was conﬁrmed as a formalized assessment for dysphagia by both the European Society for Swallowing Disorders (ESSD) and expert group on PSD and nutritional management in China (Rofes et al., 2013). Second, the severity of PSD should be further assessed to examine the factors that aﬀect the severity of dysphagia. Third, No FLAIR images were acquired which would be ideal for our study. FLAIR can more clearly show the lesions that are concealed by the high signal of cerebrospinal ﬂuid on conventional T2WI, especially in the periventricular, brain surface, and subarachnoid area, which improves the sensitivity of diagnosis. We will pay particular attention to this point in future research. LZ and XT conceived the research and wrote the main manuscript text. CW and DD participated in the recruitment of the sample population. JZ, YZ, and SD acquired the data and analyzed the results. YK and XC helped in interpreted the results and revised the manuscript. CL designed the online calculator tool to acquire PSD risks of AIS patients. YY and QF guided the process, interpreted the results, and revised the manuscript. All authors read and approved the manuscript. FUNDING This work was supported by the National Key R&D Program of China (No. 2017YFC0114300 to QF), the Shanghai Sailing Program (No. 17YF142600 to YY), the Startup Foundation of School of Public Health (No. IDF201360/009 to YY), the Fudan University (No. KBH2306002 to YY), the National Natural Science Foundation of China (No. 82001125 to XT), and the Natural Science Foundation of Jiangsu Province (No. BK20180201 to XT). REFERENCES doi: 10.1007/s00455-017- 9824-0 Rofes, L., Vilardell, N., and Clave, P. (2013). Post-stroke dysphagia: progress at last. Neurogastroenterol. Motil. 25, 278–282. doi: 10.1111/nmo. 12112 Rolls, E. T. (2016). Functions of the anterior insula in taste, autonomic, and related functions. Brain Cogn. 110, 4–19. doi: 10.1016/j.bandc.2015.07.002 Debette, S., and Markus, H. S. (2010). The clinical importance of white matter hyperintensities on brain magnetic resonance imaging: systematic review and meta-analysis. BMJ 341:c3666. doi: 10.1136/bmj.c3666 g j Saia, V., and Pantoni, L. (2009). Progressive stroke in pontine infarction. Acta Neurol. Scand. 120, 213–215. doi: 10.1111/j.1600-0404.2009.01161.x Fox, M. D. (2018). Mapping Symptoms to Brain Networks with the Human Connectome. N. Engl. J. Med. 379, 2237–2245. doi: 10.1056/NEJMra17 06158 Saito, T., Hayashi, K., Nakazawa, H., and Ota, T. (2016). Clinical Characteristics and Lesions Responsible for Swallowing Hesitation After Acute Cerebral Infarction. Dysphagia 31, 567–573. doi: 10.1007/s00455-016-9716-8 Galovic, M., Leisi, N., Muller, M., Weber, J., Abela, E., Kagi, G., et al. (2013). Lesion location predicts transient and extended risk of aspiration after supratentorial ischemic stroke. Stroke 44, 2760–2767. doi: 10.1161/STROKEAHA.113.001690 Schmidt, P., Gaser, C., Arsic, M., Buck, D., Forschler, A., Berthele, A., et al. (2012). An automated tool for detection of FLAIR-hyperintense white-matter lesions in Multiple Sclerosis. Neuroimage 59, 3774–3783. doi: 10.1016/j.neuroimage. 2011.11.032 Gonzalez-Fernandez, M., Kleinman, J. T., Ky, P. K., Palmer, J. B., and Hillis, A. E. (2008). Supratentorial regions of acute ischemia associated with clinically important swallowing disorders: a pilot study. Stroke 39, 3022–3028. doi: 10. 1161/STROKEAHA.108.518969 Smith, E. E., Kent, D. M., Bulsara, K. R., Leung, L. Y., Lichtman, J. H., Reeves, M. J., et al. (2018). Eﬀect of Dysphagia Screening Strategies on Clinical Outcomes After Stroke: A Systematic Review for the 2018 Guidelines for the Early Management of Patients With Acute Ischemic Stroke. Stroke 49, e123–e128. doi: 10.1161/STR.0000000000000159 Hinchey, J. A., Shephard, T., Furie, K., Smith, D., Wang, D., Tonn, S., et al. (2005). Formal dysphagia screening protocols prevent pneumonia. Stroke 36, 1972–1976. doi: 10.1161/01.STR.0000177529.86868.8d Smithard, D. G., Smeeton, N. C., and Wolfe, C. D. (2007). Long-term outcome after stroke: does dysphagia matter? Age Ageing 36, 90–94. doi: 10.1093/ageing/ aﬂ149 Ickenstein, G. W., Stein, J., Ambrosi, D., Goldstein, R., Horn, M., and Bogdahn, U. (2005). Predictors of survival after severe dysphagic stroke. J. Neurol. 252, 1510–1516. doi: 10.1007/s00415-005-0906-9 Suntrup, S., Kemmling, A., Warnecke, T., Hamacher, C., Oelenberg, S., Niederstadt, T., et al. (2015). REFERENCES The impact of lesion location on dysphagia incidence, pattern and complications in acute stroke. Part 1: dysphagia incidence, severity and aspiration. Eur. J. Neurol. 22, 832–838. doi: 10.1111/ ene.12670 Jeyaseelan, R. D., Vargo, M. M., and Chae, J. (2015). National Institutes of Health Stroke Scale (NIHSS) as An Early Predictor of Poststroke Dysphagia. PM R 7, 593–598. doi: 10.1016/j.pmrj.2014.12.007 Khedr, E. M., and Abo-Elfetoh, N. (2010). Therapeutic role of rTMS on recovery of dysphagia in patients with lateral medullary syndrome and brainstem infarction. J. Neurol. Neurosurg. Psychiatry 81, 495–499. doi: 10.1136/jnnp. 2009.188482 Suntrup-Krueger, S., Kemmling, A., Warnecke, T., Hamacher, C., Oelenberg, S., Niederstadt, T., et al. (2017). The impact of lesion location on dysphagia incidence, pattern and complications in acute stroke. Part 2: Oropharyngeal residue, swallow and cough response, and pneumonia. Eur. J. Neurol. 24, 867–874. doi: 10.1111/ene.13307 Labeit, B., Mueller, H., Muhle, P., Claus, I., Warnecke, T., Dziewas, R., et al. (2018). Predicting Dysphagia with National Institute of Health Stroke Scale: Distinction between Infra- and Supratentorial Region is Essential. Cerebrovasc. Dis. 46, 152–160. doi: 10.1159/000493371 Tan, Y. F., Zhan, L. X., Chen, X. H., Guo, J. J., Qin, C., and Xu, E. (2018). Risk Factors, Clinical Features and Prognosis for Subtypes of Ischemic Stroke in a Chinese Population. Curr. Med. Sci. 38, 296–303. doi: 10.1007/s11596-018- 1878-1 Leopold, N. A., and Daniels, S. K. (2010). Supranuclear control of swallowing. Dysphagia 25, 250–257. doi: 10.1007/s00455-009-9249-5 Teismann, I. K., Dziewas, R., Steinstraeter, O., and Pantev, C. (2009). Time- dependent hemispheric shift of the cortical control of volitional swallowing. Hum. Brain Mapp. 30, 92–100. doi: 10.1002/hbm.20488 Li, J., Zhang, J., Li, S., Guo, H., Qin, W., and Hu, W. L. (2014). Predictors of Percutaneous Endoscopic Gastrostomy Tube Placement after Stroke. Can J. Neurol. Sci. 41, 24–28. doi: 10.1017/S0317167100016218 Teismann, I. K., Steinstraeter, O., Schwindt, W., Ringelstein, E. B., Pantev, C., and Dziewas, R. (2010). Age-related changes in cortical swallowing processing. Neurobiol. Aging 31, 1044–1050. doi: 10.1016/j.neurobiolaging.2008. 07.001 Lin, W. S., Chou, C. L., Chang, M. H., Chung, Y. M., Lin, F. G., and Tsai, P. Y. (2018). Vagus nerve magnetic modulation facilitates dysphagia recovery in patients with stroke involving the brainstem - A proof of concept study. Brain Stimul. 11, 264–270. doi: 10.1016/j.brs.2017.10.021 Toscano, M., Cecconi, E., Capiluppi, E., Vigano, A., Bertora, P., Campiglio, L., et al. (2015). Neuroanatomical, Clinical and Cognitive Correlates of Post-Stroke Dysphagia. Eur. Neurol. 74, 171–177. REFERENCES Asaithambi, G., Tong, X., Lakshminarayan, K., Coleman King, S. M., and George, M. G. (2020). Current trends in the acute treatment of ischemic stroke: analysis from the Paul Coverdell National Acute Stroke Program. J. Neurointerv. Surg. 12, 574–578. doi: 10.1136/neurintsurg-2019-015133 Adams, H. P. Jr., Davis, P. H., Leira, E. C., Chang, K. C., Bendixen, B. H., Clarke, W. R., et al. (1999). Baseline NIH Stroke Scale score strongly predicts outcome after stroke: A report of the Trial of Org 10172 in Acute Stroke Treatment (TOAST). Neurology 53, 126–131. doi: 10.1212/WNL.53. 1.126 Ashburner, J., Andersson, J. L., and Friston, K. J. (2000). Image registration using a symmetric prior–in three dimensions. Hum. Brain Mapp. 9, 212–225. doi: 10.1002/(SICI)1097-0193(200004)9:4<212::AID-HBM3>3.0.CO;2-# Ashburner, J., Andersson, J. L., and Friston, K. J. (2000). Image registration using a symmetric prior–in three dimensions. Hum. Brain Mapp. 9, 212–225. doi: 10.1002/(SICI)1097-0193(200004)9:4<212::AID-HBM3>3.0.CO;2-# October 2021 \| Volume 13 \| Article 753364 Frontiers in Aging Neuroscience \| www.frontiersin.org 9 Predictive Model and Lesion-Symptom Mapping Zhang et al. Bates, E., Wilson, S. M., Saygin, A. P., Dick, F., Sereno, M. I., Knight, R. T., et al. (2003). Voxel-based lesion-symptom mapping. Nat. Neurosci. 6, 448–450. doi: 10.1038/nn1050 Osawa, A., Maeshima, S., and Tanahashi, N. (2013). Water-swallowing test: screening for aspiration in stroke patients. Cerebrovasc. Dis. 35, 276–281. doi: 10.1159/000348683 Campbell, B. C., and Parsons, M. W. (2018). Imaging selection for acute stroke intervention. Int. J. Stroke 13, 554–567. doi: 10.1177/1747493018765235 Piai, V., Rommers, J., and Knight, R. T. (2018). Lesion evidence for a critical role of left posterior but not frontal areas in alpha-beta power decreases during context-driven word production. Eur. J. Neurosci. 48, 2622–2629. doi: 10.1111/ ejn.13695 Daniels, S. K., and Foundas, A. L. (1999). Lesion localization in acute stroke patients with risk of aspiration. J. Neuroimaging. 9, 91–98. doi: 10.1111/ jon19999291 j Rofes, L., Muriana, D., Palomeras, E., Vilardell, N., Palomera, E., Alvarez- Berdugo, D., et al. (2018). Prevalence, risk factors and complications of oropharyngeal dysphagia in stroke patients: A cohort study. Neurogastroenterol. Motil. 2018:e13338. doi: 10.1111/nmo.13338 Daniels, S. K., Foundas, A. L., Iglesia, G. C., and Sullivan, M. A. (1996). Lesion site in unilateral stroke patients with dysphagia. J. Stroke Cerebrovasc. Dis. 6, 30–34. doi: 10.1016/S1052-3057(96)80023-1 Daniels, S. K., Pathak, S., Mukhi, S. V., Stach, C. B., Morgan, R. O., and Anderson, J. A. (2017). The Relationship Between Lesion Localization and Dysphagia in Acute Stroke. Dysphagia 32, 777–784. Frontiers in Aging Neuroscience \| www.frontiersin.org REFERENCES doi: 10.1159/000441056 Malandraki, G. A., Sutton, B. P., Perlman, A. L., Karampinos, D. C., and Conway, C. (2009). Neural activation of swallowing and swallowing-related tasks in healthy young adults: an attempt to separate the components of deglutition. Hum. Brain Mapp. 30, 3209–3226. doi: 10.1002/hbm.20743 Toscano, M., Vigano, A., Rea, A., Verzina, A., Sasso D’elia, T., Puledda, F., et al. (2019). Sapienza Global Bedside Evaluation of Swallowing after Stroke: the GLOBE-3S study. Eur. J. Neurol. 26, 596–602. doi: 10.1111/ene.13862 Marik, P. E., and Kaplan, D. (2003). Aspiration pneumonia and dysphagia in the elderly. Chest 124, 328–336. doi: 10.1378/chest.124.1.328 Vallar, G., and Calzolari, E. (2018). Unilateral spatial neglect after posterior parietal damage. Handb. Clin. Neurol. 151, 287–312. doi: 10.1016/B978-0-444-63622- 5.00014-0 Michou, E., and Hamdy, S. (2009). Cortical input in control of swallowing. Curr. Opin. Otolaryngol. Head Neck Surg. 17, 166–171. doi: 10.1097/MOO. 0b013e32832b255e Verin, E., and Leroi, A. M. (2009). Poststroke dysphagia rehabilitation by repetitive transcranial magnetic stimulation: a noncontrolled pilot study. Dysphagia 24, 204–210. doi: 10.1007/s00455-008-9195-7 Mitchell, P. H. (2016). Nursing Assessment of Depression in Stroke Survivors. Stroke 47, e1–e3. doi: 10.1161/STROKEAHA.115.008362 Okubo, P. C., Fabio, S. R., Domenis, D. R., and Takayanagui, O. M. (2012). Using the National Institute of Health Stroke Scale to predict dysphagia in acute ischemic stroke. Cerebrovasc. Dis. 33, 501–507. doi: 10.1159/000336240 Vilardell, N., Rofes, L., Arreola, V., Martin, A., Muriana, D., Palomeras, E., et al. (2017). Videoﬂuoroscopic assessment of the pathophysiology of chronic October 2021 \| Volume 13 \| Article 753364 Frontiers in Aging Neuroscience \| www.frontiersin.org 10 Zhang et al. Predictive Model and Lesion-Symptom Mapping poststroke oropharyngeal dysphagia. Neurogastroenterol. Motil. 29, 1–8. doi: 10.1111/nmo.13111 October 2021 \| Volume 13 \| Article 753364 Conﬂict of Interest: CL was employed by company Shanghai Zhiyu Software Technology Co., Ltd. Conﬂict of Interest: CL was employed by company Shanghai Zhiyu Software Technology Co., Ltd. Conﬂict of Interest: CL was employed by company Shanghai Zhiyu Software Technology Co., Ltd. poststroke oropharyngeal dysphagia. Neurogastroenterol. Motil. 29, 1–8. doi: 10.1111/nmo.13111 Wilmskoetter, J., Bonilha, L., Martin-Harris, B., Elm, J. J., Horn, J., and Bonilha, H. S. (2019). Mapping acute lesion locations to physiological swallow impairments after stroke. Neuroimage Clin. 22:101685. doi: 10.1016/j.nicl.2019. 101685 The remaining authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Wirth, R., Dziewas, R., Beck, A. M., Clave, P., Hamdy, S., Heppner, H. J., et al. (2016). Oropharyngeal dysphagia in older persons - from pathophysiology to adequate intervention: a review and summary of an international expert meeting. Clin. Interv. Aging 11, 189–208. doi: 10.2147/ CIA.S97481 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. Woodhouse, L. J., Scutt, P., Hamdy, S., Smithard, D. G., Cohen, D. L., Roﬀe, C., et al. (2018). Route of Feeding as a Proxy for Dysphagia After Stroke and the Eﬀect of Transdermal Glyceryl Trinitrate: Data from the Eﬃcacy of Nitric Oxide in Stroke Randomised Controlled Trial. Transl. Stroke Res. 9, 120–129. doi: 10.1007/s12975-017-0548-0 Copyright © 2021 Zhang, Tang, Wang, Ding, Zhu, Zhou, Diao, Kong, Cai, Li, Yao and Fang. 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. Zhang, C., Zhao, S., Zang, Y., Gu, F., Mao, S., Feng, S., et al. (2017). The eﬃcacy and safety of Dl-3n-butylphthalide on progressive cerebral infarction: A randomized controlled STROBE study. Medicine 96:e7257. doi: 10.1097/MD. 0000000000007257 October 2021 \| Volume 13 \| Article 753364 Frontiers in Aging Neuroscience \| www.frontiersin.org 11
W2036758634.txt	https://www.scielo.br/j/bjorl/a/hMJHsSZ3hHjNMX5syfSwCLg/?lang=en&format=pdf	en		Infecção oral pelo HPV em mulheres com lesão escamosa de colo uterino no sistema prisional da cidade de São Paulo, Brasil	Brazilian Journal of Otorhinolaryngology	2,012	cc-by	4,291	Braz J Otorhinolaryngol. 2012;78(2):66-72. ORIGINAL ARTICLE BJORL .org Oral infection by the Human Papilloma Virus in women with cervical lesions at a prison in São Paulo, Brazil Marco Antonio Zonta1, Jussimara Monteiro2, Gildo Santos Jr.3, Antonio Carlos Campos Pignatari4 Abstract Keywords: DNA probes, HPV, polymorphism, restriction fragment length, vaginal smears. C arcinoma of the head and neck is the 6th cause of death by cancer in the world. In recent decades the human papillomavirus (HPV) has been implicated in the etiology of this disease Objective: To characterize the types of HPV detected in the oral mucosa in women with cytological abnormalities suggesting intraepithelial squamous lesions in the uterine cervix. Methods: four-hundred-nine cervical-vaginal and oral pap-smears of women interned in a Female Prison in São Paulo were examined. The relationship between cervical and oral lesion was analyzed by PCR/RFLP and DNA sequencing. Results: Of 27 (6.67%) specimens showing cervical cytological abnormalities suggesting LSIL and HSIL, 22 (81.48%) had oncogenic high-risk HPV infection, of which HPV 59 was the most prevalent. Three (11.1%) samples showed cytological changes suggesting mild dysplasia in the oral cavity. Conclusion: Our study suggests an association between carcinoma of the oral cavity and HPV infection, regardless of the virus type. MSc in Clinical Tests – University of Santo Amaro (PhD Student of Infectology – Federal University of São Paulo - Unifesp). 2 PhD in Infectology – Federal University of São Paulo - Unifesp (Researcher of Infectology - Unifesp). 3 PhD in Infectology – Federal University of São Paulo - Unifesp (Head of the Molecular Biology Department of the Afip Lab). 4 PhD in Infectology – Federal University of São Paulo - Unifesp (Full Professor of Infectology - Unifesp). Universidade Federal de São Paulo - Departamento de Infectologia/Laboratório Especial de Microbiologia Clínica. Send correspondence to: Prof. Marco Antonio Zonta Av. Tucuruvi, 259; 2º. and. Conj 14. São Paulo - SP. CEP: 02305-000. Tel: 55 (11)-22819460/55 (11)-29508464. Email: mazonta@unifesp.br/dirgeral@incito.com.br Paper submitted to the BJORL-SGP (Publishing Management System – Brazilian Journal of Otorhinolaryngology) on February 14, 2011, 2011; and accepted on January 15, 2012. Cod. 7576. 1 Brazilian Journal of Otorhinolaryngology 78 (2) March/April 2012 http://www.bjorl.org / e-mail: revista@aborlccf.org.br 66 78(2)-ing.indb 66 27/03/2012 09:35:47 INTRODUCTION The low oncogenic risk viruses are: HPV6, HPV-11, HPV-42, HPV-43, HPV-44, HPV-54, HPV-61, HPV-70, HPV-72 and the HPV-81, which cause benign lesions, known as oral warts, papilloma and white epithelium in the oral cavity. The high oncogenic risk viruses are: HPV-16, HPV-18, HPV-31, HPV-33, HPV-45, HPV-51, HPV51, HPV-52, HPV-56, HPV-58, HPV-59, HPV-68, HPV-73 and the HPV-82. These are associated with atypical hyperplasia and oral carcinoma17,18. In these cases, HPV-16 has been more frequently found19-21. The goal of this study was to do a molecular characterization of HPV types diagnosed in the oral mucosa of the women who had cytological changes matching those of a low grade squamous lesion and a high grade one in the uterine cervix. Head and neck cancer is the 5th largest cause of cancer and the 6th largest cause of death by cancer in the world1. The largest incidence of this disease stems from the squamous cells of the upper digestive tract epithelium (oral cavity, pharynx and larynx). The main risk factors associated with the origin of this cancer are: smoking and alcoholism2. Estimates for the year 2010 showed the occurrence of 14,120 new cases of oral cavity cancer, with a total of 6,214 cases of death caused by this disease, being the largest prevalence associated with males (10,330 cases)3. In the last decades, infection by the Human Papilloma Virus (HPV) has been associated with the etiology of this disease, as it happens with uterine cervix cancer. It is believed that the oral infection may be acquired through oral or orogenital contact, or autoinfection. Sexual behavior has a direct relationship with the transmission mechanism of this viral infection, and the direct contact between the mucosae is the main route4-7. Vertical transmission can also happen8,9. HPV is a DNA virus, epitheliotropic, nonenveloped, with a double strand circular genome, and about 8,000 pb, measuring 55 nm in diameter. Its genome is broken down into three regions: a long control region (LCR) and the Early (E) and Late (L) regions. Regions E and L code viral proteins, while the LCR is called non-coding region10-12. This virus’s oncogenic action is associated with the interaction of E6 and E7 oncoproteins, produced by the early genes E6 and E7, respectively. These proteins are involved in cell proliferation control, controlled by genes p53 and pRb, respectively, responsible for cell DNA control and correction, responsible for cell transformation and the malignant evolution of these lesions13-15. HPV plays an important role in the oncogenesis of oral carcinoma, and its nomenclature is associated with its oncogenic potential. The pre-malignant lesions caused by the low-risk viruses are characterized by epithelial cells with nuclear atypias and cytoplasmic changes known as koilocyte, which have an enlarged nucleus, hyperchromia, perinuclear halo, binucleation or multinucleation and an increase in cytoplasm keratinization. These changes may also be found in high grade lesions, though less often16. MATERIALS AND METHODS The present study is a cross-sectional contemporary cohort study, in which we studied 409 cervix-vaginal and oral cavity samples from women aged between 18 and 60 years of age, sexually active, inmates of the Women’s Prison of the City of São Paulo, between February of 2006 and December of 2007. Initially, the women were submitted to an interview, aiming at obtaining clinical, sexual and social behavior information. The patients were instructed about and given the Informed Consent Form. This project was submitted to and approved by the Ethics in Research Committee of the Federal University of São Paulo (UNIFESP-CEP: 0369/06). Biological material gathering The biological material was gathered in two stages: (i) cervix-vaginal material gathering; (ii) oral material gathering. We carried out two gatherings from each one of the cavities (cervix-vaginal and oral). Initially, the gathering was carried out by the conventional method (REF) and fixed in the slide using Citofix®. Following that, we carried out another gathering in the same region, using a brush with plastic bristles from the DNA-Citoloq® gathering kit, from Digene® - Brazil and it was preserved in a proprietary liquid medium, called UMC®. The same gathering procedure was utilized in order to gather material from the oral cavity21. Brazilian Journal of Otorhinolaryngology 78 (2) March/April 2012 http://www.bjorl.org / e-mail: revista@aborlccf.org.br 67 78(2)-ing.indb 67 27/03/2012 09:35:47 Cytological analysis The cytological samples gathered by the conventional method were submitted to Papanicolau dye and analyzed under light microscopy at the Cytopathology Department of the Biomedicine Lab in São Leopoldo/”IN CITO” by two clinical examiners. The cell changes were classified according to the Bethesda 2001 system. Molecular analysis The 27 cervix-vaginal and oral cavity samples with cytology report suggestive of viral infection were confirmed as to the presence of HPV-DNA by PCR and characterized as to the type of HPV by the RFLP and sequencing methods. Of the three samples which showed cell changes associated with mild oral cavity dysplasia, all (100%) had HPV-DNA, and one (33.33%) showed concurrent infections by HPV-16 and HPV-59; and these were the most prevalent types in the premalignant lesions. Among the 27 oral and cervix samples submitted to molecular characterization, 18 (66.66%) had concurrent infection in both regions; nonetheless, only one sample (3.70%) had concurrent infections by the same type of virus, HPV-39 (Table 1). Among the 27 samples gathered from the oral mucosa, 23 (85.18%) had HPV infection, 22 (81.48%) of those were caused by high oncogenic risk HPV, one sample (3.70%) was positive concerning the presence of HPV-DNA; nonetheless, it was not possible to characterize it in a molecular level. The largest prevalence of infection in women aged between 26 and 35 years of age (44.44%) and the most prevalent type of HPV characterized was HPV59, in 11 (40.75%) oral samples. Four (14.82%) samples did not have HPV infection. Two (7.41%) samples had two different types of HPV, one sample had HPV-16 and HPV-18 and another sample had HPV-16 and HPV-59 (Table 2). When we consider the sexual behavior of the 27 inmates who had a positive result for oral cavity HPV, it was possible to notice that of the 27 positive samples for HPV, 18 (66.7%) samples , in which HPV infection was detected, were from women with heterosexual behavior. Three (11.11%) positive samples were identified in women with bisexual behavior. Two (7.41%) samples came from homosexual women (Table 3). Among the 27 samples gathered from the oral cavity, 11 (40.75%) had a greater prevalence of HPV-59, two (7.40%) samples had HPV-16, and type 16 was seen in two more cases of concurrent infections. One sample (3.70%) had HPV-18, concurrent HPV-16, three (11.11%) samples had HPV-39, five (18.53%) samples had HPV-58; and HPV–59 was the most prevalent (Table 4). Molecular detection and characterization The molecular characterization of the HPV types was carried out by the Polymerase Chain Reaction (PCR), RFLP and the Sequencing methods at the Molecular Biology Department of the Clinical Microbiology Special Lab (LEMC) of the Infectology Program of the UNIFESP and the Molecular Biology Lab from AFIP3. The HPV-DNA was detected by PCR. The HPV-DNA was extracted using the GFX® kit (GE Healthcare) used to extract the genomic DNA of epithelial cells. The HPV-DNA was amplified using the MY9 and MY11 universal primers, which amplify the large majority of HPV types. The product of such amplification was submitted to RFLP techniques, according to the protocol proposed by Bernard et al.22 and sequenced by the Big Dye Terminator Cycle Sequencing kit (Applied Biosystems, Foster City, CA). We used the ABI PRISM 310, Genetic Analyzer (Applied Biosystems, Perkin Elmer, California). The DNA sequences obtained were analyzed using the Lasergene Software Package (DNASTAR, Madison, WI) and, following that, they were submitted to comparison with the genetic data base23,24. RESULTS Cytological analysis Of the 409 cervix-vaginal samples gathered and assessed, 27 (6.67%) showed lesions in the uterine cervix, according to the cyto-pathology report. Among the 27 women who had lesions in the uterine cervix, three (11.11%) showed cell changes associated with mild dysplasia in the oral cavity, with mild dyskariosis in mature squamous cells, and 24 (88.89) showed reactive changes associated with inflammation, occasionally associated with infectious agents (Table 1). Only the 27 women who had pre-malignant and malignant lesions of the uterine cervix were assessed as to the presence of HPV in their oral cavities. Brazilian Journal of Otorhinolaryngology 78 (2) March/April 2012 http://www.bjorl.org / e-mail: revista@aborlccf.org.br 68 78(2)-ing.indb 68 27/03/2012 09:35:47 Table 1. Comparative table between the cervix-vaginal and oral samples infected by HPV. Uterine Cervix Oropharynx N Cytology PCR/RFLP Cytology Sequencing % Similarity (GeneBank) 1 HSIL – NIC II HPV – 18 Negative Negative Not applicable 2 Inflammation/AGUS NEG Negative Negative Not applicable 3 ASC-US HPV-6b Negative Negative Not applicable 4 LSIL HPV – 6b Negative HPV-59 (90%) 5 ASC-US Negative Negative HPV-39 (98%) 6 LSIL HPV – 61 Mild dysplasia HPV-59 (88%) 7 ASC-US Negative Negative HPV-59 (93%) 8 LSIL HPV – 6b Negative HPV-59 (88%) 9 LSIL HPV – 6 b Negative NEG Not applicable 10 LSIL HPV – 39 Negative HPV-39 (97%) 11 LSIL HPV – 34 Negative HPV-59 (94%) 12 LSIL HPV – 31 Negative HPV-59 (92%) 13 LSIL HPV – 6b Negative HPV-59 (89%) 14 LSIL Negative Negative HPV59 (91%) 15 LSIL HPV – 16 Negative HPV59 (90%) 16 LSIL HPV – 6b Negative HPV59 (93%) 17 LSIL HPV – 6b Mild dysplasia HPV-16/59 (85/82%) 18 LSIL HPV – 6b Negative HPV-58 (96%) 19 ASC-US Negative Negative HPV-58 (99%) 20 ASC-US Negative Negative HPV-58 (89%) 21 LSIL HPV – 16 Negative HPV-58 (98%) 22 HSIL HPV – 16 Negative HPV39 (94%) 23 LSIL HPV – 6b Negative HPV-16 (82%) 24 LSIL HPV – 16 Negative HPV-58 (94%) 25 LSIL HPV – 6b Mild dysplasia HPV-16 (91%) 26 HSIL/NIC III HPV – 33 Negative HPV-16/18 (90%) 27 Invasive CA HPV – 18 Negative undefined Not applicable LSIL: (Low-grade squamous intraepithelial lesion); ASC-US: (Squamous atypia of undetermined significance); Invasive CA: (Invasive squamous carcinoma). Table 2. Prevalence of positive oral cavity samples for HPV-DNA identified by the sequencing method and classified by age range. Table 3. Positiveness rate for HPV characterized by the molecular sequencing in the samples of women classified according to sexual behavior. Age range (years) HPV (+) HPV(-) Total Sexual orientation HPV (+) HPV(-) Total 18 to 25 4 (14.82%) 0(0%) 5 (18.52%) Heterosexual 18( 66.67%) 3 (11.11%) 21 (77.78%) 26 to 35 13 (48.14%) 3 (11.12) 15 (55.56) Bisexual 3 (11.11%) 0 (0%) 3 (11.11%) 2 (7.41%) 1 (3.70%) 3 (11.11%) 23 (85.18%) 4 (14.82%) 27(100%) 36 to 45 6 (22.22) 0 6 (6.22) Homosexual 46 to 55 0 1 (3.70%) 1 (3.70%) Total Total 23 (81.48%) 4(14.82%) 27 (100%) Among the 18 smokers, 16 (59.26%) women were infected by HPB and two (7.40%) did not have HPV-DNA. Among the non-smoking women, seven (25.95%) had oral HPV infection and two (7.40%) of these women did not have oral viral infection (Table 6). Among the 27 oral samples from the molecularly characterized women, 18 (66.67%) reported smoking and nine (33.33%) said they did not smoke. Such habit was seen more frequently among women aged between 26 and 35 years of age – 11 (40.74%) cases (Table 5). Brazilian Journal of Otorhinolaryngology 78 (2) March/April 2012 http://www.bjorl.org / e-mail: revista@aborlccf.org.br 69 78(2)-ing.indb 69 27/03/2012 09:35:48 Table 4. Prevalence of HPV types in oral samples gathered at the women’s prison of the city of São Paulo. Type of HPV Samples Percentage (%) Negative 4 14.81 HPV 16 2* 7.40 HPV 18 1 3.70 HPV 39 3 11.11 HPV 58 5 18.53 * HPV 59 11 40.75 Undefined 1 3.70 Total 27 100 cohol increase virus penetration in the oral mucosa, thus increasing the likelihood of pre-malignant and malignant lesions. Studies showed the association between this infection and oral lesions and oral carcinoma, varying between 25% and 80% of high risk HPV prevalence in samples gathered from oral cavity lavage or brushed25. Our study showed 81.48% of HPV in the oral mucosa of women inmates of the women’s prison of the city of São Paulo who had concurrent infections and lesions in the uterine cervix, suggesting an internal transmission. Rintala et al.26 reported 42% of HPV oral infection in the mucosae of children, having a 10% persistence within a 3-year interval. The correlation with the genital infection was 36%. Our study suggested a good correlation among the women who had uterine cervix infection; nonetheless, the similarity between the viral types in the two sites was low, and only one patient had the same type of HPV in the oral cavity and uterine cervix, which suggested a low frequency of autoinfection. Saini et al.27 reported the occurrence of 5.71% of concurrent infection by the HPV in women who had uterine cervix cancer. Beaudenon et al.28 had associated the occurrence of multiple infections by HPV types 13 and 32 in cases of focal epithelial hyperplasia in the oral cavity. Our study found three oral samples which had pre-neoplastic cell changes, mild dysplasia and also infection by high-oncogenic risk HPV, and in one of them had concurrent infections by HPV types 16 and 59. Insofar as the age range is concerned, we found a higher frequency of HPV in the oral cavities of women between 20 and 30 years of age, mean age of 25 years, encompassing 40.74% of infection cases. Smith et al.29 found 47.10% of HPV infection in women with the mean age of 24 years. The study showed a greater prevalence of HPV59, and in two cases there was oral dysplasia and one case of multiple infections associated with the HPV 16. Kreimer et al.30 found a greater prevalence of HPV-16 in samples obtained from the oral cavity lavage in HIV-positive patients; HPV 58 and 59 were found in a lesser frequency. Hanson et al.31 found HPV-16 and HPV-59 infections in samples obtained from the oral cavity lavage of patients with oral and oropharynx cancer in Sweden. Hennessey et al.32 reported the presence of infections by HPV-58 and 59 in pre-malignant and malignant lesions in the oral cavity and suggested * HPV 16 was found in two isolated samples and also in two samples with simultaneous infections. Table 5. The table shows the occurrence rate of women smokers per age range. SMOKER YES AGE NO GENERAL % GENERAL % 18-25 3 11.11% 1 3.70% 26-35 11 40.74% 4 14.81% 36-45 4 14.81% 2 7.41% 46-55 0 0.00% 2 7.41% Total 18 66.66% 9 33.33% Table 6. This table shows the occurrence of HPV infection and pre-malignant lesions in women smokers and non-smokers from the women’s prison of the city of São Paulo. Samples HPV + HPV - MILD DYSPLASIA Smokers 18 (66.66%) 16 (59.26%) 2 (7.40%) 2 (7.40%) Non-smokers 9 (33.34%) 7 (25.93%) 2 (7.40%) 1 (3.70%) Total 27 (100%) DISCUSSION The relationship between HPV infection and lesions on the oral mucosa and oropharynx still is controversial. Numerous studies showed the HPV in the intact oral mucosa, and infected by the pre-malignant and malignant lesions; the tonsil area seems to be the area of the greatest penetration and cell transformation induced by the HPV. More sensitive molecular techniques, such as molecular characterization by sequencing, enable a better quality in the identification of the viral type in its infection site. Sexual behavior, associated or not to risk factors such as cigarette smoking and al- Brazilian Journal of Otorhinolaryngology 78 (2) March/April 2012 http://www.bjorl.org / e-mail: revista@aborlccf.org.br 70 78(2)-ing.indb 70 27/03/2012 09:35:48 ACKNOWLEDGEMENT that these types of viruses have a high oncogenic potential for the development of neoplasia in the oropharynx mucosa. The greater rate of HPV oral infection happened to heterosexual women, showing, in this case, that the deviation in behavior does not have any direct relation to the viral transmission mechanism. Our results corroborate the findings from D’Souza et al.33, who found a greater occurrence of HPV oral infection in heterosexual women; nonetheless, they found a low prevalence of infections in smoking women. Xavier et al.34 found a greater occurrence of HPV oral infection in heterosexual and monogamist men; nonetheless, the prevalence of oral infection was found in one case only. During a fair amount of time, cigarette smoking and alcohol ingestion were considered the main cofactors in the occurrence of oral cavity and oropharynx lesions. Today, it is believed that the HPV plays an important role in the development of these lesions. Our study showed that 66.66% of the women, prison inmates, were smokers; among them, 59.26% were infected by the high risk HPV. Studies have shown an association between 0 and 55% of HPV infection in smokers. It is believed that this risk factor causes sequential repairs to the oral mucosa, making it more susceptible for viral infection. Chen et al.35, studied tissues through in situ hybridization, and found 78% of high risk HPV infection in smokers with squamous papilloma. Thus, following up the inmates of the women’s prison of the city of São Paulo by means of preventive gynecological exams and clinical and molecular evaluation of the oral cavity may clarify the possible relationship between HPV infection and oral cavity lesions in women who have simultaneous infection of the female genital tract. We appreciate the help from the technicians from the “IN CITO” - Citologia Diagnóstica Ltda laboratory and those from the AFIP - Medicina Laboratorial for producing the cytological and molecular samples, respectively. We would also like to thank the warden of the women’s prison for the support in this study. REFERENCE 1. www.inca.gov.br 2. Venturi BRM, Cabral MG, Lourenço SQC. Carcinoma de células escamosas oral – contribuição de vírus oncogênico e alguns marcadores moleculares no desenvolvimento e prognóstico das lesões: uma revisão. Rev Bras Otorrinolaringol. 2004;70(3):385-92. 3. www.ebi.ac.uk/fasta33 4. Castro TPPG, Bussoloti Filho I. Prevalência do papilomavírus humano (HPV) na cavidade oral e na orofaringe. Braz J Otorhinolaryngol. 2006;72(2):272-82. 5. Chaudhary AK, Pandya S, Mehotra R, Bharti AC, Singh M, Singh M. Comparative study between the Hybride Capture II test and PCR based assay for the detection of human papillomavirus DNA in oral submucous fibrosis and oral squamous cell carcinoma. Virol J. 2010;7:253. 6. Fernandes JV, Meissner RV, Carvalho MGF, Fernandes TAAM, Azevedo PRM, et al. Prevalence of HPV infection by cervical cytologic status in Brazil. Int J Gynecol Obstet. 2009;105:21-4. 7. Gonzáles-Losa MR, Manzano-Cabrera L, Ruenda Gordilo F, Hernándes Solís SE, Puerto Solís M. Low prevalence of high risk Human Papilomavirus in normal oral mucosa by hybrid capture 2. Braz J Microbiology. 2008;39(1):32-4. 8. Feller L, Khammissa RA, Wood NH, Lemmer J. Epithelial maturation and molecular biology of oral HPV. Infect Agent Cancer. 2009;4:16. 9. Rose Ragin CC, Taioli E. Second primary head and neck tumor risk in patients with cervical cancer–SERR data analysis. Head Neck. 2008;30(1):58-66. 10. Ault KA. Epidemiology and natural history of papillomavirus infections in female genital tract. Infect Dis Obstetr Gynecol. 2006;Suppl 40470. 11. Bosch FX, Rohan T, Schneider A, Frazer H, Castellsagué X, de Sanjosé S, et al. Papillomavirus research update: highlights of the Barcelona HPV 2000 international papillomavirus conference. J Clin Pathol. 2001;54(3):163-75. 12. Giovannelli L, Campisi G, Lama A, Giambalvo O, Osborn J, Margiotta V, et al. Human papillomavirus DNA in oral mucosal lesion. J Infect Dis. 2000;185(6):833-6. 13. Furniss CS, Mcclean MD, Smith JF, Bryan J, Nelson HH, Peters ES, et al. Human papillomavirus 16 and head and neck squamous cell carcinoma. Int J Cancer. 2007;120(11):2386-92. 14. Lopes F, Latorre MRSO, Pignatari ACC, Buchalla CM. Prevalência de HIV, papilomavírus humano e sífilis na Penitenciária Feminina da Capital, São Paulo, 1997-1998. Cad Saúde Pública. 2001;17(6):1473-80. 15. Magi JC, Brito EMS, Grecco ETO, Pereira SMMP, Formiga GJS. Prevalência de Papilomavírus Humano (HPV) Anal, Genital e Oral em Ambulatório Geral de Coloproctologia. Rev Bras Colo-proctol. 26(3):233-8. CONCLUSION According to our study, we may suggest the existence of an association between the development of oral cavity lesions and HPV infection; however, it did not show a direct association between the types of viruses present in the uterine cervix and in the oral cavity, and still a weak association with the sexual behavior of the inmates of the women’s prison of the city of São Paulo. Brazilian Journal of Otorhinolaryngology 78 (2) March/April 2012 http://www.bjorl.org / e-mail: revista@aborlccf.org.br 71 78(2)-ing.indb 71 27/03/2012 09:35:48 16. Brunotto M, Zárate AM, Cismondi A, Fernández MdelC, Noher de Halac RI. Valoración de la citologia exfoliativa como factor de predicción en lesiones de la mucosa oral. Med Oral Patol Oral Cir Bucal. 2005;10 Suppl 2:E92-102. 17. Rocha DAP, Souza LB, Pinto LP. Análise comparativa da proliferação celular entre carcinomas de células escamosas orais HPV-positivos e HPV-negativos. J Bras Patol Med Lab. 2007;43(4):269-74. 18. Syrjänen S. Human papillomavirus in head and neck carcinomas. N Engl J Med. 2007;356(19):1993-5. 19. Nichols AC, Faquin WC, Westra WH, Mroz EA, Begun S, Clark JR, et al. HPV-16 infection predicts treatment outcome in oropharyngeal squamous cell carcinoma. Otolaryngol Head Neck Surg. 2009;140(2):228-34. 20. Syrjänen S. Human papillomavirus infections and oral tumors. Med Microbiol Immunol. 2003;192(3):123-8. 21. Goon PK, Stanley MS, Ebmeyer J, Steinsträsser L, Upile T, Jerjes W, et al. HPV & head and neck cancer: a descriptive update. Head Neck Oncol. 2009;1:36. 22. Bernard HU, Chan SY, Manos MM, Ong CK, Villa LL, Delius H, et al. Identification and assessment of known and novel human papillomavirus by polimerase chain reaction amplification, restriction fragment length polymorphisms, nucleotide sequence, and phylogenic algorithms. J Infect Dis. 1994;170(5):1077-85. 23. www.ncbi.nlm.nih.gov/blast/ 24. www.who.int/hpvcenter/statiscs/dynamic/ico/contry.pdf/ bra/pdf 25. Varnai AD, Bollmann M, Bankfalvi A, Kovacs K, Heller H, Schmitt C, et al. The prevalence and distribution of human papillomavirus genotypes in oral epithelial hyperplasia: proposal of a concept. J Oral Pathol Med. 2009;38(2):181-7. 26. Rintala MA, Grénman SE, Järvenkylä ME, Syrjänen KJ, Syrjänen SM. High-risk types of papillomavirus (HPV) DNA in oral and genital mucosa of infants during their first 3 years of life: experience from the Finnish HPV Family Study. Clin Infect Dis. 2005;41(12)1728-33. 27. Saini R, Khim TP, Rahman SA, Ismail M, Tang TH. High-risk human papillomavirus in the oral cavity of women with cervical cancer, and their children. Virol J. 2010;7:131. 28. Beaudenon S, Praetorius F, Kremsdorf D, Lutzner M, Worsaae N, Pehau-Arnaudet G, et al. A new type of human papillomavirus associated with oral focal epitelial hyperplasia. J Invest Dermatol. 1987;88(2):130-5. 29. Smith EM, Ritchie JM, Yankowitz J, Wang D, Turek LP, Haugen TH. HPV prevalence and concordance in the cervix and oral cavity of pregnant women. Infect Dis Obstet Gynecol. 2004;12(2):43-56. 30. Kreimer AR, Alberg AJ, Daniel R, Gravitt PE, Viscidi R, Garret ES, et al. Oral human papillomavirus infection in adults is associated with sexual behavior and HIV serostatus. J Infect Dis. 2004;189(4):686-98. 31. Hansson BG, Rosenquist K, Antonsson A, Wennerberg J, Schildt EB, Bladström A, et al. G. Strong association between infection with human pappilomavirus and oral and oropharyngeal squamous cell carcinoma: A population-based case control study in southern Sweden. Acta Otolaryngol. 2005;125(12):1337-44. 32. Hennessey PT, Westra WH, Califano JA. Human papillomavirus and head and neck squamous cell carcinoma: recent evidence and clinical implications. J. Dent Res. 2009;88(4):300-6. 33. D’Souza G, Agrawal Y, Halpern J, Bodison S, Gillison MM. Oral sexual behaviors associated with prevalent oral human papillomavirus infection. J Infect Dis. 2009;199(9):1263-9. 34. Xavier SD, Bussoloti Filho I, de Carvalho JM, Castro TM, Framil VMS, Syrjänen KJ. Prevalence of human papillomaviurs (HPV) DNA in oral mucosa of men with anogenital HPV infection. Oral Surg Oral Med Oral Pathol Oral Raiol Endod. 2009;108(5):732-7. 35. Chen PC, Pan CC, Kuo C, Lin CP. Risk of oral nonmalignant lesion associated with human papillomavirus infection, betel quid chewing, and cigarette smoking in Taiwan: an integrated molecular and epidemiologic study. Arch Pathol Lab Med. 2006;130(1):57-61. Brazilian Journal of Otorhinolaryngology 78 (2) March/April 2012 http://www.bjorl.org / e-mail: revista@aborlccf.org.br 72 78(2)-ing.indb 72 27/03/2012 09:35:48
https://openalex.org/W3209414850	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0258658&type=printable	English	null	Hollow-tree super: A directional and scalable approach for feature importance in boosted tree models	PloS one	2,021	cc-by	9,175	PLOS ONE PLOS ONE RESEARCH ARTICLE OPEN ACCESS Current limitations in methodologies used throughout machine-learning to investigate fea- ture importance in boosted tree modelling prevent the effective scaling to datasets with a large number of features, particularly when one is investigating both the magnitude and directionality of various features on the classification into a positive or negative class. This manuscript presents a novel methodology, “Hollow-tree Super” (HOTS), designed to resolve and visualize feature importance in boosted tree models involving a large number of features. Further, this methodology allows for accurate investigation of the directionality and magnitude various features have on classification and incorporates cross-validation to improve the accuracy and validity of the determined features of importance. Citation: Doyen S, Taylor H, Nicholas P, Crawford L, Young I, Sughrue ME (2021) Hollow-tree super: A directional and scalable approach for feature importance in boosted tree models. PLoS ONE 16(10): e0258658. https://doi.org/10.1371/journal. pone.0258658 Editor: Sriparna Saha, Indian Institute of Technology Patna, INDIA Editor: Sriparna Saha, Indian Institute of Technology Patna, INDIA Hollow-tree super: A directional and scalable approach for feature importance in boosted tree models Stephane Doyen☯, Hugh Taylor‡, Peter Nicholas‡, Lewis CrawfordID‡, Isabella Young‡, Michael E. Sughrue☯ Omniscient Neurotechnology, Sydney, Australia Stephane Doyen☯, Hugh Taylor‡, Peter Nicholas‡, Lewis CrawfordID‡, Isabella Young‡, Michael E. Sughrue☯ Omniscient Neurotechnology, Sydney, Australia ☯These authors contributed equally to this work. ‡ These authors also contributed equally to this work. * stephane.doyen@o8t.com a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Methods Published: October 25, 2021 Using the Iris dataset, we first highlight the characteristics of HOTS by comparing it to other commonly used techniques for feature importance, including Gini Importance, Partial Dependence Plots, and Permutation Importance, and explain how HOTS resolves the weaknesses present in these three strategies for investigating feature importance. We then demonstrate how HOTS can be utilized in high dimensional spaces such as neuroscientific setting, by taking 60 Schizophrenic subjects from the publicly available SchizConnect data- base and applying the method to determine which regions of the brain were most important for the positive and negative classification of schizophrenia as determined by the positive and negative syndrome scale (PANSS). Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here: https://doi.org/10.1371/journal.pone.0258658 Copyright: © 2021 Doyen 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. Conclusion HOTS effectively overcomes previous challenges of identifying feature importance at scale, and can be utilized across a swathe of disciplines. As computational power and data quan- tity continues to expand, it is imperative that a methodology is developed that is able to han- dle the demands of working with large datasets that contain a large number of features. This approach represents a unique way to investigate both the directionality and magnitude of feature importance when working at scale within a boosted tree model that can be easily visualized within commonly used software. Competing interests: All authors (SD, HT, PN, LC, IY, MS) are employees of Omniscient Neurotechnology Pty Ltd. The study is consistent with the objectives of Omniscient Neurotechnology Pty Ltd in furthering the scientific understanding of machine learning models in data analytics and neuroscience. Related innovative elements of the methodology outlined in the Study are the basis for patent protection sought by Omniscient Neurotechnology Pty Ltd. This does not alter our adherence to PLOS ONE policies on sharing data and materials expressly discussed in the Study. Results HOTS effectively replicated and supported the findings of feature importance for classifica- tion of the Iris dataset when compared to Gini importance, Partial Dependence Plots and Permutation importance, determining ‘petal length’ as the most important feature for positive and negative classification. When applied to the Schizconnect dataset, HOTS was able to Data Availability Statement: All Iris files are available from the UCI Machine Learning Repository (https://archive.ics.uci.edu/ml/datasets/ iris) All Schizconnect files are available upon 1 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0258658 October 25, 2021 PLOS ONE Hollow-tree super: A directional and scalable approach for feature importance in boosted tree models resolve from 379 independent features, the top 10 most important features for classification, as well as their directionality for classification and magnitude compared to other features. Cross-validation supported that these same 10 features were consistently used in the deci- sion-making process across multiple trees, and these features were localised primarily to the occipital and parietal cortices, commonly disturbed brain regions in those afflicted with Schizophrenia. creating an account and loading the dataset from the Collaborative Informatics and Neuroimaging Suite (COINS) (https://coins.trendscenter.org/). After an account has been made with COINS, researchers can locate the Schizconnect data to download in its entirety by typing “COBRE” into the main search panel. Funding: The authors SD & MS are co-founders of Omniscient Neurotechnology Pty Ltd. All authors (SD, HT, PN, LC, IY, MS) are employees of Omniscient Neurotechnology Pty Ltd. Omniscient Neurotechnology Pty Ltd provided support in the form of salaries for all authors (SD, HT, PN, LC, IY, MS). The study design, data collection and analysis, decision to publish, or preparation of the manuscript was decided collectively by the authors. The specific roles of these authors are articulated in the ‘author contributions’ section. The study is consistent with the objectives of Omniscient Neurotechnology Pty Ltd in furthering the scientific understanding of machine learning models in data analytics and neuroscience. PLOS ONE \| https://doi.org/10.1371/journal.pone.0258658 October 25, 2021 1. Introduction Tree based models, a category of supervised machine learning algorithms, have become widely used to perform regression or classification. Among the reasons for their popularity is the abil- ity to perform predictions on data with high dimensionality, mixed type variables and com- plex, non-linear relationships—better so than linear methods [1]. In many real-life applications however, model interpretation is equally as valuable as the prediction output. Yet understanding why a prediction was made can be a non-trivial exercise given that tree-based models can become extremely complex (e.g. deep trees) and difficult to interpret at scale. Interpretability becomes even more complex in the case of boosted trees such as XGBoost [2] where numerous different trees are bagged together and weighted with different importance (boosting). Some methods exist to understand why a model makes these predictions, such as Gini importance, partial dependence plots and permutation analysis [3–5]. Whilst those techniques provide a certain degree of useful insights into the model, they each lack one or more impor- tant properties for explainability of the model, specifically: (1) the direction in the relationship between features and the response variable (e.g., whether feature X1 is predictive of the nega- tive/positive outcome) and (2) magnitude (e.g., how much feature X1 influences the prediction towards the positive or negative outcome). It is important to note that a successful technique would need to achieve (1) and (2) in a way that would scale to a larger number of features, thus providing a truly commensurable understanding of datasets, particularly those that have high dimensionality. Recently, a method was proposed to linearize tree-based model nodes to provide an answer to both (1) and (2) [6]. However, this method has some limitations when it comes to applying it on boosted trees as each instance of the model added to the ensemble can have a different tree structure. In this paper, we first explore the current predominant methods in the field which exist to determine feature importance in decision trees, before addressing a common problem within these methodologies: the ability to jointly derive magnitude and directionality of classification PLOS ONE \| https://doi.org/10.1371/journal.pone.0258658 October 25, 2021 2 / 16 PLOS ONE Hollow-tree super: A directional and scalable approach for feature importance in boosted tree models between features in these models. 1.1 Common approaches to feature importance for single trees Before demonstrating our feature contribution method on boosted trees, it is important to first discuss the most common methods currently available for calculating feature importance. Whilst there exist many feature importance algorithms, here we examine three popular meth- ods in the field: Gini importance, partial dependence plots and permutation importance. We do this on single decision trees—the simplest of the tree-based approaches and thus the most readily interpretable. For this (and throughout the worked example), we use the well-known iris dataset [7]—a simple classification problem with four input features relating to plant dimensions: petal length, petal width, sepal length and sepal width. Typically, the iris dataset categorizes plants as one of; iris versicolor, iris virginica and iris setosa. To keep things simple, we removed the iris setosa group to make this a binary classifica- tion problem (iris versicolor being the negative class (= 0) and iris virginica as the positive class (= 1). Using the sklearn DecisionTreeClassifier package [8] we constructed a single decision tree with a max depth of 4 (Fig 1a). 1.1.1 Gini importance. A standard approach to determining feature importance is to score features based on the number of times or probability a variable is utilized by the model for splitting, weighted by some other value. This could be the criterion used to select split points (Gini or entropy), or some other metric such as the squared improvement to the mod- el’s F-score. Fig 1b shows the feature importance’s for our binarized decision tree, computed using sklearn’s “feature_importances_” property. This function calculates feature importance using the “normalized” total reduction of the criterion brought by that feature, also referred to as the “Gini importance”. This approach attributes a score to each feature, where a higher value indicates greater influence on the output prediction. From our simple decision tree, the most important feature for outcome prediction was petal length, with petal width, sepal length and sepal width show- ing no significant difference in their influence for predicting outcomes through this tree. Despite receiving scrutiny for biasing against variables with higher numbers of categories [9] the use of Gini importance has seen a resurgence in recent years, particularly when analyz- ing genomic datasets [10,11]. 1. Introduction We then present a novel feature contribution method, “Hol- low-Tree Super” which extends the linearization methods available for single and ensemble trees to include boosted trees. Finally, we demonstrate and provide an example for how this new method can be used to analyse high-dimensionality data, such as in the case of human neuroimaging datasets investigating brain pathology. PLOS ONE \| https://doi.org/10.1371/journal.pone.0258658 October 25, 2021 1.1 Common approaches to feature importance for single trees Whilst it remains an informative metric for assessing the relative importance of different features used in a model by providing them a relative ranking and is scalable, it lacks the ability to provide directional information on classification between fea- tures. Indeed, nothing is said for the variable “petal length” to be more predictive of the posi- tive or negative outcome. 1.1.2 Partial dependence plots. As an alternative, partial dependence plots (PDP) are often used to visualize decision boundaries. They help to describe relationships with non-lin- ear effects, and show interactions between features. To construct such a plot, the PDP function is calculated at each possible value of a feature, representing the average model prediction out- put at that value. Without the requirement for linearity, PDPs have shown great advantages in ecological studies, where features often interact non-linearly to influence species classification [12]. PDP functions can be calculated for one or two features [13], such that one can visualise how the values for one feature influence classification, or how values between two distinct PLOS ONE \| https://doi.org/10.1371/journal.pone.0258658 October 25, 2021 3 / 16 Hollow-tree super: A directional and scalable approach for feature importance in boosted tree models PLOS ONE Fig 1. Single binarized decision tree created in SKlearn to classify the Iris dataset. (a) A single binarized decision tree was created in the sklearn DecisionTreeClassifier package to effectively delineate the Iris dataset. The depth four tree used Iris versicolor as the negative class (= 0), and Iris Virginica as the positive class (= 1). Iris Setosa was removed from the classification system to allow the binarization of the dataset. (b) We calculated the Gini Importance for our decision tree using sklearn’s “feature_importances_” property. This revealed ‘petal length’ to be the most important feature in the model for classification into the positive or negative class. (c) When performing permutation importance on our four features included in our simple decision tree, we found that permuting the values of petal width had the greatest impact on the model prediction error when attempting to classify data to the positive or negative class. Unlike Gini importance, this analysis revealed that petal length was also significantly important in the classification process, whilst sepal length and width again were found to be relatively unimportant in the decision making process, and permutation of these features did not greatly impact model prediction error. 1.1 Common approaches to feature importance for single trees (d) A one feature partial dependence plot (PDP) for ‘petal length’ revealed that positive classification as Iris Virginica (partial dependence) was non-linearly related to ‘petal length’, with a critical point at 5cm marking certain negative (Iris Versicolor) classification. (e) By introducing ‘petal width’ and conducting a two feature PDP, we were able to determine that these two features were increasingly dependent for positive classification at values lower than 5cm and 1.6cm for petal length and width respectively, giving us directional and magnitudinal inferences between two features (For two feature PDP’s, a colour map is added to help visualise dependencies such that green indicates a greater partial dependency than purple). https://doi.org/10.1371/journal.pone.0258658.g001 ree created in SKlearn to classify the Iris dataset. (a) A single binarized decision tree was created in the sklearn Fig 1. Single binarized decision tree created in SKlearn to classify the Iris dataset. (a) A single binarized decision tree was created in the sklearn DecisionTreeClassifier package to effectively delineate the Iris dataset. The depth four tree used Iris versicolor as the negative class (= 0), and Iris Virginica as the positive class (= 1). Iris Setosa was removed from the classification system to allow the binarization of the dataset. (b) We calculated the Gini Importance for our decision tree using sklearn’s “feature_importances_” property. This revealed ‘petal length’ to be the most important feature in the model for classification into the positive or negative class. (c) When performing permutation importance on our four features included in our simple decision tree, we found that permuting the values of petal width had the greatest impact on the model prediction error when attempting to classify data to the positive or negative class. Unlike Gini importance, this analysis revealed that petal length was also significantly important in the classification process, whilst sepal length and width again were found to be relatively unimportant in the decision making process, and permutation of these features did not greatly impact model prediction error. (d) A one feature partial dependence plot (PDP) for ‘petal length’ revealed that positive classification as Iris Virginica (partial dependence) was non-linearly related to ‘petal length’, with a critical point at 5cm marking certain negative (Iris Versicolor) classification. PLOS ONE \| https://doi.org/10.1371/journal.pone.0258658 October 25, 2021 1.2 Direction and magnitude in feature importance coefficients As shown, none of the three methods already described are able to provide information about both outcome directionality and magnitude, in a way which could be efficiently scaled to a large number of features. Linearizing decision trees [6] offers the advantage of giving feature importance coefficients that have a direction, which magnitude can be compared and further scaled to larger datasets. A general linear equation can be derived from the model by considering that each decision in the tree stems from a feature and these decisions either increase or decrease the value from the parent node. Thus, it is possible to consider the final prediction as the sum of each feature’s contribution within the tree plus a bias value (typically the topmost sample average). To achieve this for a given prediction, the decision tree that led to that prediction is navi- gated and the local increments of feature contributions at each node (positive or negative) are identified. In this way, each prediction can be mathematically described using Eq (1). f ðxÞ ¼ bias XK k¼1 contribution ðx; kÞ ð1Þ ð1Þ f ðxÞ ¼ bias XK k¼1 contribution ðx; kÞ Where; K is the number of features, bias is the value at the root of the node and contribution(x, k) is the contribution from the k-th feature in the feature vector x [15]. This approach is similar to linear regression in a dynamic sense, and so by borrowing from regression models in this way we can achieve a similar level of interpretability as linear models. More specifically, this view of trees enables us to isolate the contributions of each feature for each prediction. It should be noted though that this linearization technique is limited in use to boosted trees whose input feature vectors only contain linearizable variables [16]. Here we provide a worked example, showing how the feature contributions are calculated for one prediction. Fig 2 depicts the decision tree, and Table 1 outlines the values of each fea- ture for a sample iris plant, as well as the feature contribution score. Such score can easily be calculated programmatically though the Python package eli5 package [17] provides a conve- nient implementation which Fig 2 was derived form. 1.1 Common approaches to feature importance for single trees We can use sklearn’s permutation importance package to perform this calculation on the Iris dataset (Fig 1c). Whilst this is another useful approach to determine the magnitude of feature importance, much like Gini importance, this method lacks information about directionality. In applying permutation importance analysis to the Iris dataset, we are able to glean that permuting ‘petal width’ had the largest effect on the model’s prediction error, and was therefore the most ‘important’ feature. However, we are again unable to say whether ‘petal width’ was more pre- dictive of the positive or negative outcome. 1.1 Common approaches to feature importance for single trees (e) By introducing ‘petal width’ and conducting a two feature PDP, we were able to determine that these two features were increasingly dependent for positive classification at values lower than 5cm and 1.6cm for petal length and width respectively, giving us directional and magnitudinal inferences between two features (For two feature PDP’s, a colour map is added to help visualise dependencies such that green indicates a greater partial dependency than purple). htt //d i /10 1371/j l 0258658 001 https://doi.org/10.1371/journal.pone.0258658.g001 features interact together to influence classification. Fig 1d shows the PDP for ‘petal length’ alone, and Fig 1e demonstrates the interaction between ‘petal length’ and ‘petal width’ and the predicted outcome of the constructed simple decision tree. These two PDP’s together reveal that petal length and petal width become dependent fea- tures for classification at values less than 5cm for petal length and 1.6cm for petal width respec- tively, and at greater values for either feature they are largely independent for classification. The benefits of PDP’s are that they are easy to implement, interpret and provide a measure of directionality for feature importance. This would scale nicely for the iris dataset, which has four features. However, the main disadvantage is that the 2D representation of PDPs limits observations to two variables at a time [13]. This makes PDPs difficult to interpret at the scale of a dataset which comprises hundreds of variables, as each feature or pair of features at most, would require their own plot and subsequent analysis to ultimately determine and inform fea- ture importance. 1.1.3 Permutation importance. Finally, permutation importance of features can be used to measure the change in the model’s prediction error as the value of the feature is ‘permuted’. Permutation is the process of shuffling the data points for one feature whilst retaining the 4 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0258658 October 25, 2021 PLOS ONE Hollow-tree super: A directional and scalable approach for feature importance in boosted tree models order for all other features to measure which variable most greatly affects model prediction error [14]. From this perspective, a feature is unimportant if changing its values has little effect on the model’s error—implying that the model did not rely strongly on this feature to make predictions. Conversely, an ‘important’ feature would increase the model error when its values are shuffled. Contributionpetal length ðcmÞ ¼ ð0:051 0:493Þ þ ð0:25 0:051Þ ¼ 0:243 1.2 Direction and magnitude in feature importance coefficients If we have a flower with the attribute values described in Table 1 (sepal length = 6.9, sepal width = 3.1 and petal length = 4.9), the model estimates the likelihood of this being in the posi- tive class (y = 1, an iris virginica) at 1.0 (i.e. 100%). Since this is a simple single decision tree, we can easily follow the path through the tree (Fig 2) for this prediction and isolate the relative contribution of each feature; Contributionpetal length ðcmÞ ¼ ð0:051 0:493Þ þ ð0:25 0:051Þ ¼ 0:243 ð2Þ 5 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0258658 October 25, 2021 PLOS ONE Hollow-tree super: A directional and scalable approach for feature importance in boosted tree models Fig 2. Single decision tree linearized using the eli5 package. By employing a general linear equation to define the relative contributions of each feature as decisions are made at an increasing depth in the tree, it is possible to derive feature importance values which lead to a positive (Iris Virginica) or negative (Iris versicolor) classification. This analysis offers a unique advantage over Gini importance, partial dependence, and permutation importance such that feature importance coefficients provide both a directionality and magnitude for each feature in delineating data into the positive and negative classes. Fig 2. Single decision tree linearized using the eli5 package. By employing a general linear equation to define the relative contributions of each feature as decisions are made at an increasing depth in the tree, it is possible to derive feature importance values which lead to a positive (Iris Virginica) or negative (Iris versicolor) classification. This analysis offers a unique advantage over Gini importance, partial dependence, and permutation importance such that feature importance coefficients provide both a directionality and magnitude for each feature in delineating data into the positive and negative classes. Contributionsepal width ðcmÞ ¼ ð0:143 0:25Þ ¼ 0:107 ð3Þ Contributionsepal length ðcmÞ ¼ ð1:0 0:143Þ ¼ 0:857 ð4Þ Contributionsepal width ðcmÞ ¼ ð0:143 0:25Þ ¼ 0:107 ð3Þ ð3Þ Contributionsepal length ðcmÞ ¼ ð1:0 0:143Þ ¼ 0:857 ð4Þ ð4Þ Table 1. Sample prediction and feature contribution score. Class = 1, probability = 1.0 Contribution Feature Value +0.857 Sepal length (cm) 6.90 +0.493 BIAS 1.00 -0.107 Sepal width (cm) 3.10 -0.243 Petal length (cm) 4.90 https://doi.org/10.1371/journal.pone.0258658.t001 Table 1. Sample prediction and feature contribution score. 1.2 Direction and magnitude in feature importance coefficients PLOS ONE \| https://doi.org/10.1371/journal.pone.0258658 October 25, 2021 6 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0258658 October 25, 2021 PLOS ONE Hollow-tree super: A directional and scalable approach for feature importance in boosted tree models The model is telling us that sepal length of 6.9cm makes it much more likely that this is a iris virginica (85.7% more likely, to be exact), whilst the petal length of 4.9 somewhat lowers the chances of this being a iris virginica. This has made our single decision tree fully interpret- able, in the sense that we can readily say exactly how each feature has influenced the prediction. The final prediction (probability of being in the positive class) is the sum of the feature con- tributions towards the prediction (0.857–0.107–0.243 = 0.507) plus the bias value (= 0.493), in this case, 1. Since we don’t want to study feature importance at a prediction level, we can average the contributions of each feature across all predictions to obtain a global picture of importance. The method to achieve this involves three steps: Since we don’t want to study feature importance at a prediction level, we can average the contributions of each feature across all predictions to obtain a global picture of importance. The method to achieve this involves three steps: 1. Filter out incorrect and low probability predictions. 1. Filter out incorrect and low probability predictions. 2. Separate the feature contributions towards the positive and negative classes. 3. Sum the contributions of each feature across all predictions and normalize these values by dividing by the number of predictions made. 3. Sum the contributions of each feature across all predictions and normalize these values by dividing by the number of predictions made. After performing this process on the Iris dataset, we found the top feature for predicting both the positive and negative classes to be petal length (Fig 2). Unsurprisingly, petal length also had the greatest Gini importance and the second greatest permutation importance, sug- gesting this method correctly captures feature importance within a decision model and sup- ports the findings of alternative, less robust, analyses. 1.3 Scaling feature extraction from single tree to boosted ensembles Ensemble trees and particularly boosted ensemble trees often provide a superior prediction ability than single trees [12]. Unfortunately, the linearization method described above falls short as each tree of the boosted ensemble leads to a different succession of split and group mean for each node due to the random start for the generation of each tree used in the ensem- ble. As a matter of fact, running the prediction function provided in the eli5 package would lead to a different linear equation for each tree of the ensemble. To circumvent this issue, we propose here an aggregation method across several boosted tree instances within a model to provide directional, proportional, and interpretable feature importance. In addition, this method has the advantage of working across multiple cycles of cross-validation dealing more positively with boosted trees’ tendency to overfit. PLOS ONE \| https://doi.org/10.1371/journal.pone.0258658 October 25, 2021 2.1 Gradient-boosting decision trees using XGB classifier We use XGBClassifier [2] to fit the model. Table 2 below shows the improvement in model accuracy, ROC AUC and F1 score achieved by the Gradient boosted approach over single Decision trees. Accuracy is the proportion of correct predictions to total predictions. Accuracy can be represented in the following equation: Where TP is true positive predictions, TN is true Table 2. [Iris dataset] model performance metrics. Measure Decision Tree XGBoost Accuracy 0.920 0.940 ± 0.075 ROC AUC 0.923 0.940 ± 0.075 F1 Score 0.917 0.939 ± 0.081 https://doi.org/10.1371/journal.pone.0258658.t002 Table 2. [Iris dataset] model performance metrics. Table 2. [Iris dataset] model performance metrics. 7 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0258658 October 25, 2021 PLOS ONE Hollow-tree super: A directional and scalable approach for feature importance in boosted tree models negative predictions, FP is false positive predictions, and FN is false negative predictions. “Pre- dictions” refers to the number of items for which a classification was made by XGBClassifier, for example here, the number of plants in the iris dataset. Accuracy ¼ TP þ TN TP þ TN þ FP þ FN ð5Þ ð5Þ F1 score (or F score) is the weighted average of precision and recall and takes into account both false positives and false negatives. Precision is a measure of the number of predictions of the positive class that actually belong to the positive class. It is calculated as the number of true positives as a proportion of the sum of true positives and false positives. Recall is a measure of the number of positive class predictions out of all positive classes. It is calculated as the number of true positives as a proportion of the sum of true positives and false negatives. ROC AUC provides an aggregate measure of performance across all possible classification thresholds. It is measured as the area under the receiver operating characteristic curve, which is a plot of the true positive rate (i.e. recall) to the false positive rate. Note that a greater positive value indicates a greater contribution towards positive (Iris Virginica) or negative (Iris Versicolor ositive value indicates a greater contribution towards positive (Iris Virginica) or negative (Iris Versicolor) classification. 2.2 Performing Hollow-Tree Super (HOTS) 1. For each subject, we can use eli5 explain_prediction [17] to obtain each feature’s contribu- tion to each prediction (ignoring the bias value). Note that when extracting feature contri- bution from a boosted tree model, the ‘weights’ become the log odds contribution of each feature (as opposed to the probabilities shown under a single tree model). 1. For each subject, we can use eli5 explain_prediction [17] to obtain each feature’s contribu- tion to each prediction (ignoring the bias value). Note that when extracting feature contri- bution from a boosted tree model, the ‘weights’ become the log odds contribution of each feature (as opposed to the probabilities shown under a single tree model). 2. We separate the weights contributing towards the positive and negative class cases. 3. Incorrect predictions, and those with a prediction probability of less than 70% are filtered out, keeping only the subjects that were correctly predicted by the model with confidence. 4. The weights across all the remaining predictions are aggregated by feature and divided by the number of predictions, obtaining an average weight of each feature per prediction. 5. As mentioned in (1), the weights provided are the log odds of being in the class that is ulti- mately predicted (i.e. for positive class predictions, the weights are the log odds of being in the positive class). Additionally, the weights are the log odds at the value of the feature cur- rently being predicted. Thus, to extract the directionality desired here, it is necessary to infer the sign of these log odds for each feature. This is achieved by identifying whether the mean value for each feature in this positive class is greater or less than the mean of each Table 3. 5-fold feature contribution average for positive and negative classification of the Iris dataset using HOTS. Feature Classification Fold 1 Fold 2 Fold 3 Fold 4 Fold 5 Petal Length Positive -4.04 -1.54 -1.90 -2.63 -2.51 Negative +2.97 +1.69 +1.24 +2.90 +2.60 Petal Width Positive -1.09 -3.86 -2.31 -2.42 -2.26 Negative +1.61 +2.83 +2.64 +2.11 +2.20 Sepal Length Positive -0.13 -0.13 -0.11 +0.14 -0.04 Negative +0.27 +0.63 +0.25 -0.25 +0.15 Sepal Width Positive +0.02 +0.13 +0.01 +0.02 -0.25 Negative -0.12 -0.24 +0.07 -0.27 -0.08 le 3. 5-fold feature contribution average for positive and negative classification of the Iris dataset using HOTS. 2.2 Performing Hollow-Tree Super (HOTS) PLOS ONE \| https://doi.org/10.1371/journal.pone.0258658 October 25, 2021 8 / 16 PLOS ONE Hollow-tree super: A directional and scalable approach for feature importance in boosted tree models corresponding feature in the negative class. The log odds of each feature in the positive class are multiplied by the sign of the mean value for the positive class less the mean value for the negative class, whilst the log odds of each feature in the negative class are multiplied by the inverse sign of this same calculation. The ‘weights’ now become the log odds with a standard view of directionality. However, it is important to note that this approach assumes linearity between the feature values. 6. As mentioned, one challenge to this approach is the inherent instability of feature impor- tance over different runs caused by the overfitting and random factor dependence of gradi- ent boosted tree models. Different cuts of data for training and testing produce different results. This problem can be solved by performing cross-validation to arrive at a stable set of features. This allows for greater confidence when interpreting the model as it highlights only features that are consistent across runs. Here we use 5-fold cross-validation to obtain the mean feature importance’s, with an average accuracy across all 5 folds = 0.94. 6. As mentioned, one challenge to this approach is the inherent instability of feature impor- tance over different runs caused by the overfitting and random factor dependence of gradi- ent boosted tree models. Different cuts of data for training and testing produce different results. This problem can be solved by performing cross-validation to arrive at a stable set of features. This allows for greater confidence when interpreting the model as it highlights only features that are consistent across runs. Here we use 5-fold cross-validation to obtain the mean feature importance’s, with an average accuracy across all 5 folds = 0.94. 3.1 Iris dataset Using HOTS, A high degree of concordance with the features determined by Gini and permu- tation importance is maintained, with petal length and petal width both identified as the top two most predictive features (Fig 3). Since these weights are effectively the log odds for the respective classes, we can interpret them as such. The large negative value for petal length in the positive class (= -0.45) indicates that as petal length increases, the odds of being in the posi- tive class decreases. A similar conclusion can be made for petal width. Further, we can see that the sepal dimensions had little to no predictive power. We are also able to perform a count of how many folds each feature appears during the cross-validation process (Fig 4a). We see that all four features of the iris dataset were used to make predictions in each of the 5 folds (Fig 4b). This is particularly useful when modelling on data with a large number of features, as it highlights the features that may only appear sporadically (low fold count in Fig 4b), but which have a large weight in those instances (large average feature importance weighting in Fig 4a). This methodology unveils a way to achieve a similar outcome as calculating feature impor- tance by Gini importance or improvement to F-score, whilst also making use of ensemble methods like gradient boosting which provide a superior fit. Further, it does this in a way that better quantifies the impact of the variable since the weights in Fig 4a represent the averaged feature importance contributions after cross-validation towards the respective final predictions. This method is optimized for approximating the contribution of each feature to the classifi- cation outcome and while it is limited to one observation at a time, it can be easily scaled. 3.2 Case study: HOTS feature importance in Schizophrenic brain data Furthermore, we propose this method as a suitable way of tying clinically observed behaviours to functional brain regions, or “parcellations”. A “parcellation atlas” in simple terms is a map of the brain. It delineates regions of the neo- cortex that exhibit similar properties across individuals, such as functional activity, structural connectivity, or cellular composition. Thus, a ‘parcellation’ is a region of the brain that expresses similar properties in a population, even if the exact boundaries or topological loca- tion may differ between individuals. 9 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0258658 October 25, 2021 PLOS ONE Hollow-tree super: A directional and scalable approach for feature importance in boosted tree models Fig 3. Average feature contribution weight per prediction in a single decision tree. After entering the Iris dataset into our linearized decision tree, we found the most important feature for successfully determining positive or negative class to be ‘petal length’. Importantly, the outputs provided in this analysis are given a magnitude and direction for their respective involvement in classification compared to other features. These metrics offer a significant improvement on previous analyses, whilst remaining consistent with the findings of Gini and permutation importance, where ‘petal length’ had the highest and second highest weightings respectively. Note that this represents the output data from a single decision tree (fold) prior to cross- validation. Fig 3. Average feature contribution weight per prediction in a single decision tree. After entering the Iris dataset into our linearized decision tree, we found the most important feature for successfully determining positive or negative class to be ‘petal length’. Importantly, the outputs provided in this analysis are given a magnitude and direction for their respective involvement in classification compared to other features. These metrics offer a significant improvement on previous analyses, whilst remaining consistent with the findings of Gini and permutation importance, where ‘petal length’ had the highest and second highest weightings respectively. Note that this represents the output data from a single decision tree (fold) prior to cross- validation Fig 3. Average feature contribution weight per prediction in a single decision tree. After entering the Iris dataset into our linearized decision tree, we found the most important feature for successfully determining positive or negative class to be ‘petal length’. Importantly, the outputs provided in this analysis are given a magnitude and direction for their respective involvement in classification compared to other features. PLOS ONE \| https://doi.org/10.1371/journal.pone.0258658 October 25, 2021 3.2 Case study: HOTS feature importance in Schizophrenic brain data Average feature contributions and count number over 5-fold cross validation. (a) By investigating the five folds used for cross-validation shown in Table 3, we once again determined ‘petal width’ and ‘length’ to be the most important features for positive and negative classification within our decision tree. (b) A count of the number of folds each feature appeared in during the cross-validation process revealed that all four features appeared equally throughout the decision making process—suggesting that despite being significantly less important features for positive and negative classification than ‘petal length’ and ‘width’, sepal features are utilised equally regularly to make predictions within the model across multiple folds. Fig 4. Average feature contributions and count number over 5-fold cross validation. (a) By investigating the five folds used for cross-validation shown in Table 3, we once again determined ‘petal width’ and ‘length’ to be the most important features for positive and negative classification within our decision tree. (b) A count of the number of folds each feature appeared in during the cross-validation process revealed that all four features appeared equally throughout the decision making process—suggesting that despite being significantly less important features for positive and negative classification than ‘petal length’ and ‘width’, sepal features are utilised equally regularly to make predictions within the model across multiple folds. https://doi.org/10.1371/journal.pone.0258658.g004 https://doi.org/10.1371/journal.pone.0258658.g004 functional brain areas would not only need to provide direction and magnitude, but also be scalable to a large number of features. Here we present a case example of the feature contribution for boosted trees method described throughout this manuscript, demonstrating its ability to effectively tie functional regions of the brain to clinically observed behaviours. We performed this analysis using the SchizConnect Center for Biomedical Research Excel- lence (COBRE) dataset [20]. The dataset consists of the necessary brain MRI data for mapping, as well as neuropsychological assessment scores for 60 patients diagnosed with Schizophrenia. The analysis was designed as a binary classification problem—predicting the presence or absence of a specific symptom. Specifically, we use item N4 from the patient’s Positive and Negative Syndrome Scale (PANSS) [21], which measures the degree to which patient’s show “passive/apathetic social withdrawal”. Each patient recorded their response on a 7 point Likert scale, representing increasing levels of psychopathology (1 = absent, 7 = extreme), with a sensi- ble binarization point determined at a score of 2 (i.e. 3.2 Case study: HOTS feature importance in Schizophrenic brain data These metrics offer a significant improvement on previous analyses, whilst remaining consistent with the findings of Gini and permutation importance, where ‘petal length’ had the highest and second highest weightings respectively. Note that this represents the output data from a single decision tree (fold) prior to cross- validation. https://doi.org/10.1371/journal.pone.0258658.g003 Parcellation atlases are particularly useful when analyzing functional magnetic resonance imaging (fMRI) data, which through recording changes in blood flow over time, can produce a representation of neural activity. This information can further be used to show functional connectivity between regions of the brain if the activity recorded between them exhibits a sta- tistical relationship. A parcellation atlas can be used to reduce this complexity of pairwise cor- relations by reducing the comparisons to a finite number of regions, assumed to perform somewhat uniform functions. Recently, Doyen et al. (forthcoming) developed a machine learning-based technique for parcellating the brain in a way that is both subject specific, and comparable between subjects [2,18]. Using this technique, we are able to generate adjacency matrices representing the corre- lation between every pair of parcellations—described for the remainder of this paper as ‘con- nectomic features’. The applied atlas contains 379 parcellations in Glassian nomenclature [19] equating to 71,631 input features, and so any method used to tie clinically observed behaviour to these PLOS ONE \| https://doi.org/10.1371/journal.pone.0258658 October 25, 2021 10 / 16 PLOS ONE Hollow-tree super: A directional and scalable approach for feature importance in boosted tree models Fig 4. Average feature contributions and count number over 5-fold cross validation. (a) By investigating the five folds used for cross-validation shown in Table 3, we once again determined ‘petal width’ and ‘length’ to be the most important features for positive and negative classification within our decision tree. (b) A count of the number of folds each feature appeared in during the cross-validation process revealed that all four features appeared equally throughout the decision making process—suggesting that despite being significantly less important features for positive and negative classification than ‘petal length’ and ‘width’, sepal features are utilised equally regularly to make predictions within the model across multiple folds. https://doi.org/10.1371/journal.pone.0258658.g004 Fig 4. Average feature contributions and count number over 5-fold cross validation. (a) By investigating the five f ld d f l d h T bl d d ‘ l d h’ d ‘l h’ b h Fig 4. PLOS ONE \| https://doi.org/10.1371/journal.pone.0258658 October 25, 2021 Fig 5. Predictive brain regions for passive/apathetic social withdrawal. Applying our methodology to a cohort of 60 subjects from the Schizconnect COBRE dataset revealed brain regions most predictive for item N4 of the Positive and Negative Syndrome Scale (PANSS). Connectivity matrices were generated between 379 cortical and subcortical parcellations using a scheme derived from Glasser and colleagues (2015), and feature importance was carried out on connectivity measures extracted from each individual parcellation. After performing cross-validation and averaging the weights of feature importance, we determined R_POS2, R_IFSa, R_6mp, R_FST, R_AAIC, R_1, R_PFcm, R_PGs, and L_LIPd to be most predictive for positive classification of PANSS N4. Positive class indicates a score > 2 on item N4 of the PANSS. X-axis values are provided in log odds to more easily visualise the features of importance on a logarithmic scale. L_ = left side, R_ = right side, POS2 = area 2 of the parietal-occipital sulcus, IFSa = anterior inferior frontal sulcus, 6mp = medial posterior aspect of area 6, FST = lateral occipital visual area, AAIC = anterior agranular insular cortex, 1 = primary sensory area, PFcm = centromedian part of parietal area F, PGs = superior aspect of parietal area G, LIPd = dorsal aspect of the lateral intraparietal area. https://doi.org/10.1371/journal.pone.0258658.g005 https://doi.org/10.1371/journal.pone.0258658.g005 https://doi.org/10.1371/journal.pone.0258658.g005 Further, cross-validation revealed that the R_POS2 was used by the boosted tree model to make predictions in 3 out of 5 folds (Fig 6). This suggests that this feature was consistently important for making predictions towards the positive class. 3.2 Case study: HOTS feature importance in Schizophrenic brain data subjects with a N4 score > 2 were in the positive class, and 2 were in the negative class). As described above, the input features used to predict the presence or absence of this symptom were the subject’s pairwise functional cor- relation between the 379 regions of the brain atlas which together form a large adjacency matrix. As with the iris dataset, we use XGBClassifier [2] to fit the model and perform 5-fold cross- validation with an average accuracy of 0.71. Using the same feature contribution method described for the iris dataset, we are able to generate a list of the features (parcellations) that were most predictive of the positive and negative class symptom (Fig 6). The positive and neg- ative class importance’s are complementary, and so only the positive class (target) impor- tance’s are shown here. As Fig 5 shows, the region of the brain most predictive of the presence of ‘passive/apathetic social withdrawal’ is area 2 of the right parieto occipital sulcus (R_POS2). Since these values are represented as log odds, the value for R_POS2 (-1.33) tells us that as the values for this parcellation increased, the probability of being in the positive class (having greater “passive/apathetic social withdrawal”) decreased. Conversely, we can see that increases in the value for the dorsal aspect of the left lateral intraparietal lobule (L_LIPd) indicated an increased probability of being in the positive class (+0.48). PLOS ONE \| https://doi.org/10.1371/journal.pone.0258658 October 25, 2021 11 / 16 PLOS ONE Hollow-tree super: A directional and scalable approach for feature importance in boosted tree models PLOS ONE \| https://doi.org/10.1371/journal.pone.0258658 October 25, 2021 4. Discussion Throughout this manuscript we detailed and validated a novel method, “Hollow-tree super” (HOTS), which accurately provides metrics of directionality and magnitude between features in boosted tree models. Further, the ability to discern between a large number of features and be scaled to large datasets makes HOTS an easily implementable method which can be utilized by a range of data-driven disciplines. When applied to the Iris dataset, HOTS showed high 12 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0258658 October 25, 2021 PLOS ONE Hollow-tree super: A directional and scalable approach for feature importance in boosted tree models Fig 6. Count of recurring features across folds. By performing a count of feature appearance during the cross validation process, we determined that the same features (parcellations) responsible for positive class classification were also consistently used in 2 (R_PGs, R_PFcm, R_1, L_LIPd), 3 (R_POS2, R_IFSa, R_AAIC, R_6mp), or 4 (R_FST) out of the 5 folds, suggesting that these same features were regularly used throughout the decision making process. Note that this plot was abbreviated to only show features with a count of greater than 1. https://doi.org/10.1371/journal.pone.0258658.g006 Fig 6. Count of recurring features across folds. By performing a count of feature appearance during the cross validation process, we determined that the same features (parcellations) responsible for positive class classification were also consistently used in 2 (R_PGs, R_PFcm, R_1, L_LIPd), 3 (R_POS2, R_IFSa, R_AAIC, R_6mp), or 4 (R_FST) out of the 5 folds, suggesting that these same features were regularly used throughout the decision making process. Note that this plot was abbreviated to only show features with a count of greater than 1. https://doi.org/10.1371/journal.pone.0258658.g006 https://doi.org/10.1371/journal.pone.0258658.g006 https://doi.org/10.1371/journal.pone.0258658.g006 concordance with other commonly used methods for investigating feature importance: Gini importance, partial dependence plots and permutation importance. In a neuroscientific set- ting, HOTS was able to accurately identify between 379 features, the most responsible brain regions for positive classification of Schizophrenia from item N4 on the PANSS. Cross-valida- tion supported that these same features were consistently utilized throughout the decision- making process, improving our confidence that the chosen model had an accurate fit for diag- nosis. Modern neuroscientific investigations further bolster our findings, supporting that abnormal activity and connectivity between occipital and parietal brain regions occurs during Schizophrenia [22,23]. That is, our use of HOTS supports the current understanding of the determinants of this pathology and provides neuroscientists with a novel method for investi- gating similar pathologies of the brain. 5. Study limitations Importantly, this study involves a few key limitations that require addressing. First, our investi- gation involved benchmarking HOTS as a feature importance method by testing its concor- dance with Gini Importance, Partial Dependence Plots and Permutation Importance, as we felt those were the three most common methods used throughout the field. Whilst HOTS was able to effectively reproduce the results of these three methods using the Iris dataset, we cannot with certainty assert that HOTS would reproduce the results of other methodologies such as Information Gain or Chi-squared tests for feature selection. Additionally, as with any machine-learning model, increasing the size of the dataset would improve the ability of HOTS 13 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0258658 October 25, 2021 PLOS ONE Hollow-tree super: A directional and scalable approach for feature importance in boosted tree models to make accurate inferences about feature contribution and classification into positive and negative classes [24], and whilst the Iris dataset remains widely-accepted a valid tool for testing machine-learning algorithms [25,26], our observations on the Schizophrenic brain data may be improved with greater volumes of imaging data which we aim to collect and test in future investigations. Finally, an inherent limitation to boosted-tree modelling extends to our method in that instability can arise due to random factor dependence occurring between trees. Whilst we did consider this problem, we believe the incorporation of cross-validation when perform- ing HOTS provides an effective regulating step to best minimize this, and further promotes this method as superior for investigating feature importance in boosted-tree models. 6. Conclusion and future directions These results support that HOTS offers superior metrics for investigating feature importance than previously used methodologies. At least in the case of boosted-tree models, we suggest that HOTS could be well incorporated into the modelling process and provide thorough and easily interpretable metrics for the most responsible features which are also consistently uti- lized for classification. Overall, the potential to visualise directionality and magnitude of fea- ture contribution within a single methodology streamlines the process of performing boosted- tree modelling, and offers a unique approach which can be applied to a range of different types of data, including high dimensional datasets such as those encountered in the study of neuro- imaging data. Further investigations should test the applicability of this method on other data- sets, for example in neuroscience, where HOTS could be further utilized to investigate which brain regions are most responsible for other disorders of the brain such as Major Depressive Disorder, Alzheimer’s and Parkinson’s disease. A longitudinal dataset of pre- and post-treat- ment scans with corresponding symptom scores could be used to verify the results of these neuroscientific applications. An interesting future extension of the method could include designing an approach to deliver HOTS feature importance scores in regression models, as well as a method for working with categorical input data. References 1. Klosterman, S. Why decision trees are more flexible than linear models, explains Stephen Klosterman. 2019 Dec. https://hub.packtpub.com/why-decision-trees-are-more-flexible-than-linear-models- explains-stephen-klosterman/. 2. Chen T, Guestrin C. XGBoost: A Scalable Tree Boosting Method. Association for Computing Machin- ery, NY, USA. 2016. 3. Breiman L, Friedman JH, Olshen RA, and Stone CJ., Classification and regression trees: Wadsworth, Inc. 1984. 4. Greenwell BM. pdp: An R Package for Constructing Partial Dependence Plots. R J., 2017; 9(1), 421. 5. Ojala M, Garriga GC. Permutation tests for studying classifier performance. Journal of Machine Learn- ing Research, 2010; 11(6). 6. Palczewska A, Palczewski J, Robinson R, Neagu D. Interpreting random forest models using a feature contribution method. 2013 IEEE 14th International Conference on Information Reuse & Integration (IRI), 2013; 112–119. 7. Fisher RA. The Use Of Multiple Measurements in Taxonomic Problems. Annals of Eugenics, 1936; 7 (2), 179–188. https://doi.org/10.1111/j.1469-1809.1936.tb02137.x 8. Pedregosa F, Varoquaux G, Gramfort A, Michel V, Thirion B, Grisel O, et al. Scikit-learn: Machine learn- ing in Python. the Journal of machine Learning research, 2011; 12, 2825–2830. 9. Strobl C, Boulesteix AL, Zeileis A, Hothorn T. Bias in random forest variable importance measures: Illus- trations, sources and a solution. BMC Bioinformatics. 2007; 8(1):1–21. https://doi.org/10.1186/1471- 2105-8-25 PMID: 17254353 10. Dı´az-Uriarte R, Alvarez de Andre´s S. Gene selection and classification of microarray data using random forest. BMC Bioinformatics. 2006; 7(1):1–13. https://doi.org/10.1186/1471-2105-7-3 PMID: 16398926 11. Nembrini S, Ko¨nig IR, Wright MN. The revival of the Gini importance? Bioinformatics. 2018; 34 (21):3711–3718. https://doi.org/10.1093/bioinformatics/bty373 PMID: 29757357 12. Cutler DR, Edwards TC, Beard KH, Cutler A, Hess KT, Gibson J, et al. Random forests for classification in ecology. Ecology. 2007; 88(11):2783–2792. https://doi.org/10.1890/07-0539.1 PMID: 18051647 13. 4.1. Partial Dependence and Individual Conditional Expectation plots—scikit-learn 0.24.2 documenta- tion. Accessed June 13, 2021. https://scikit-learn.org/stable/modules/partial_dependence.html. 14. Altmann A, Toloşi L, Sander O, Lengauer T. Permutation importance: a correctedfeature importance measure. Bioinformatics, 2010; 26(10), 1340–1347. https://doi.org/10.1093/bioinformatics/btq134 PMID: 20385727 15. Saabas A. Interpreting Random Forests. 2014 Oct. http://blog.datadive.net/interpreting-random- forests/. 16. Friedman J. Greedy Function Approximation: A Gradient Boosting Machine. The Annals of Statistics, 2000; 29. https://doi.org/10.1214/aos/1013203451 17. Tulio Ribeiro M, Singh S, Guestrin C. " Why Should I Trust You?": Explaining the Predictions of Any Classifier. arXiv e-prints, 2016;arXiv-1602. 18. Omniscient Neurotechnology Pty. Ltd. (o8t). Infinitome [Software] 2020. https://www.o8t.com/ neurologistresearchers. 19. Glasser MF, Coalson TS, Robinson EC, Hacker CD, Harwell J, Yacoub E, et al. A multi-modal parcella- tion of human cerebral cortex. Nature, 2016; 536(7615), 171–178. Author Contributions Conceptualization: Stephane Doyen, Isabella Young, Michael E. Sughrue. Conceptualization: Stephane Doyen, Isabella Young, Michael E. Sughrue. Data curation: Stephane Doyen, Hugh Taylor, Peter Nicholas. Formal analysis: Stephane Doyen, Hugh Taylor, Peter Nicholas. Investigation: Stephane Doyen, Hugh Taylor, Peter Nicholas, Isabella Young. Methodology: Stephane Doyen, Hugh Taylor, Peter Nicholas, Lewis Crawford, Isabella Young. Formal analysis: Stephane Doyen, Hugh Taylor, Peter Nicholas. Investigation: Stephane Doyen, Hugh Taylor, Peter Nicholas, Isabella Young. Methodology: Stephane Doyen, Hugh Taylor, Peter Nicholas, Lewis Crawford, Isabella Young. Investigation: Stephane Doyen, Hugh Taylor, Peter Nicholas, Isabella Young. Methodology: Stephane Doyen, Hugh Taylor, Peter Nicholas, Lewis Crawford, Isabella Young. Resources: Isabella Young, Michael E. Sughrue. Resources: Isabella Young, Michael E. Sughrue. Software: Stephane Doyen, Peter Nicholas. Supervision: Stephane Doyen, Michael E. Sughrue. Validation: Stephane Doyen, Michael E. Sughrue. Validation: Stephane Doyen, Michael E. Sughrue. Visualization: Stephane Doyen, Lewis Crawford. Writing – original draft: Stephane Doyen, Lewis Crawford. Writing – review & editing: Stephane Doyen, Hugh Taylor, Lewis Crawford. 14 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0258658 October 25, 2021 PLOS ONE \| https://doi.org/10.1371/journal.pone.0258658 October 25, 2021 PLOS ONE Hollow-tree super: A directional and scalable approach for feature importance in boosted tree models 25. Schutten M, Wiering MA. An analysis on better testing than training performances on the Iris dataset. InProceedings of Belgian Dutch Artificial Intelligence Conference, Amsterdam, The Netherlands 2016 Nov (pp. 10–11). 26. Wu Y, He J, Ji Y, Huang G, Yao H, Zhang P, et al. Enhanced classification models for iris dataset. Pro- cedia Computer Science. 2019 Jan 1; 162:946–54. Hollow-tree super: A directional and scalable approach for feature importance in boosted tree models PLOS ONE \| https://doi.org/10.1371/journal.pone.0258658 October 25, 2021 References https://doi.org/10.1038/nature18933 PMID: 27437579 20. Mayer AR, Ruhl D, Merideth F, Ling J, Hanlon FM, Bustillo J, Cañive J. Functional imaging of the hemo- dynamic sensory gating response in schizophrenia. Human brain mapping, 2013; 34(9), 2302–2312. https://doi.org/10.1002/hbm.22065 PMID: 22461278 21. Kay SR, Fiszbein A, Opler LA. The positive and negative syndrome scale (PANSS) for schizophrenia. Schizophr Bull. 1987; 13(2):261–76. https://doi.org/10.1093/schbul/13.2.261 PMID: 3616518. 22. Danckert J, Saoud M, Maruff P. Attention, motor control and motor imagery in schizophrenia: implica- tions for the role of the parietal cortex. Schizophrenia Research, 2004; 70(2), 241–261. https://doi.org/ 10.1016/j.schres.2003.12.007 PMID: 15329301 23. Tohid H, Faizan M, Faizan U. Alterations of the occipital lobe in schizophrenia. Neurosciences (Riyadh, Saudi Arabia), 2015; 20(3), 213–224. https://doi.org/10.17712/nsj.2015.3.20140757 PMID: 26166588 24. Mola F. Classification and Regression Trees Software and New Developments. In: Rizzi A., Vichi M., Bock HH. (eds) Advances in Data Science and Classification. Studies in Classification, Data Analysis, and Knowledge Organization. Springer, Berlin, Heidelberg. 1998. 15 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0258658 October 25, 2021 PLOS ONE 16 / 16
https://openalex.org/W4283017500	https://www.researchsquare.com/article/rs-1728327/latest.pdf	English	null	The CT delta-radiomics based machine learning approach in evaluating multiple primary lung carcinoma	Research Square (Research Square)	2,022	cc-by	4,279	Yanqing Ma Yanqing Ma Zhejiang Provincial People’s Hospital, Affiliated People’s Hospital, Hangzhou Medical College Jie Li Zhejiang Provincial People’s Hospital, Affiliated People’s Hospital, Hangzhou Medical College Xiren Xu Zhejiang Provincial People’s Hospital, Affiliated People’s Hospital, Hangzhou Medical College Yang Zhang Zhejiang Provincial People’s Hospital, Affiliated People’s Hospital, Hangzhou Medical College Yi Lin (  Linyi197001@163.com ) Zhejiang Provincial People’s Hospital, Affiliated People’s Hospital, Hangzhou Medical College Zhejiang Provincial Peoples Hospital, Affiliated Peoples Hospital, Hangzhou Medical College Jie Li Zhejiang Provincial People’s Hospital, Affiliated People’s Hospital, Hangzhou Medical College Xiren Xu Zhejiang Provincial People’s Hospital, Affiliated People’s Hospital, Hangzhou Medical College Yang Zhang Zhejiang Provincial People’s Hospital, Affiliated People’s Hospital, Hangzhou Medical College Yi Lin (  Linyi197001@163.com ) Zhejiang Provincial People’s Hospital, Affiliated People’s Hospital, Hangzhou Medical College Results To radiomics analysis, the forest classifier (FC-radio) with the minimal RSD showed the better stability was chosen with AUCs of 0.840 (95%CI, 0.810–0.867) and 0.670 (95%CI, 0.611–0.724) in the training and validation set. The AUCs of the forest classifier based on delta-radiomics (FC-delta) were higher than those of FC-radio. In addition, with the extension of follow-up duration, the performance of FC-delta in Group C were the best with AUCs of 0.998 (95%CI, 0.993-1.000) in the training set and 0.853 (95%CI, 0.752–0.940) in the validation set. Conclusions The machine-learning approach based on radiomics and delta-radiomics helped to differentiate SPLCs from MPLCs. The FC-delta with longer follow-up duration better differentiated SPLCs from MPLCs. Methods A cohort of 1094 patients containing 268 MPLCs and 826 SPLCs were enrolled for radiomic study. After the segmentation of volume of interest, the radiomic features were automatically calculated. The patients were divided into the training set and validation set with a random proportion of 7:3. After the methods of feature selection, the relevant classifiers were constructed by the machine learning algorithms of Bayes, forest, k-nearest neighbor, logistic regression, support vector machine, and decision tree. The relative standard deviation (RSD) was calculated and the classification model with minimal RSD was chosen for delta-radiomics analysis to explore the variation of tumor during follow-up surveillance in the cohort of 225 MPLCs and 320 SPLCs. According to the different follow-up duration, three groups were divided into group A (3–12 months), group B (13–24 months), and group C (25–48 months). Then the corresponding delta-radiomics classifiers were developed to evaluate MPLCs. The area under the receiver operator characteristic curve (AUC) with 95% confidence interval (CI) was quantified to evaluate the efficiency of the model. Abstract Object: To evaluate the difference between multiple primary lung carcinoma (MPLC) and solitary primary lung carcinoma (SPLC) by delta-radiomics based machine learning algorithms in CT images. Research Article Keywords: Lung, carcinoma, multiple, computed tomography, radiomics Posted Date: June 17th, 2022 DOI: https://doi.org/10.21203/rs.3.rs-1728327/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Posted Date: June 17th, 2022 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Page 1/14 Page 1/14 Page 1/14 Introduction Page 2/14 Lung carcinoma is the most common cause of cancer-related death in china and is also a major health challenge worldwide[1]. The frequency and detection of lung carcinoma has descended gradually in the US, while it is remarkably increased in china over recent years[2]. Patients who survive one occurrence of non-small-cell lung carcinoma are at high risk of a second malignancy[3]. Cases of multiple primary lung carcinoma (MPLCs) are increasing, mainly leading to the improved diagnostic strategies, surveillance modalities, and the aging population[4]. The diagnostic criteria of MPLCs was firstly established by Martini and Melamed in the year of 1975[5] and was renewed by the American College and Chest Physicians (ACCP) in 2007[6]. The MPLC is classified into synchronous phenotype (sMPLC) when the second lung carcinoma was simultaneously diagnosed within 2 years after the primary lesion, and metachronous phenotype (mMPLC) when it was separately diagnosed more than 2 years after the initial surgery[7]. Duchateau et al. firstly indicated that 25% of patients accompanied with MPLCs, and patients with and without MPLCs had different growth habits[8]. Evaluation the MPLCs from SPLCs by empiric radiological experience is difficult[7]. Further analysis is therefore needed to comprehend more clearly. Radiomics converts the traditional radiological images into a large amount of minable high-dimensional data to explore the potential imaging biomarkers and support decision making[9]. And the delta-radiomics is the change of radiomic features after treatment or surveillance[10]. Undoubtedly, a delta-radiomics approach will have a growing impact on distinction the MPLCs and SPLCs, which will enable optimized management of patients with MPLCs. To best of our knowledge, there is no study focused on the delta-radiomics difference between MPLCs and SPLCs. The purpose of our study is to evaluate the delta-radiomic influence of MPLCs on prognosis to help us better understand their difference with SPLCs. Materials And Methods This retrospective study was approved by the Institutional Review Board of our hospital (NO. 2020QT108), which waived the informed consent of all patients. CT examination and Volume of interest segmentation All the patients underwent CT unenhanced examinations in Somanton Definition AS 64 or 128 CT (Siemens Medical Solutions, Germany). The scan parameters were as follows: tube voltage, 120 kVp; tube current, 200mA; rotation speed, 0.75s; beam pitch, 1.375; pixel matrix, 512512; detector collimation, 640.625mm; slice thickness, 2.0mm; reconstruction interval, 2.0mm; width of lung window, 1500HU; level of lung window, -600HU. The volume of interest (VOI) of tumor was depicted in software of “ITK-snap 3.8.0” (http://www.itksnap.org/pmwiki/) by two radiologists with 10 and 12 years of experience, manually (Fig. 2a, b). Then, the radiomic features were automatically calculated in software of “A.K. 3.0.0” (GE Healthcare) after steps of preprocessing involved resampling images to be 1.0mm at X/Y/Z space, reducing the image noise by a method of Gaussian, and discretizing the gray level to the range of 1 to 32. The intra-class correlation coefficients (ICCs) of radiomic features from two radiologists were calculated to evaluate the agreement between different observers. The radiomic features with ICCs greater than 0.75 were selected and the mean values of two radiologists were calculated for further analysis. Patients screening This retrospective study enrolled 1094 patients, which were pathologically diagnosed as lung adenocarcinoma after 6 years follow-up surveillance, including 826 patients with SPLCs and 268 MPLCs, between January 2014 to December 2020. Among these patients there were 320 SPLCs patents and 225 MPLCs patents with regular surveillance were incorporated for delta-radiomics analysis (Fig. 1). The inclusion criteria were as follows: (1) tumors were classified to be MPLCs according to the criteria of the 2nd edition of ACCP evidence-based clinical practice guidelines[6] (Table 1); (2) patients had only one primary lung tumor at the time cut-off of inclusion were classified to be SPLCs; (3) patients were pathologically proved to be minimally invasive (MIA) or invasive adenocarcinoma (IAC) of lung; (4) patients underwent CT examinations with the same protocol. The exclusion criteria were as follows: (1) patients were pathologically confirmed to be atypical adenomatous hyperplasia, in situ adenocarcinoma, or pulmonary squamous carcinoma; (2) patients was pathologically confirmed by needle biopsy; (3) patients were treated with the methods of radiation, chemotherapy, or radio-chemotherapy. Page 3/14 Page 3/14 Table 1 The criteria to diagnose MPLCs according to ACCP. MPLCs Same histology, anatomically separated Carcinomas in different lobes And no N2,3 involvement And no systemic metastases Same histology, temporally separated ≥ 4-yr interval between carcinomas And no systemic metastases from either carcinoma Different histology Different histology type Or different molecular genetic characteristics Or arising separately from foci of carcinoma in situ Table 1 Radiomics and delta-radiomics analysis Radiomics and delta-radiomics analysis Prior to radiomic analysis, the steps of excluding variables with zero variance, replacing abnormal values by median values, and standardization were adopted to normalize radiomic features. Then the cohort was randomly divided into the training set and validation set with a random proportion of 7:3. In order to eliminate the influence of unbalanced sample size, the way of synthetic minority over-sampling technique Page 4/14 Page 4/14 (SMOTE) was carried out[11]. After the methods of analysis of variance, correlation analysis with a threshold of 0.7, and gradient boosting decision tree (GBDT), the optimal radiomic features were extracted. Ultimately, the corresponding machine learning based classifiers including Bayes, forest, k- nearest neighbor, logistic regression, support vector machine, and decision tree algorithms were developed to identify MPLCs and SPLCs. The relative standard deviation (RSD) was calculated and the classification model with minimal RSD was chosen for further analysis. The area under the curve (AUC) with 95% confidence interval (95%CI) of receiver operator characteristic curve (ROC) was quantified to evaluated the efficiency of the machine learning based classifiers. With consideration of the different progression during regular follow-up surveillance between MPLCs and SPLCs, the delta-radiomics was utilized. The delta-radiomics was defined as the change of radiomic features between baseline and follow-up surveillance, including Group A (3–12 months), Group B (13–24 months), and Group C (25–48 months). The equation of delta-radiomics was: (follow-up radiomics - baseline radiomics)/follow-up interval. The specific information of radiomics and delta-radiomics analysis were listed in Supplementary Material. Statistics The general clinical characteristics were analysis by software of “SPSS 22.0” with methods of student’s t- test or chi-square test. The methods of radiomic feature selection including variance, correlation analysis, GBDT, and machine learning algorithms were performed by the software of “Python 3.5”. The ROC curve was delineated by the software of “MedCalc 15.8”. A p-value less than 0.05 indicates statistical significance. Radiomics analysis The radiomic features between the patients of MPLCs and SPLCs were differentiated. There were 27 radiomic features remained after feature selection of GBDT (Fig. 3) and the machine learning based classifiers including Bayes, forest, k-nearest neighbor, logistic regression, support vector machine, and decision tree were constructed in the training set and confirmed in the validation set. The forest classifier of radiomics (FC-radio) with minimal RSD of 1.82 was chosen for further analysis (Supplementary Material, Table 1). The specific AUC values of six machine-learning algorithms from 100 Bootstrap replication in the training set were listed in Supplementary Material, Table 2. The AUC of this FC-radio in the training set was 0.840 (95%CI, 0.810–0.867) and that of the validation set was 0.670 (95%CI, 0.611– 0.724). The low discrimination efficiency of this model indicates that the radiomic difference between tumors of SPLCs and MPLCs was inconspicuous. Patient’s general information There were 1094 patients with 268 MPLCs and 826 SPLCs. The general information of all patients were listed in Table 2. The general information included gender, age, location, and pathology. The variables of gender (p = 0.279) and age (p = 0.575) had no statistical significance, while the variables of location (p < 0.05) and pathology (p < 0.05) showed the significant difference. The tumors of SPLCs were more likely to locate in the right lung than these of MPLCs (62.2% vs. 60.1%). The pathological type of MPLCs was easier to be MIA (45.9% vs. 32.7%), while that of SPLCs was more prone to be IAC (67.3% vs. 54.1%). Page 5/14 Table 2 Patients’ general information MPLCs (n = 268) SPLCs (n = 826) p Gender 0.279 Female (%) 168 (62.7%) 487 (59.0%) Male (%) 100 (37.3%) 339 (41.0%) Age (mean ± standard) 57.4 ± 10.6 56.9 ± 12.9 0.575 Location < 0.05 Right lung 161 (60.1%) 514 (62.2%) Superior lobe 76 (28.4%) 298 (36.1%) Middle lobe 40 (14.9%) 54 (6.5%) Inferior lobe 45 (16.8%) 162 (19.6%) Left lung 107 (39.9%) 312(37.8%) Superior lobe 71 (26.5%) 199 (24.1%) Lnferior lobe 36 (13.4%) 113 (13.7%) Pathology < 0.05 MIA 123 (45.9%) 270 (32.7%) IAC 145 (54.1%) 556 (67.3%) ics analysis Table 2 Table 2 Patients’ general information Delta-radiomics analysis Regardless of the poor efficiency of the FC-radio in distinguishing MPLCs and SPLCs, a further forest machine learning algorithm of delta-radiomics (FC-delta) was conducted. Depending on the different Page 6/14 Page 6/14 duration of follow-up, we divided the patients into four groups: group A with a follow-up intervals of 3–12 months (105 MPLCs vs. 145 SPLCs), group B with a follow-up intervals of 13–24 months (68 MPLCs vs. 96 SPLCs ), and group C with a follow-up intervals of 25–48 months (52 MPLCs vs. 79 SPLCs). duration of follow-up, we divided the patients into four groups: group A with a follow-up intervals of 3–12 months (105 MPLCs vs. 145 SPLCs), group B with a follow-up intervals of 13–24 months (68 MPLCs vs. 96 SPLCs ), and group C with a follow-up intervals of 25–48 months (52 MPLCs vs. 79 SPLCs). The AUC of FC-delta in group A was 0.972 (95%CI, 0.951–0.989) in the training set and was 0.798 (95%CI, 0.704–0.892) in the validation set. The AUC of FC-delta in group B was 0.989 (95%CI, 0.978–0.997) in the training set and was 0.821 (95%CI, 0.708–0.915) in the validation set. The AUC of FC-delta in group C was 0.998 (95%CI, 0.993-1.000) in the training set and was 0.853 (95%CI, 0.752–0.940) in the validation set. With the extension of follow-up intervals, the difference between MPLCs and SPLCs was more obvious (Fig. 4). Discussions We reported a single-institution experience on radiomic differentiation on MPLCs from SPLCs, especially emphasis on their long-term variation. The reported incidence of synchronous lung carcinoma is variably between 0.2–20%[12]. In our study, the incidence of MPLCs was 24.5%, which was slightly higher than the reported incidence. It may be related to the universality of chest computed tomography screening programs. There were no statistical difference between the characteristics of gender and age. Slightly different from the outcome in the past study, female gender and smoke free statue were more frequent in MPLCs[7]. And the MPLCs were more easier to the pathological type of MIA compared with SPLCs (45.9% vs. 32.7%, p<0.05). This result supported the previous view that the proportion of MIA and adenocarcinoma in situ is high in sMPLCs[13]. This also may be due to timely detection of MPLCs at a early stage with regular surveillance for the first primary carcinoma[14]. However, present clinical and traditional methods are unable to understand the different evolution between MPLCs and SPLCs. Thus, studies of novel factors that differ significantly between patients with MPLCs and SPLCs are necessary vehicles for identifying subtleties in two diseases. Patients with first primary cancer remain at risk of developing a secondary tumor at a distant site through metastasis via the lymphatic or circulatory system[15]. Second primary carcinomas showed specific associations with the first one and their nature course were not the same[16]. The etiology of MPLCs is ambiguous[17]. Our radiomics analysis between MPLCs and SPLCs found of interest that radiomics could identify the difference between two groups with the AUC of 0.840 (95%CI, 0.810–0.867) in the training set and that of 0.670 (95%CI, 0.611–0.724) in the validation set. Nevertheless, this low discrimination efficiency was insufficient to supplied accurate information to better understand the difference between MPLCs and SPLCs. To best of our knowledge, it is the first article focused on the distinction between MPLCs and SPLCs from the point view of radiomics and delta-radiomics. The crucial challenge regarding MPLCs is what they differed with SPLCs in the course of development, on which both the treatment strategies and prognosis are based[18]. The possible of difference between MPLCs and SPLCs should always be considered during the follow-up surveillance, which determines the Page 7/14 Page 7/14 subsequent management strategy[19]. It has conclusively been suggested that the overall survival of MPLCs was better than SPLCs with intrapulmonary metastasis[20]. Conclusion In conclusion, our study revealed that the approaches of radiomics and delta-radiomics help to differentiate MPLCs and SPLCs. The radiomic difference between SPLCs and MPLCs was faint and the delta-radiomics better differentiate these patients. Moreover, with the extension of follow-up duration, the delta-radiomics difference between SPLCs and MPLCs appeared more distinctly. Discussions Therefore, a delta-radiomics approach studied the variation of radiomic features during baseline examination and follow-up duration[21]. The AUC of FC-delta of group C was the highest both in the training set (0.998 vs. 0.989 and 0.972) and the validation set (0.853 vs. 0.821 and 0.798). With the extension of follow-up intervals, the difference between MPLCs and SPLCs was more obvious. The literature on survival difference between sMPLCs and SPLCs has quantified and highlighted that the prognosis of sMPLCs was poorer and resembled that of SPLCs of a higher stage[22]. Our results suggested that the nature course of two diseases was inconsistent and the delta-radiomics could better distinct the MPLCs and SPLCs than radiomics. We firstly reported the difference of two diseases in terms of both radiomics and delta-radiomics to help us make decision on individual therapy and predict the prognosis of diseases. Our present study has several limitations. Currently, there are no definitive guideline for the diagnosis and treatment of MPLCs. In 2003, the American College of Chest Physicians (ACCP) developed a new diagnostic criteria for MPLCs with evaluations of lymphatic and systemic metastasis and the interval between mMPLC was extended to at least 4 years[23]. Antakli et al. revised the criteria of Martini and Melamed by adding DNA ploidy validation for distinction[24]. However, they have not widely applied to clinical practice due to its disadvantages of expensive, time consuming, and low sensitivity. Hence, we adopted the most cited criteria of the 2nd edition of ACCP in our research. Second, the MPLCs can be subdivided into mMPLCs and sMPLCs. Due to the limitation of incidence and sample size, we performed a general analysis of MPLCs which may lead to a biased result. Third, we only enrolled the cohort with pathological types of MIA and IAC to analysis and neglected other pathological types of lung carcinomas. The MPLCs and SPLCs with pathological types of adenocarcinoma in situ, squamous carcinoma[25], and so on should further be studied after collecting enough cases. Declarations Ethics approval and consent to participate: This retrospective study was approved by the Medical Ethics Committee of Zhejiang Provincial People’s Hospital (NO. 2020QT108) and in conformity to the Declaration of Helsinki. The informed consent was waived for this retrospective study by the Medical Ethics Committee of Zhejiang Provincial People’s Hospital (NO. 2020QT108). Competing interests: No competing interests. Competing interests: No competing interests. Funding: The study was supported by the fund of Medical and Health Research Project of Health Commission of Zhejiang Province (No. 2022492695). Funding: The study was supported by the fund of Medical and Health Research Project of Health Commission of Zhejiang Province (No. 2022492695). Authors’ contribution: Yanqing Ma: Conceptualization, Methodology, Writing-Original Draft. Yi Lin: Writing- Review & Editing, Supervision. Xiren Xu: : Software, Formal analysis, Data Curation. Jie Li: Validation, Resources. Yang Zhang: Validation, Resources, statistics, and software. Acknowledgements: not applicable. Acknowledgements: not applicable. Acknowledgements: not applicable. Consent for publication: NA. Consent for publication: NA. Page 8/14 Page 8/14 Availability of data and materials: The datasets used and analyzed in this article is available from the corresponding author on reasonable request. The code used in this study is available at GitHub (https://github.com/mayq1988/GGN). References 1. Chen W, Zheng R, Baade P D, et al. Cancer statistics in China, 2015[J]. CA: A Cancer Journal for Clinicians, 2016, 66(2):115–32. 1. Chen W, Zheng R, Baade P D, et al. Cancer statistics in China, 2015[J]. CA: A Cancer Journal for Clinicians, 2016, 66(2):115–32. 2. Yang D, Liu Y, Bai C, et al. Epidemiology of lung cancer and lung cancer screening programs in China and the United States[J]. Cancer Letters, 2020, 468(82–87. 3. Haraguchi S, Hioki M, Koizumi K, et al. Characteristics of multiple primary malignancies associated with lung cancer by gender[J]. Respiration, 2007, 74(2):192–195. 3. Haraguchi S, Hioki M, Koizumi K, et al. Characteristics of multiple primary malignancies associated with lung cancer by gender[J]. Respiration, 2007, 74(2):192–195. 4. Mariotto A B, Rowland J H, Ries L A, et al. Multiple cancer prevalence: a growing challenge in long- term survivorship[J]. Cancer Epidemiology, Biomarkers and Prevention, 2007, 16(3):566–571. 5. Martini N,Melamed M R. Multiple primary lung cancers[J]. Journal of Thoracic and Cardiovascular Surgery, 1975, 70(4):606–612. 6. Shen K R, Meyers B F, Larner J M, et al. Special treatment issues in lung cancer: ACCP evidence- based clinical practice guidelines (2nd edition)[J]. Chest, 2007, 132(3 Suppl):290S-305S. 7. Shintani Y, Okami J, Ito H, et al. Clinical features and outcomes of patients with stage I multiple primary lung cancers[J]. Cancer Sci, 2021, 112(5):1924–1935. 8. Duchateau C S,Stokkel M P. Second primary tumors involving non-small cell lung cancer: prevalence and its influence on survival[J]. Chest, 2005, 127(4):1152–1158. 9. Gillies R J, Kinahan P E,Hricak H. Radiomics: Images Are More than Pictures, They Are Data[J]. Radiology, 2016, 278(2):563–577. 9. Gillies R J, Kinahan P E,Hricak H. Radiomics: Images Are More than Pictures, They Are Data[J]. Radiology, 2016, 278(2):563–577. 10. Peeken J C, Asadpour R, Specht K, et al. MRI-based delta-radiomics predicts pathologic complete response in high-grade soft-tissue sarcoma patients treated with neoadjuvant therapy[J]. Radiotherapy and Oncology, 2021, 164(73–82. 10. Peeken J C, Asadpour R, Specht K, et al. MRI-based delta-radiomics predicts pathologic complete response in high-grade soft-tissue sarcoma patients treated with neoadjuvant therapy[J]. Radiotherapy and Oncology, 2021, 164(73–82. 11. Koivu A, Sairanen M, Airola A, et al. Synthetic minority oversampling of vital statistics data with generative adversarial networks[J]. J Am Med Inform Assoc, 2020, 27(11):1667–1674. 11. Koivu A, Sairanen M, Airola A, et al. Synthetic minority oversampling of vital statistics data with generative adversarial networks[J]. J Am Med Inform Assoc, 2020, 27(11):1667–1674. References Page 9/14 Page 9/14 12. Jung E J, Lee J H, Jeon K, et al. Treatment outcomes for patients with synchronous multiple primary non-small cell lung cancer[J]. Lung Cancer, 2011, 73(2):237–242. 13. Chen D, Mei L, Zhou Y, et al. A novel differential diagnostic model for multiple primary lung cancer: Differentially-expressed gene analysis of multiple primary lung cancer and intrapulmonary metastasis[J]. Oncol Lett, 2015, 9(3):1081–1088. 13. Chen D, Mei L, Zhou Y, et al. A novel differential diagnostic model for multiple primary lung cancer: Differentially-expressed gene analysis of multiple primary lung cancer and intrapulmonary metastasis[J]. Oncol Lett, 2015, 9(3):1081–1088. 14. Komatsu H, Izumi N, Tsukioka T, et al. Prognosis associated with synchronous or metachronous multiple primary malignancies in patients with completely resected non-small cell lung cancer[J]. Surgery Today, 2019, 49(4):343–349. 14. Komatsu H, Izumi N, Tsukioka T, et al. Prognosis associated with synchronous or metachronous multiple primary malignancies in patients with completely resected non-small cell lung cancer[J]. Surgery Today, 2019, 49(4):343–349. 15. Tanjak P, Suktitipat B, Vorasan N, et al. Risks and cancer associations of metachronous and synchronous multiple primary cancers: a 25-year retrospective study[J]. BMC Cancer, 2021, 21(1):1045. 15. Tanjak P, Suktitipat B, Vorasan N, et al. Risks and cancer associations of metachronous and synchronous multiple primary cancers: a 25-year retrospective study[J]. BMC Cancer, 2021, 21(1):1045. 16. Utada M, Ohno Y, Hori M, et al. Incidence of multiple primary cancers and interval between first and second primary cancers[J]. Cancer Sci, 2014, 105(7):890–896. 17. Girard N, Ostrovnaya I, Lau C, et al. Genomic and mutational profiling to assess clonal relationships between multiple non-small cell lung cancers[J]. Clinical Cancer Research, 2009, 15(16):5184–5190. 18. Romaszko A M,Doboszyńska A. Multiple primary lung cancer: A literature review[J]. Adv Clin Exp Med, 2018, 27(5):725–730. 19. Fleckenstein J, Petroff A, Schäfers H J, et al. Long-term outcomes in radically treated synchronous vs. metachronous oligometastatic non-small-cell lung cancer[J]. BMC Cancer, 2016, 16(348. 20. Jiang L, He J, Shi X, et al. Prognosis of synchronous and metachronous multiple primary lung cancers: systematic review and meta-analysis[J]. Lung Cancer, 2015, 87(3):303–310. 21. Ma Y, Ma W, Xu X, et al. How Does the Delta-Radiomics Better Differentiate Pre-Invasive GGNs From Invasive GGNs?[J]. Frontiers in Oncology, 2020, 16(10):DOI:10.3389/fonc.2020.01017. 22. van Rens M T, Zanen P, Brutel de La Rivière A, et al. Survival in synchronous vs. single lung cancer: upstaging better reflects prognosis[J]. Chest, 2000, 118(4):952–958. 23. References Detterbeck F C, Jones D R, Kernstine K H, et al. Lung cancer. Special treatment issues[J]. Chest, 2003, 123(1 Suppl):244S-258S. 23. Detterbeck F C, Jones D R, Kernstine K H, et al. Lung cancer. Special treatment issues[J]. Chest, 2003, 123(1 Suppl):244S-258S. 24. Antakli T, Schaefer R F, Rutherford J E, et al. Second primary lung cancer[J]. Annals of Thoracic Surgery, 1995, 59(4):863–866. 24. Antakli T, Schaefer R F, Rutherford J E, et al. Second primary lung cancer[J]. Annals of Thoracic Surgery, 1995, 59(4):863–866. 25. Ullah A, Munagala R, Mishra P, et al. Small cell carcinoma of the lung in a patient with previously treated synchronous adenocarcinoma and squamous cell carcinoma[J]. Lung India, 2021, 38(3):263–265. 25. Ullah A, Munagala R, Mishra P, et al. Small cell carcinoma of the lung in a patient with previously treated synchronous adenocarcinoma and squamous cell carcinoma[J]. Lung India, 2021, 38(3):263–265. Figures igure 1 he flow diagram of patients selection. Figures Page 10/14 Figure 1 The flow diagram of patients selection. Page 11/14 Figure 2 The VOI of tumor was manually depicted in the software of “ITK-snap”. Figure 2 The VOI of tumor was manually depicted in the software of “ITK-snap”. Page 12/14 Figure 3 Figure 4 The comparison of AUCs of FC-radio and FC-delta of Group A, B, and C in the training and validation set. S l t Fil The comparison of AUCs of FC-radio and FC-delta of Group A, B, and C in the training and validation set. The comparison of AUCs of FC-radio and FC-delta of Group A, B, and C in the training and validation set. Figure 3 Figure 3 The heatmap of radiomics analysis after feature selection of GBDT, and there were 27 radiomic features selected. Page 13/14 Page 13/14 Page 13/14 Figure 4 The comparison of AUCs of FC-radio and FC-delta of Group A, B, and C in the training and validation set. Supplementary Files Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. This is a list of supplementary files associated with this preprint. Click to download. SupplementMaterial.doc Page 14/14
https://openalex.org/W2901314953	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0207797&type=printable	English	null	Fucoidans inhibit the formation of post-operative abdominal adhesions in a rat model	PloS one	2,018	public-domain	6,971	RESEARCH ARTICLE Methods and materials Editor: Jung Weon Lee, Seoul National University College of Pharmacy, REPUBLIC OF KOREA Received: September 13, 2018 Accepted: November 6, 2018 Published: November 21, 2018 Editor: Jung Weon Lee, Seoul National University College of Pharmacy, REPUBLIC OF KOREA Received: September 13, 2018 Accepted: November 6, 2018 Published: November 21, 2018 Editor: Jung Weon Lee, Seoul National University College of Pharmacy, REPUBLIC OF KOREA Female Sprague Dawley rats were studied. A 2.5x2.5cm full thickness segment of abdomi- nal wall was excised. The skin edges were approximated. This model induces extensive adhesions and allows objective quantitation. Three fucoidans were evaluated- Sigma Fucoi- dan Crude (SFC), Fucus vesiculosis 95% (Sigma) and, Peridan. One protocol involved con- tinuous infusion into the abdomen from a subcutaneous osmotic pump. Alternatively, boluses of the solutions were injected IP at the end of the operation. Rats were sacrificed a week later. Adhesion extent was scored. Systemic coagulation effects of fucoidans were also evaluated. INR and aPTT were measured following IP injection of the fucoidan solu- tions and after 7 days of continuous infusion. Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. OPEN ACCESS Citation: Charboneau AJ, Delaney JP, Beilman G (2018) Fucoidans inhibit the formation of post- operative abdominal adhesions in a rat model. PLoS ONE 13(11): e0207797. https://doi.org/ 10.1371/journal.pone.0207797 Alex J. CharboneauID1☯, John P. Delaney2☯, Greg Beilman2‡ 1 University of Minnesota Medical School, Minneapolis, Minnesota, United States of America, 2 Department of Surgery, University of Minnesota, Minneapolis, Minnesota, United States of America 1 University of Minnesota Medical School, Minneapolis, Minnesota, United States of America, 2 Department of Surgery, University of Minnesota, Minneapolis, Minnesota, United States of America ☯These authors contributed equally to this work. ‡ These authors also contributed equally to this work. char0247@umn.edu a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Purpose Fibrin clot is essential for post-operative abdominal adhesion formation. Fucoidans, sulfated polysaccharides, inhibit fibrin clot formation. In addition, they inhibit inflammation and fibro- sis, which also play important roles in adhesion formation. The purpose of this study was to evaluate fucoidans’ potential for inhibiting post-operative abdominal adhesions and mea- sure their effects on systemic coagulation parameters when administered intraperitoneally (IP). Fucoidans inhibit the formation of post- operative abdominal adhesions in a rat model Alex J. CharboneauID1☯*, John P. Delaney2☯, Greg Beilman2‡ Introduction Adhesions following abdominal or pelvic operations occur in the great majority, 65% to 95%, of cases. Adhesive intestinal obstruction later develops in as many as 15% of such individuals and requires operative relief in almost a third of these [1, 2]. Adhesions are responsible for up to 20% of infertility cases [3]. A common after-effect of pelvic operations is chronic pain, thought to be due to adhesions. Adhesions impede later abdominal operations by interfering with exposure and by increasing the risk of inadvertent enterotomies. Beginning in the early 1900s, the quest began for means to prevent post-operative abdomi- nal adhesions [4, 5]. Subsequent publications number in the thousands. Yet, today, only 3 commercial products have been approved by the FDA for this purpose, Adept (Baxter Interna- tional), Interceed (Johnson and Johnson), and Seprafilm (Genzyme Corporation). Numerous excellent reviews have been published on the subject [1, 6, 7], most of which address the issue of prevention. The reviews generally end with a statement to the effect that, we have learned much about the pathogenesis of postoperative adhesions, but that there is no convincing evidence that any currently available agents actually provide clinical benefit. Cochrane meta-analyses [8, 9, 10] similarly conclude that there is no useful fluid borne phar- macologic agent for preventing post-operative abdominal adhesions. Clinically used barrier methods of preventing adhesions have yielded modest reduction in adhesions and no known reduction in adhesion-related bowel obstruction to date [9, 11–14] The conduct of an abdominal operative procedure involves inevitable and unpredictable peritoneal injuries remote from the surgical site, leading to adhesions. Omentum, which is highly mobile, participates in random patterns of adhesion formation. Protection of these scat- tered sites requires agents which can circulate throughout the abdomen to bathe its surfaces. It should be noted that fluid placed anywhere in the peritoneal cavity does circulate throughout [15]. The sequence of events leading to adhesion formation is well documented. Injury to the peritoneum generates an inflammatory reaction with accompanying exudation of fibrin-rich plasma and leukocytes, initially polymorphs. In short order monocyte-derived fibrocytes invade the fibrin clot. The resulting matrix serves to hold two injured surfaces in approxima- tion, providing a scaffold for conversion of fibrin to a collagen adhesive attachment. In 2011, Springate, Cashman, and colleagues submitted patent applications regarding Fucoidans for adhesion prevention [16, 17]. Fucoidans inhibit abdominal adhesions Conclusions Competing interests: Competing interest statement: This work was supported by private investment of G.D.N. Holdings and Anhese, LLC. Author J.P.D. was a board member of Anhese, LLC at the time of these experiments. However, no authors received direct payment from either company. Dr. Delaney did participate in manuscript preparation, experimental design, and the decision to publish. Anhese, LLC applied for and held a provisional patent for the use of fucoidans for adhesion prevention, but the provisional patent has since lapsed. Anhese, LLC has since dissolved. G. D.N. Holdings did not have any role in experimental design, data collection, or preparation of this manuscript. There is currently no financial stake in the outcome of this manuscript or the use of fucoidans for adhesion prevention for the authors of this manuscript or the companies that funded the work. This does not alter our adherence to PLOS ONE policies on sharing data and materials. These findings indicate that selected fucoidans infused intraperitoneally for a week after abdominal operations reduce adhesion extent by up to 90%. Results Data Availability Statement: All relevant data are within the manuscript and its Supporting Information file. Animals given a continuous infusion of either SFC or Peridan yielded adhesion reduction of 80 to 90% from control. Bolus Peridan had no discernable influence on adhesion formation, but a single bolus of SFC caused significant adhesion reductions. Peridan resulted in prompt aPTT elevations which fell to nearly normal by 5 hours. The maximum peak value after SFC injection was seen in 15 hours. The maximal INR elevations were around 2. Mea- surement of INR and aPTT after a week of continuous infusion of either Peridan or SFC, were always in the normal control range. The third agent, Sigma, frequently yielded intraper- itoneal infection found at autopsy. Funding: This work was supported by private investment by G.D.N. Holdings and Anhese, LLC. Author J.P.D. was a board member of Anhese, LLC at the time of these experiments. He participated in manuscript preparation, experimental design, and the decision to publish. G.D.N. Holdings did not have any role in experimental design, data collection, or preparation of this manuscript. 1 / 13 PLOS ONE \| https://doi.org/10.1371/journal.pone.0207797 November 21, 2018 PLOS ONE \| https://doi.org/10.1371/journal.pone.0207797 November 21, 2018 Animals This study was approved by the University of Minnesota Institutional Animal Care and Use Committee (IACUC). The protocol was approved by IACUC (Protocol Number: 1101A95274). Mature, female Sprague-Dawley rats, weighing between 300 and 400 grams, were obtained from Harlan Laboratories (Madison, WI). The animals were cared for by the University of Minnesota Research Animal Resources Department in accordance with the prin- ciples in the NIH Guide for Care and Use of Laboratory Animals [21]. They were acclimated for at least a week before entering the study. Euthanasia was accomplished with carbon dioxide asphyxiation or exsanguination from the heart under anesthesia. Agent preparation and delivery Sigma Fucoidan Crude (SFC), purchased from the Sigma-Aldrich Corporation as a powder, had been extracted from Fucus vesiculosis. Peridan Concentrate solution, supplied by ARC Medical Devices, was derived from Laminara japonica and in a concentration of 50 mg/ml. Doses were chosen empirically based, in part, on the ranges reported in company literature or in patents. For each fucoidan these studies involved dose escalation seeking a level to inhibit adhesions without causing bleeding. An Alzet 2ML1 osmotic pump (Durect Corporation, Cupertino, CA) was used for the continuous perfusion studies. This model is calibrated to deliver 2 ml of liquid at a rate of 10 microliters per hour over the course of seven days. The pumps were aseptically prepared prior to implantation in a subcutaneous pocket. A 5 cm length of 5Fr PVC Feeding Tube (Covidien, Mansfield, MA) served as the conduit for delivery into the abdomen. The tube was filled with the drug solution at the outset to ensure that deliv- ery would start promptly. SFC powder was dissolved in sterile water and diluted such that the intended one-week dose was contained in a 2cc volume. This solution was centrifuged for 10 minutes, leaving a small plug of visible debris. Supernatant was withdrawn and 2cc injected into the pump. Peridan Concentrate solution was diluted with water to get the chosen one- week dose of fucoidan in 2cc volume. Introduction These documents include extensive experimental data, much of which is not to be found in the scientific literature. This group of investigators tested a total of 75 potential anti-adhesion agents in rat and rabbit models, including various fucoidan preparations. Their fundamental conclusion was that fucoidans were the most effec- tive anti-adhesion agents among the many candidates tested. Fucoidans had not been previ- ously reported as abdominal antiadhesive agents. Their efforts were first published as an abstract which described promising results from the use of a single intraperitoneal (IP) dose of a fucoidan solution in a rat cecal sidewall model [18]. A subsequent publication described excellent adhesion reductions in a different rat cecal sidewall model. Here the fucoidans were embedded in a hyaluronic glycerol film which was interposed between the injured cecum and the sidewall defect and so served additionally as a 2 / 13 PLOS ONE \| https://doi.org/10.1371/journal.pone.0207797 November 21, 2018 Fucoidans inhibit abdominal adhesions mechanical barrier [19]. These investigators also reported successful adhesion reductions in two rabbit uterine horn models [20]. Importantly, they found no significant toxicities, gross or biochemical, in any of their multiple different fucoidan studies, whether the agents were applied as a circulating fluid or fixed in a barrier material. PLOS ONE \| https://doi.org/10.1371/journal.pone.0207797 November 21, 2018 Fucoidans inhibit abdominal adhesions muscle and peritoneum, were exposed to the abdominal viscera. Postoperative analgesia (Ketoprofen 5 mg/kg) was given for two days. For single bolus experiments the operative preparation was identical, but no pump was employed. Two cc containing the chosen dose of fucoidan was injected into the abdomen as the incision was being closed. PLOS ONE \| https://doi.org/10.1371/journal.pone.0207797 November 21, 2018 Experimental model The rat adhesion model utilized in all the present experiments was described by Gaertner [22] and has been used extensively in our lab. Anesthesia was induced with inhaled Isoflurane (5%) and oxygen (1 L/min) and maintained for the duration of the operation. The surgical site was aseptically prepared, and a 3 cm abdominal midline skin incision was made. A 2.5 by 2.5 cm full thickness square of abdominal muscle, fascia, and peritoneum was excised, preserving the skin. Hemostasis was achieved using clamps or digital pressure to avoid suture or cautery inju- ries. A second transverse incision was made in the right flank and a subcutaneous pocket was developed with the pump placed therein. The delivery catheter, tunneled through the abdominal wall, protruded about 2–3 cm into the abdomen with the tip lying in the right lateral gutter. One control group had no pump. A second control group got a continuous one-week water infusion. Skin incisions were closed with a single layer of running 4–0 poly- glactin 910 suture. In this way, the subcutaneous layer, as well as the defect edge, cut fascia, 3 / 13 PLOS ONE \| https://doi.org/10.1371/journal.pone.0207797 November 21, 2018 https://doi.org/10.1371/journal.pone.0207797.g001 Adhesion reduction after continuous IP infusion Seven-day continuous infusion of SFC fucoidan at doses of 4.3 mg/day (n = 3), 8.3 mg/day (n = 5), and 17.2 mg/day (n = 6) yielded statistically significant reduction in adhesion scores from water control and no pump controls (Fig 2 and Table 1). Peridan infusion at doses of 5.3 mg/day (n = 5) and 10.6 mg/day (n = 6) also yielded significantly lower adhesion scores as compared to control experiments (Fig 2 and Table 1). Adhesion studies Animals were sacrificed after 7 days. Gross observations regarding the general health as well as animal weight were recorded daily. Following euthanasia, a U-shaped incision was made extending from the suprapubic area bilaterally to the costal margins to fold back the ventral abdominal wall and expose the midline defect. The defect was visually divided into four quad- rants to facilitate the scorer’s estimation of the percent of exposed subcutaneous tissue covered by adhesions. Adhesions to the defect cut edges were recorded as the estimated percent of the circumference involved. The percentage of the defect surface and percentage of edge circum- ference were averaged to provide a composite score (0–100). The field was photographed for later review (Fig 1). The scorer was not blinded. The pump was removed and weighed. Any solution remaining was expelled by compressing the outer casing with pliers and then reweighing the pump. Results were thereby calculated and expressed based on the actual delivered dose of fucoidan. The total volume delivered was generally 1.5 to 1.7 cc. or 75–85% of the original dose of fucoidan. Fig 1. Diagram describing score derivation for each animal. The red circle represents the full thickness defect cut edge and the beige fill represents the sewn together subcutaneous tissue overlying the defect. Black lines were visualized by the scorer to assist quantifying percent adhesion coverage (A). Figure displays an adhesion score of 20. Representative photos of adhesion scores include 0 (B), 7.5 (C), and 45 (D). https://doi org/10 1371/journal pone 0207797 g001 Fig 1. Diagram describing score derivation for each animal. The red circle represents the full thickness defect cut edge and the beige fill represents the sewn together subcutaneous tissue overlying the defect. Black lines were visualized by the scorer to assist quantifying percent adhesion coverage (A). Figure displays an adhesion score of 20. Representative photos of adhesion scores include 0 (B), 7.5 (C), and 45 (D). https://doi.org/10.1371/journal.pone.0207797.g001 https://doi.org/10.1371/journal.pone.0207797.g001 4 / 13 PLOS ONE \| https://doi.org/10.1371/journal.pone.0207797 November 21, 2018 Fucoidans inhibit abdominal adhesions Coagulation studies Systemic coagulation parameters, International Normalized Ratio (INR) and Activated Partial Thromboplastin time (aPTT), were measured after intraperitoneal injections of 50 mg of either of the fucoidan solutions, two hours after injection and at intervals thereafter up to 40 hours. Blood was obtained from anesthetized animals by terminal cardiac puncture. Systemic coagu- lation values were similarly assayed at the time of sacrifice in the rats that had received contin- uous infusions of IP drug or water for a week. Control values were established by obtaining blood from rats that had received no drugs. Statistical analysis Statistical differences within the adhesion inhibition data were determined using single factor one-way analysis of variance (ANOVA) tests. P < 0.05 was considered evidence of significance between experimental groups. Post-hoc analysis was done using two-tailed Student’s t-tests assuming unequal variance. These calculations were performed using Microsoft Excel 2010 Data Analysis Tools (Microsoft Corporation, Redmond, WA). Fucoidans inhibit abdominal adhesions injection led to a substantial reduction in adhesion extent. This effect was not quite as large as that obtained with continuous infusion. In contrast, Peridan given as a single dose had no measurable influence on adhesions in this model. Table 1. Effects of varying fucoidan doses on adhesion extent when delivered via subcutaneous pump. 7 Day Continuous Infusion Source Dose (mg/day) n Adhesion Score SEM (+/-) SFC 4.3a 3 7.5 2.50 8.3a,b 5 6.8 2.42 17.2a,b 6 0.8 0.616 Peridan 5.3a,b 5 3.3 2.08 10.6a,b 6 3.8 1.21 Control Water 13 43.1 5.21 No Pump 23 49.2 5.67 a = significantly less than control (p < 0.05) b = significantly less than water control (p < 0.05) https://doi.org/10.1371/journal.pone.0207797.t001 Table 1. Effects of varying fucoidan doses on adhesion extent when delivered via subcutaneous pump. https://doi.org/10.1371/journal.pone.0207797.t001 injection led to a substantial reduction in adhesion extent. This effect was not quite as large as that obtained with continuous infusion. In contrast, Peridan given as a single dose had no measurable influence on adhesions in this model. injection led to a substantial reduction in adhesion extent. This effect was not quite as large as that obtained with continuous infusion. In contrast, Peridan given as a single dose had no measurable influence on adhesions in this model. PLOS ONE \| https://doi.org/10.1371/journal.pone.0207797 November 21, 2018 Adhesion reduction following bolus injection Fig 3 shows adhesion scores a week following a single IP dose of 80 mg of either agent (SFC n = 5; Peridan n = 4) and 40 mg of Peridan (n = 3) injected at the time of skin closure. SFC n = 5; Peridan n = 4) and 40 mg of Peridan (n = 3) injected at the time of skin closure. SFC Fig 2. Comparison of mean adhesion score after 7-day continuous IP infusion between experimental groups. Error bars indicate standard error of the mean. a = significantly less than control p < 0.05; b = significantly less than water control (p < 0.05). https://doi.org/10.1371/journal.pone.0207797.g002 PLOS ONE \| https://doi.org/10.1371/journal.pone.0207797 November 21, 2018 5 / 13 n 5; Peridan n 4) and 40 mg of Peridan (n 3) injected at the time of skin closure. SFC Fig 2. Comparison of mean adhesion score after 7-day continuous IP infusion between experimental groups. Error bars indicate standard error of the mean. a = significantly less than control p < 0.05; b = significantly less than water control (p < 0.05). https://doi.org/10.1371/journal.pone.0207797.g002 Fig 2. Comparison of mean adhesion score after 7-day continuous IP infusion between experimental groups. Error bars indicate standard error of the mean. a = significantly less than control p < 0.05; b = significantly less than water control (p < 0.05). https://doi.org/10.1371/journal.pone.0207797.g002 Fig 2. Comparison of mean adhesion score after 7-day continuous IP infusion between experimental groups. Error bars indicate standard error of the mean. a = significantly less than control p < 0.05; b = significantly less than water control (p < 0.05). https://doi.org/10.1371/journal.pone.0207797.g002 PLOS ONE \| https://doi.org/10.1371/journal.pone.0207797 November 21, 2018 5 / 13 Discussion Our studies in a rat model have focused on pharmacologic fluid administration to protect the entire abdomen rather than on site specific mechanical barrier agents. Over a number of years, we tested 40 agents administered in fluid form, either as a single bolus dose at the end of the operative preparation, or as a one-week continuous IP infusion from a subcutaneous osmotic pump. Most of these compounds were examined because they had previously been reported to have anti-adhesive capabilities. Very few had been evaluated by means of prolonged continu- ous infusion. We did indeed observe modest adhesion reductions with a number of these fluid borne agents. In general, the results with one-week IP continuous infusions were more effec- tive than single bolus delivery. For differing reasons, all except fucoidans fell short as potential candidates for clinical use. Some had no detectable anti-adhesive effects in our model. Others resulted in statistically sig- nificant reductions in adhesion formation but of insufficient magnitude to warrant clinical consideration. A few proved to be toxic, or even fatal, when delivered into the peritoneal cavity. Fucoidans are a class of fucose rich, sulfated, branched polysaccharides derived from vari- ous species of brown seaweeds. Among the many fucoidan extracts there are extensive varia- tions with respect to constituents and related biologic activities. Structural, chemical and functional differences between fucoidan extracts depend on the specific seaweed source, extraction methods, and degree of purification. These structural variables are reflected in dif- fering pharmacologic functions [23, 24, 25, 26, 27]. Fucoidans, as a group, have a broad spec- trum of biologic activities. Information regarding parenteral administration of fucoidans in the human is not available. No such product has satisfied the requirements needed to obtain FDA approval for human use. A great deal of effort has been expended to develop clean, consistent extracts suitable for systemic human usage, thus far without success. Fitton [24] has suggested that the production of well-characterized reproducible fucoidan fractions is within reach, and that therapies from fucoidans are a realizable goal. For present considerations, the most pertinent biologic capabil- ities of fucoidans are anti-inflammatory, anti-coagulant, anti-fibrotic and adhesolytic. Each of these latter processes plays a role in the genesis of abdominal adhesion formation. To date, information regarding systemic effects of fucoidans has been derived entirely from studies in experimental animals using intravenous or intraperitoneal injections. Coagulation studies Shortly following intraperitoneal Peridan injection, values of aPTT exceeded the maximum level measured in our laboratory. At 5 hours aPTT was approaching normal and was consis- tently near control levels after 15 hours. The maximum effects of SFC on aPTT were not reached until 15 hours post injection and fell abruptly thereafter. With either agent, INR values rose only to levels that would be considered therapeutic for human patients being treated with warfarin. These observations are in accord with previous information regarding the anti-coag- ulant properties of various fucoidan extracts. Activated PTT proved to be the more sensitive measure of systemic coagulation changes (Fig 4). By contrast systemic INR and aPTT values measured at sacrifice after a week of continuous infusion of similar total doses of the two fucoidans were normal in every instance. Apparently local concentrations at the sites of operative injury are enough to inhibit fibrin clot formation, but the slow constant infusion does not alter systemic coagulation parameters. Regarding the issue of potential bleeding, using fucoidan doses sufficient for anti-adhesive effects, there was no evidence of intraperitoneal bleeding apparent at the time of sacrifice. Fig 3. Comparison of mean adhesion score at 7 days post-operation after single intraoperative bolus between experimental groups. Error bars indicate standard error of the mean. a = significantly less than Control group (p < 0.05). https://doi org/10 1371/journal pone 0207797 g003 Fig 3. Comparison of mean adhesion score at 7 days post-operation after single intraoperative bolus between experimental groups. Error bars indicate standard error of the mean. a = significantly less than Control group (p < 0.05). https://doi.org/10.1371/journal.pone.0207797.g003 PLOS ONE \| https://doi.org/10.1371/journal.pone.0207797 November 21, 2018 6 / 13 Fucoidans inhibit abdominal adhesions Fig 4. Systemic INR and aPTT at timed sacrifice after IP injection of 50 mg doses of fucoidans. aPTT maximum laboratory reporting value was 300 seconds. Fig 4. Systemic INR and aPTT at timed sacrifice after IP injection of 50 mg doses of fucoidans. aPTT maximum laboratory reporting value was 300 seconds. https://doi.org/10.1371/journal.pone.0207797.g004 https://doi.org/10.1371/journal.pone.0207797.g004 PLOS ONE \| https://doi.org/10.1371/journal.pone.0207797 November 21, 2018 Anticoagulants Historically, many attempts to avert postoperative abdominal adhesions were based on inhibi- tion of clot formation by means of local anticoagulant applications aimed at blocking conver- sion of soluble fibrinogen to fibrin. This concept continues to find favor today and is likely a central factor for our observed efficacy of fucoidans. Through the years virtually every newly developed anticoagulant agent has been tested with the goal of preventing abdominal adhe- sions. Much of the early interest in fucoidans was based on the idea that they might provide a substitute for heparin. They have been thoroughly studied and found to be effective anticoagu- lants [26, 34]. Intraperitoneal use of anticoagulants for adhesion prevention has not proved to be clinically applicable partly because of inconsistent adhesion effects or, in some instances, excessive bleeding [35, 36]. Inescapable facts are that conventional anticoagulants act briefly, but adhe- sions continue to form for a week after injury. Meaningful adhesion reduction with a single topical application at the outset is not to be expected. Timing With respect to the time course of post traumatic adhesion formation, we previously reported sequential laparoscopic observations in this rat model [28]. Gross adhesions were seen within 24 hours of operation. Additional adhesions continued to form for up to a week, but no new ones were seen to develop thereafter. Sequential biopsies of the surgically injured surfaces were examined by means of scanning electron microscopy. Plentiful new mesothelial cells were identified on such surfaces by the third day. By seven days, the defect was completely carpeted with mesothelial cells. Thus, surface healing coincided with cessation of new adhesion forma- tion. Subsequent laparoscopic studies in the same animals, weeks and months later, showed adhesion extent and distribution identical to that seen at one week. Our observation that the adhesion process starts immediately after injury and is complete within 7 days is supported by a number of other reports [29, 30]. An important practical principle suggested by these findings is that an antiadhesive agent should be introduced into the abdomen near the end of the operative procedure. A second is that the preventive effects must continue, possibly for a full week. The progression of adhesion formation is time-dependent. The normal peritoneal fibrino- lytic capability is promptly depressed in association with operative trauma. Human and experi- mental animal studies of peritoneal tissues or of fluid have shown rapid reduction in tissue plasminogen activator (tPA), accompanied by an increase in plasminogen activator inhibitor (PAI-1) associated with operative injury, thereby disrupting the normal ability to lyse newly formed fibrin clots [31, 32, 33]. These circumstances, present at the outset, are optimal for fibrin clot-matrix formation. They revert toward normal in succeeding days. Fucoidans inhibit abdominal adhesions local topical effects. Fucoidan concentrations in or on the peritoneal surface, the locus of adhe- sion formation, are no doubt far higher than what could be attained with delivery via the sys- temic circulation. local topical effects. Fucoidan concentrations in or on the peritoneal surface, the locus of adhe- sion formation, are no doubt far higher than what could be attained with delivery via the sys- temic circulation. Discussion Our observa- tions of elevated systemic coagulation parameters shortly after IP administration of Peridan or of SFC confirm rapid trans-peritoneal systemic absorption. Intraperitoneal delivery also causes 7 / 13 PLOS ONE \| https://doi.org/10.1371/journal.pone.0207797 November 21, 2018 Fucoidans inhibit abdominal adhesions thrombosis lead to a doubling of bleeding time. The investigators estimated that, on a weight basis, fucoidan had 20 to 30 times less anticoagulant effect than did heparin. In terms of anti- thrombosis efficacy versus bleeding risk, fucoidans are measurably superior to conventional anticoagulants, specifically to heparin. In the present studies, we observed that appropriate doses of slowly infused intraperitoneal fucoidans induced major adhesion reductions without causing problem bleeding. But, when the effective total dose was raised by a factor of four or more, we saw major bleeding. These observations relate to the role of timing for preventing abdominal adhesions follow- ing operative trauma. For optimal results, the anti-thrombotic agent is best applied early. This, however, is when the surgically damaged small blood vessels, recently clotted, might resume bleeding when exposed to anticoagulants. In the present studies, such bleeding was not induced by fucoidans given in doses sufficient to inhibit adhesions. This efficacy might be explained by the fortuitous combination of fucoidan biologic capabilities. Anti-inflammation depletes the amount of the fibrin-rich exudate. Prevention of soluble fibrinogen conversion to insoluble fibrin clots, anticoagulation, plays a key role. Antifibrosis delays maturation of fibrin clot to collagen, thereby extending the time available for lysis to take place. In addition to their anticoagulant properties, fucoidans have been shown to have fibrinolytic activity [40]. Adhesion reduction with Peridan in the present studies was accomplished only with pro- longed constant delivery. A single bolus of Peridan, given at the end of the operation, had no effect on adhesion formation. By contrast, SFC, given as a single IP bolus, resulted in adhesion reductions of about 75%. Why the difference? That these two fucoidans are derived from dif- ferent seaweed species and have different molecular constituents no doubt accounts for their differing behaviors, but the molecular specifics are unknown. PLOS ONE \| https://doi.org/10.1371/journal.pone.0207797 November 21, 2018 Thrombosis versus clot Animal experiments have established important distinctions between hemorrhagic and antith- rombotic pharmacological drug effects [37, 38, 39]. Studies in rat and in rabbit models of thrombosis prevention have examined bleeding times and systemic coagulation parameters. Bleeding times were only slightly affected by systemic fucoidan, given in doses which pre- vented or delayed thrombus formation. On the other hand, doses of heparin sufficient to avert 8 / 13 PLOS ONE \| https://doi.org/10.1371/journal.pone.0207797 November 21, 2018 Fucoidans inhibit abdominal adhesions large mammal have special importance with respect to potential human use. The investigators describe no infections or any adverse effects whatsoever. The possibility that the complication of peritoneal infection might be limited to specific fucoidan preparations and doses will require further study. The Peridan horse studies and our rat studies indicate that certain fucoi- dans in reasonable doses would likely be well tolerated in humans with regards to potential bleeding, infusion reactions, and healing interference. None of these were seen in past animal studies. However, this can only be confirmed after a singular source of well characterized and clean fucoidan becomes available for further study in both animals and humans. As for human clinical use, the matter of single bolus dose at operation versus several days of continuous infusion is important. Bolus application of an effective anti-adhesion fluid would encourage relatively routine use. On the other hand, if success requires the use of external por- table pump drug infusion for some days following the operation, nuisance factors and addi- tional expense might limit its use to high risk situations. Development of a dependable bioresorbable delayed drug delivery vehicle could avoid the need for prolonged IP infusion and encourage common application. There are limitations to the presently described study that bear consideration. Firstly, the evaluator of adhesion scores was not blinded. Unblinded scoring is a potential source of bias. However, the scorer had scored thousands of similar experiments prior to the work presented in this manuscript, which favors consistency. Secondly, sample sizes were variable and small within experimental groups. While statistical significance was achieved in many cases, larger sample sizes would decrease the variance and paint a clearer picture of the effect fucoidan preparations had on adhesion formation. Finally, there will need to be substantial follow-up studies to this work to characterize additional effects of adhesion administration in both ani- mals and humans. For example, we do not know if fucoidan or fucoidan complexes are subject to molecular mimicry and the development of immune responses that would cause harm after repeat exposure. This is just one of many potential side effects that cannot be commented on with any certainty due to the limited scope of this study. Conclusions In summary, two laboratories, working independently, have tested a total of more than one-hundred candidate fluid agents as to their capacity to inhibit peritoneal adhesions follow- ing abdominal operations. Our lab found that certain fucoidan extracts, applied intraperitone- ally, yielded anti-adhesive results superior to the many other proposed agents that were similarly evaluated. What is the explanation? Almost all candidate agents studied over the years were aimed at countering a single aspect of the adhesion process, such as inflammation, thrombosis, or fibrosis. Based on the results of coagulation studies done in our lab, we can sur- mise that thrombosis is attenuated in the presence of fucoidans. Other studies have shown their anti-inflammatory and anti-fibrotic effects. Together, this combination of effects allows doses sufficient to provide major adhesion reduction but small enough to avoid potential complications. At present the possibility of clinical use is hindered by two barriers. The first is lack of clean and consistent products that could be considered for human use. The second is the potential for engendering infections. Both issues deserve further investigation. S1 File. Data collected during studies reported in this manuscript. (XLSX) Infection As noted above, we studied a third, much more highly purified fucoidan, also derived from Fucus vesiculosis. This agent is currently marketed as Sigma Fucoidan 95% (Sigma). The same rat model and protocols were employed. Anti-adhesive effects were similar to those of SFC, with mean adhe- sion scores of 7.1 (2.7 mg/day), 5.1 (5.3 mg/day), and 2.8 (8 mg/day). However, in a significant portion of these rats (30%) we found and cultured unexplained bacterial infections in the perito- neal fluid (S1 File). This complication was seen only twice in the many SFC experiments and not at all with Peridan. The infections were noted over the course of experiments by decreased rat activity and responsiveness, but they were ultimately identified at sacrifice. The infections were not lethal during the seven-day experiment. The presence of peritonitis obviates rational interpre- tation of adhesion results. These observations will be the subject of a separate communication, focused on the infection issue. As a practical matter, the threat of peritonitis eliminates this fucoi- dan from clinical consideration and also raises concerns regarding other fucoidans. The explanation for a particular fucoidan enhancing infection is not clear. A well-estab- lished property of fucoidans, in general, is anti-inflammation. A critical aspect of this effect is selectin blocking on leucocytes. The leucocytes are thereby functionally inhibited from passing into tissue spaces. One relevant experimental observation was made in a model of sterile, chemically-induced peritonitis. There was a dearth of leucocytes in the peritoneal fluid of those animals given systemic fucoidan [41]. A plausible hypothesis for our finding of infections is that peritoneal leucopenia prevailed in the rats that received Sigma Fucoidan 95% and allowed infection by otherwise harmless numbers of translocated bacteria. Others who have studied fucoidans for adhesion prevention have not reported any gross complications [19, 20] Two reports affirm the safety of Peridan solution given into the horse abdomen during operation [42, 43]. While not convincing as to anti-adhesion effects, these observations in a PLOS ONE \| https://doi.org/10.1371/journal.pone.0207797 November 21, 2018 9 / 13 Author Contributions Conceptualization: Alex J. Charboneau, John P. Delaney, Greg Beilman. Conceptualization: Alex J. Charboneau, John P. Delaney, Greg Beilman. Project administration: Alex J. Charboneau, John P. Delaney. Resources: John P. Delaney, Greg Beilman. Resources: John P. Delaney, Greg Beilman. Software: Greg Beilman. Supervision: John P. Delaney, Greg Beilman. Validation: Alex J. Charboneau. Writing – original draft: Alex J. Charboneau, John P. Delaney, Greg Beilman. Writing – original draft: Alex J. Charboneau, John P. Delaney, Greg Beilman. Supporting information S1 File. Data collected during studies reported in this manuscript. (XLSX) 10 / 13 PLOS ONE \| https://doi.org/10.1371/journal.pone.0207797 November 21, 2018 Fucoidans inhibit abdominal adhesions Acknowledgments We would like to acknowledge Kristine Mulier for her technical assistance and support. References 1. Arung W, Meurisse M, Detry O. Pathophysiology and prevention of postoperative peritoneal adhesions. World J Gastroenterol 2011; 17:4545. https://doi.org/10.3748/wjg.v17.i41.4545 PMID: 22147959 2. Okabayashi K, Ashrafian H, Zacharakis E, Hasegawa H, Kitagawa Y, Athanasiou T et al. Adhesions after abdominal surgery: A systematic review of the incidence, distribution and severity. Surgery Today 2014; 44:405.http://link.springer.com/article/10.1007/s00595-013-0591-8-page-1 PMID: 23657643 3. Marana R, Muzii L. Infertility and adhesions. Peritoneal Surgery. New York: Springer, 2000: 329. 4. Richardson EH. Studies on peritoneal adhesions: With a contribution the treatment of denuded bowel surfaces. Ann Surg 1911; 54:758. PMID: 17862775 5. Claypool JR, Vance BM, Robertson PR, Field CW. A study in the prevention of adhesions. J Am Med Assoc 1910; 55:312. 6. Ward BC, Panitch A. Abdominal adhesions: Current and novel therapies. J Surg Res 2011; 165: 91. https://doi.org/10.1016/j.jss.2009.09.015 PMID: 20036389 7. Maciver AH, McCall M, Shapiro AMJ. Intra-abdominal adhesions: Cellular mechanisms and strategies for prevention. Int J Surg 2011; 9:589. https://doi.org/10.1016/j.ijsu.2011.08.008 PMID: 21964216 8. Hindocha A, Beere L, Dias S, Watson A, Ahmad G. Adhesion prevention agents for gynaecological sur- gery: An overview of Cochrane reviews. Cochrane Database Syst Rev 2015. 6:1:CD011254, Epub 2015 Jan 6. 9. Kumar S, Wong PF, Leaper DJ. Intra-peritoneal prophylactic agents for preventing adhesions and adhesive intestinal obstruction after non-gynaecological abdominal surgery. Cochrane Database Syst Rev 2009 Jan 21(1). http://onlinelibrary.wiley.com/doi/10.1002/14651858.CD005080.pub2/full 10. Ahmad G, Mackie FL, Iles DA, O’Flynn H, Metwally M, Watson A. Fluid and pharmacological agents for adhesion prevention after gynaecological surgery. Cochrane Database of Syst Rev 2014 (7). 11. Ahmad G, O’Flynn H, Hindocha A, Watson A. Barrier agents for adhesion prevention after gynaecologi- cal surgery. Cochrane Database Syst Rev 2015(30).http://www.cochrane.org/CD000475/MENSTR_ barrier-agents-for-adhesion-prevention-after-gynaecological-surgery 12. Felemovicius I, Bonsack ME, Hagerman G, Delaney JP. Prevention of adhesions to polypropylene mesh. J Am Coll Surg 2004; 198:543. https://doi.org/10.1016/j.jamcollsurg.2003.12.004 PMID: 15051006 13. Becker JM, Dayton MT, Fazio VW, Beck DE, Stryker SJ, Wexner SD et al. Prevention of postoperative abdominal adhesions by a sodium hyaluronate-based bioresorbable membrane: A prospective, ran- domized, double-blind multicenter study. J Am Col Surg 1996; 183:297. PLOS ONE \| https://doi.org/10.1371/journal.pone.0207797 November 21, 2018 11 / 13 Fucoidans inhibit abdominal adhesions 14. Fazio VW, Cohen Z, Fleshman JW, Van Goor H, Bauer JJ, Wolff BG et al. Reduction in adhesive small- bowel obstruction by Seprafilm adhesion barrier after intestinal resection. Dis Colon Rectum 2006; 49:1. https://doi.org/10.1007/s10350-005-0268-5 PMID: 16320005 15. Imai A, Suzuki N. References Topical non-barrier agents for postoperative adhesion prevention in animal models. Eur J Obstet Gynecol Reprod Biol 2010; 149:131. https://doi.org/10.1016/j.ejogrb.2009.12.026 PMID: 20074848 16. Springate, CMK. Pharmaceutical compositions comprising modified fucans and methods relating thereto. United States Patent Application 20110021457, Kind Code: A1, Application Number; 12/ 843992, Publication Date: 01/27/2011; Filing Date: 07/27/2010. 17. Cashman J, Springate C, Winternitz C. Assignee: Pharmaceutical compositions and methods relating to inhibiting fibrous adhesions or inflammatory disease using low sulphate Fucans. ARC Medical Devices, Inc. Vancouver CA. United States Patent Application: 20110250275, Kind Code: A1; Serial No.: 084660, Filed: April 12, 2011. 18. Cashman JD, Kennah E, Shuto C, Winternitz C, Springate CMK. PERIDAN gel and solution safely inhibit postsurgical adhesions in two rat models. Proc. Eur Soc Human Reproduction and Embryology. ARC Pharmaceuticals, Inc., 102–2386 East Mall, Vancouver, British Columbia, Canada, V6T 1Z3, 2009. 19. Cashman JD, Kennah E, Shuto A, Winternitz C, Springate CM. Fucoidan film safely inhibits surgical adhesions in a rat model. J Surg Res 2011; 171:495. https://doi.org/10.1016/j.jss.2010.04.043 PMID: 20638689 20. Cashman J, Winternitz C, Springate C. Fucoidan solution safely inhibits postsurgical adhesions in two rabbit uterine horn models. Fertil Steril 2009; 92:S58. 21. National Research Council. Guide for the care and use of laboratory animals. National Academies Press, 2010. 22. Gaertner WB, Hagerman GF, Felomovicius I, Bonsack ME, Delaney JP. Two experimental models for generating abdominal adhesions. J Surg Res 2008; 146:241. https://doi.org/10.1016/j.jss.2007.08.012 PMID: 18314140 23. Peridan Technical Reference Guide. Bioniche Pharma USA: Rockford, Illinois, 2012 24. Fitton JH, Stringer DN, Karpiniec SS. Therapies from Fucoidan: An update. Mar Drugs 2015; 13:5920. https://doi.org/10.3390/md13095920 PMID: 26389927 25. Poman VH. Review: an overview about the structure-function relationship of marine sulfated polysac- charides with regular chemical structures. Biopolymers 2009; 93:496. 26. Cumashi A, Ushakova NA, Preobrazhenskaya ME, D’inecco A, Piccoli A, Totani L et al. A comparative study of the anti-inflammatory, anticoagulant, antiangiogenic, and antiadhesive activities of nine differ- ent fucoidans from brown seaweeds. Glycobiology 2007; 17:541. https://doi.org/10.1093/glycob/ cwm014 PMID: 17296677 27. Jiao G, Yu G, Zhang J, Ewart H. Chemical structures and bioactivities of sulfated polysaccharides from marine algae. Mar Drugs 2011; 9:196. https://doi.org/10.3390/md9020196 PMID: 21566795 28. Baptista ML, Bonsack ME, Felemovicius I, Delaney JP. Abdominal adhesions to prosthetic mesh evalu- ated by laparoscopy and electron microscopy. J Am Coll Surg 2000; 190:271. PMID: 10703851 29. diZerega G. The peritoneum and its response to surgical injury. References Progress in Clinical and Biological Research 1990; 358:1. 30. Raftery AT. Regeneration of parietal and visceral peritoneum: An electron microscopical study. J Anat 1973; 115:375. PMID: 4762132 31. Hellebrekers BWJ, Kooistra T. Pathogenesis of postoperative adhesion formation. Br J Surg 2011; 98:1503. https://doi.org/10.1002/bjs.7657 PMID: 21877324 32. Hellebrekers BWJ, Trimbos-Kemper TCM, Trimbos JB, Emeis JJ, Kooistra T. Use of fibrinolytic agents in the prevention of postoperative adhesion formation. Fertility and Sterility 2000; 74:203. PMID: 10927033 33. Holmdahl L, Eriksson E, Eriksson BI, Risberg B. Depression of peritoneal fibrinolysis during operation is a local response to trauma. Surgery 1998; 123:539. https://doi.org/10.1067/msy.1998.86984 PMID: 9591006 34. Jin W, Zhang Q, Wang J, Zhang W. A comparative study of the anticoagulant activities of eleven fucoi- dans. Carbohydrate Polymers 2013; 91:1. https://doi.org/10.1016/j.carbpol.2012.07.067 PMID: 23044098 35. Lehman EP, Boys F. Heparin in the prevention of peritoneal adhesions: Report of progress. Ann Surg 1940; 125:909. 12 / 13 PLOS ONE \| https://doi.org/10.1371/journal.pone.0207797 November 21, 2018 Fucoidans inhibit abdominal adhesions 36. Chandy J. Use of heparin in the prevention of peritoneal adhesions. Arch Surg1950; 60:1151. PMID: 15414118 37. Colliec-Jouault S, Millet J, Helley D, Sinquin C, Fischer AM. Effect of low-molecular-weight fucoidan on experimental arterial thrombosis in the rabbit and rat. J Thromb Haemost 2003; 1:1114. PMID: 12871385 38. Millet J, Jouault SC, Mauray S, Theveniaux J, Sternberg C, Vidal BC. Antithrombotic and anticoagulant activities of a low molecular weight fucoidan by the subcutaneous route. Thromb Haemost 1999; 81:391. PMID: 10102467 39. Min SK, Kwon OC, Lee S, Park KH, Kim JK. An antithrombotic fucoidan, unlike heparin, does not pro- long bleeding time in a marine arterial thrombosis model: A comparative study of Undaria pinnatafida sporophylls and Fucus vesciculosis. Phytother Res 2012; 26:752. https://doi.org/10.1002/ptr.3628 PMID: 22084059 40. Min SK, Han SM, Kim HT, Kwon OC, Lee S, Kim JK. Algal fucoidan, unlike heparin, has thrombolytic activity in a murine arterial thrombosis model. Blood Coagulation & Fibrinolysis 2012; 23:359. 41. Preobrazhenskaya ME, Berman AE, Mikhailov VI, Ushakova NA, Mazurov AV, Semenov AV et al. Fucoidan inhibits leukocyte recruitment in a model peritoneal inflammation in rat and blocks interaction of P-selectin with its carbohydrate ligand. Biochem Mol Biol Int 1997; 43:443. PMID: 9350352 42. Morello S, Southwood LL, Engiles J, Slack J, Crack A, Springate CM. Effect of intraperitoneal PERIDAN concentrate adhesion reduction device on clinical findings, infection, and tissue healing in an adult horse jejunojejunostomy model. Vet Surgery 2012; 41:5689. PLOS ONE \| https://doi.org/10.1371/journal.pone.0207797 November 21, 2018 References 43. Alonso JD, Alves AL, Watanabe MJ, Rodrigues CA, Hussni CA. Peritoneal response to abdominal sur- gery: the role of equine abdominal adhesions and current prophylactic strategies. Vet Med Int 2014. 13 / 13
https://openalex.org/W2768032712	https://www.nature.com/articles/s41526-017-0032-x.pdf	English	null	Coculture of meniscus cells and mesenchymal stem cells in simulated microgravity	NPJ microgravity	2,017	cc-by	13,940	INTRODUCTION mechanical stimuli, including compressive load,26 simulated microgravity (SMG),30 low-intensity pulsed ultrasound,27,28,31 and hydrostatic pressure has been reported to induce or regulate the differentiation of MSCs through TGF-β signaling pathway.32 Hypoxia stimulated chondrogenic differentiation of bone marrow-derived MSCs by induction of TGF-β1 gene expression and protein production.33 The menisci of the knee are a pair of ﬁbrocartilaginous tissues.1 They primarily serve as mechanical load distributors within the knee joint.2 Traumatic tears in the avascular region of the tissue are common and do not heal.1 Treatment options for these tears are currently limited to partial meniscectomy.3,4 However, partial meniscectomy is a major risk factor for the early development of knee osteoarthritis.3,4 Tissue engineering using cells presents a potential option to create functional tissues to replace damaged meniscus.5–15 Primary meniscus cells (MC) are the ideal cell sources as they closely resemble the in vivo phenotype of MC and can form the biomechanically functional extracellular matrix (ECM) of the meniscus.1,16,17 Obtaining sufﬁcient numbers of primary MC for meniscus tissue engineering is often impossible and imprac- tical.18,19 To circumvent this limitation, primary MC are expanded in in vitro culture. However, expanded MC suffer from dediffer- entiation and loss of the functional matrix-forming phenotype of native MC.18,19 Supplementation of primary MC through direct coculture with bone marrow mesenchymal stem cells (MSCs) has been demonstrated as a strategy to retain the differentiated phenotype of primary MC with the additional beneﬁts of synergistic production of the functional matrix components of the meniscus20–22 and downregulation of hypertrophic differen- tiation of MSCs.21,23 However, the use of bone marrow MSCs potentially presents another anatomical site other than the knee for MSCs harvest. Supplementation of primary MC with knee infrapatellar fat pad derived MSCs may be a better anatomical option. www.nature.com/npjmgrav ARTICLE OPEN Coculture of meniscus cells and mesenchymal stem cells in simulated microgravity William M. Weiss1,2, Aillette Mulet-Sierra1, Melanie Kunze1, Nadr M. Jomha1 and Adetola B. Adesida 1 Simulated microgravity has been shown to enhance cartilaginous matrix formation by chondrocytes and chondrogenesis of mesenchymal stem cells (MSCs). Similarly, coculture of primary chondrocytes with MSCs has been shown as a strategy to simultaneously retain the differentiated phenotype of chondrocytes and enhance cartilaginous matrix formation. In this study, we investigated the effect of simulated microgravity on cocultures of primary human meniscus cells and adipose-derived MSCs. We used biochemical, qPCR, and immunoﬂuorescence assays to conduct our investigation. Simulated microgravity signiﬁcantly enhanced cartilaginous matrix formation in cocultures of primary meniscus cells and adipose-derived MSCs. The enhancement was accompanied by increased hypertrophic differentiation markers, COL10A1 and MMP-13, and suppression of hypertrophic differentiation inhibitor, gremlin 1 (GREM1). npj Microgravity (2017) 3:28 ; doi:10.1038/s41526-017-0032-x Received: 13 May 2017 Revised: 17 September 2017 Accepted: 22 September 2017 1Laboratory of Stem Cell Biology and Orthopaedic Tissue Engineering, Department of Surgery, University of Alberta, Li Ka Shing Centre for Health Research Innovation, Edmonton, AB, Canada and 2Department of Orthopedic Surgery and Rehabilitation, Sports Medicine, Reconstruction and Trauma, Texas Tech University Health Sciences Center, Lubbock TX USA Correspondence: Adetola B. Adesida (adesida@ualberta.ca) Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA Received: 13 May 2017 Revised: 17 September 2017 Accepted: 22 September 2017 INTRODUCTION The adherent cell populations were positive for the panel of cell surface markers characteristic of MSCs;38,39 CD13, CD29, CD44, CD73, CD90, CD105, and CD151 (Fig. 1d). Their relative mean ﬂuorescence intensities (MFI) ± SD are as in the parentheses: CD13 (29.26 ± 10.79), CD29 (2.83 ± 0.35), CD44 (6.40 ± 2.40), CD73 (8.63 ± 3.79), CD90 (113.62 ± 14.90), CD105 (1.87 ± 0.05), and CD151 (8.57 ± 0.16)—Fig. 1e. The proportion (%) MSCs (Fig. 1b). We determined the clonogenicity of the MNCs to be 10.65 ± 4.15% (Fig. 1c). The adherent cell populations were positive for the panel of cell surface markers characteristic of MSCs;38,39 CD13, CD29, CD44, CD73, CD90, CD105, and CD151 (Fig. 1d). Their relative mean ﬂuorescence intensities (MFI) ± SD are as in the parentheses: CD13 (29.26 ± 10.79), CD29 (2.83 ± 0.35), CD44 (6.40 ± 2.40), CD73 (8.63 ± 3.79), CD90 (113.62 ± 14.90), CD105 (1.87 ± 0.05), and CD151 (8.57 ± 0.16)—Fig. 1e. The proportion (%) INTRODUCTION p p The rotary cell culture system (RCCS) bioreactor, developed by the National Aeronautics and Space Administration (NASA), as a tool to simulate microgravity has been shown to provide a relatively well-deﬁned ﬂuid dynamic environment with efﬁcient mass transfer for nutrients and gases and low shear stress for tissue growth.34 RCCS has been reported to produce hydrody- namic forces supporting the development of tissue structures resembling cartilage35,36 and meniscus.37 Simulated microgravity facilitated the retention of the differentiated phenotype of chondrocytes within 3D porous scaffolds.36 Moreover, RCCS synergistically enhanced TGF-β1-mediated chondrogenesis of human adipose-derived MSCs.30 However, to the best of our knowledge, no studies has investigated the effect of SMG on TGF- β-mediated chondrogenesis in cocultures of primary human MC and infrapatellar fat pad-derived MSCs. p p In the present study, primary human MC were cocultured with infrapatellar fat pad-derived MSCs on a 3D porous collagen scaffold in the presence of TGF-β3, and cultured under SMG using the RCCS bioreactor. The goals of the present study were (a) to investigate the effect of SMG on human MC, (b) to determine the effect of SMG on the interaction of MC and MSCs in coculture and (c) to assess the potential beneﬁt of using SMG to tissue engineer meniscus constructs in 3D porous type I collagen scaffolds. We hypothesized that SMG will augment the synergistic interaction between primary human MC and MSCs, and result in increased matrix production with suppression of hypertrophic differentiation. The differentiation of multiple types of MSCs is inﬂuenced by a wide range of biochemical,24 microenvironmental,25 and mechan- ical factors.26–28 TGF-β1 and TGF-β3, members of the TGF-β superfamily, that promote ﬁbro-chondrogenic differentiation of MSCs29 and production of meniscus-like ECM. Diverse types of Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA Simulated microgravity enhances chondro-induction WM Weiss et al. 2 RESULTS Colony forming characteristics and immuno-phenotype of adipose stem cells (ASC) A proportion of the seeded adipose tissue-derived mononu- cleated cells (MNCs) formed plastic adherent cell populations with distinct colonies. The developed colonies stained with crystal violet (Fig. 1a). Microscopic visuals of the colonies revealed cells with ﬁbroblastic morphologies characteristics of plastic adherent MSCs (Fig. 1b). We determined the clonogenicity of the MNCs to be 10.65 ± 4.15% (Fig. 1c). Simulated microgravity increases DNA content in MC and cocultures Except for pure MC constructs, the characteristic phenotype of hypertrophic chondrocytes of enlarged size/lacunae was evident in groups containing ASC. Quantitative analysis for the GAG contents of the constructs via DMB assay demonstrated that GAG contents were higher in constructs containing cocultured cells regardless of whether the constructs were cultured under static or SMG conditions (Fig. 4a). The constructs formed under static conditions from the coculture of 25% MC and 75% ASC had a measured GAG content of 195.25 ± 38.73 μg (mean ± SD) compared to an expected GAG content of 148.50 ± 38.36 μg. Wilcoxon signed rank pairwise comparison test between the measured and expected GAG quantities conﬁrmed a signiﬁcantly higher measured GAG content than the expected values (p = 0.003; Fig. 4a). Similarly, there were signiﬁcant differences between the measured and expected GAG content of cocultured constructs containing 25% MC and 75% ASC after culture under SMG. The SMG cultured constructs had a measured GAG content of 279.39 ± 124.09 μg relative to an expected GAG content of 167.78 ± 75.39 μg. Pairwise comparison test using Wilcoxon signed rank test revealed a signiﬁcant difference between the measured and expected GAG values (p = 0.003; Fig. 4a). After static culture, the constructs containing 50% MC and 50% ASC had a measured GAG content of 190.53 ± 47.84 μg. The expected GAG values for these constructs were 172.21 ± 54.80 μg. However, there was no signiﬁcant difference between the measured and expected GAG values as per Wilcoxon signed rank test (p = 0.109; Fig. 4a). In contrast, after SMG culture, the constructs containing 50% MC and 50% ASC measured a signiﬁcantly different GAG content of 251.63 ± 121.92 μg com- pared to an expected GAG content of 173.04 ± 62.29. The p-value Effect of SMG on gene expression of chondrogenically stimulated MC, ASC, and cocultures of MC and ASCs We used quantitative polymearse chain reaction (qPCR) to investigate the gene expression of chondrogenically stimulated MC, ASC and cocultures of MC and ASCs after static and SMG culture conditions. Data followed normal distribution as assessed by Shapiro Wilk test. However, the Levene’ assessment of error variance proved highly signiﬁcant. To this end, pairwise compar- ison between gene expression data of static and SMG cultured constructs were performed using paired Student’s t test with unequal variance. Simulated microgravity increases DNA content in MC and cocultures We next explored the possibility that cell proliferation may accompany the enhanced chondro-induction in cocultures of MC and ASCs after culture under SMG. Shapiro Wilk test for DNA content distribution proved to be normal. However, the Levene’s test for homogeneity of data variance proved negative. To that end, a paired one-tailed Student t test with unequal variance was used to assess differences between parallel static and SMG groups. First, we compared the DNA contents of constructs containing pure MC and pure ASC after static culture and SMG culture conditions. The DNA content of constructs containing pure MCs was 4.53 ± 0.81 µg after SMG culture compared to 3.73 ± 0.63 µg after static culture. These values were signiﬁcantly different (p = 0.03; Fig. 4c). In contrast, the DNA content of constructs containing pure ASCs after static culture and SMG culture approached signiﬁcant difference (p = 0.05). The DNA contents of pure ASC containing constructs after static culture and SMG culture were 3.35 ± 0.56 µg and 2.67 ± 0.84 µg, respectively (Fig. 4c). The constructs containing 25%MC and 75% ASC had a DNA content of 5.46 ± 2.11 µg after SMG culture and 4.20 ± 0.57 µg after static culture. These values were not signiﬁcantly different (p = 0.08) (Fig. 4c). In contrast, constructs containing 50% MC and 50% ASC were signiﬁcantly different (p = 0.03) in DNA contents after static culture and SMG culture. The constructs had a DNA content of 5.33 ± 1.49 µg after SMG culture and those after static culture had a DNA content of 4.06 ± 0.41 µg (Fig. 4c). Chondro-induction is enhanced in SMG cocultures of primary MC and ASC Cell-seeded scaffold constructs of pure MC, pure ASC, and cocultures of MC and ASC were cultured in serum-free chondro- genic media for up to 28 days in the presence of TGF-β3 under static or SMG conditions. Qualitative histological analysis of the constructs with safranin O staining demonstrated intense positive glycosaminoglycan (GAG) staining after the 28 days of culture under static and SMG conditions (Fig. 3). All groups were intensely positive for safranin O and nuclei content, an indication of signiﬁcant synthesis and deposition of sulfated GAG matrix in the constructs (Fig. 3a–h). However, the deposition of GAG matrix appears to be more homogenously distributed in the constructs cultured under SMG (Fig. 3e–h). 3 for the pairwise comparison was 0.026 as per Wilcoxon signed rank test (Fig. 4a). of cells that were positive for these markers varied considerably; CD13 (99.67 ± 0.15), CD29 (93.18 ± 3.26), CD44 (98.06 ± 0.08), CD73 (99.67 ± 0.09), CD90 (90.97 ± 8.86), CD105 (57.94 ± 11.38), and CD151 (98.86 ± 0.16), an indication of heterogeneity (Fig. 1f). In contrast to the MFI associated with mesenchymal markers, the cells displayed lower MFIs for markers of hematopoietic lineage; CD34 (1.29 ± 0.02) and CD45 (1.92 ± 0.50). The proportion (%) of cells that were positive for CD34 and CD45 were 4.93 ± 0.39 and 3.16 ± 1.10, respectively. Interestingly, the cells had a low MFI (0.73 ± 0.52) for the C-X-C chemokine receptor type 4 (CXCR-4) also known as CD184, which has been reported to be expressed on synovial ﬂuid-derived MSCs.40 The proportion of CD184+ cells was 1.29 ± 0.05%. ( g ) After calculating the interaction index as a ratio of the measured and expected GAG contents of the constructs containing cocultured cells, we conﬁrmed that the interaction indices were greater than 1, signifying that chondro-induction had occurred (Fig. 4b). Shapiro Wilk test for normality of data distribution proved positive. The Levene’s test was signiﬁcant and lacked homogeneity of variances. For cocultured cells at 25% MC and 75% ASC, the interaction index was 1.35 ± 0.16 after culture under static conditions and 1.72 ± 0.39 after culture under SMG (Fig. 4b). Paired one-tailed Student’s t test (with unequal variance) comparison of the interaction indices revealed a signiﬁcantly higher chondro-induction under SMG conditions (p = 0.006; Fig. 4b). For cocultured cells at 50% MC and 50% ASC ratio, the interaction index was 1.16 ± 0.14 after static culture and 1.49 ± 0.44 after SMG culture. Similarly, the level of chondro-induction after SMG culture conditions was signiﬁcantly higher (p = 0.022; Fig. 4b). Fluorescent-labeled cells reveal coculture of primary MC and ASC Pure ASC and pure MC were labeled successfully with cell membrane ﬂuorescent dyes PKH26 (red; Fig. 2a) and PKH67 (green; Fig. 2b), respectively. Pure ASC (Fig. 2c), pure MC (Fig. 2d), and cocultured cells at 25% MC and 75% ASC (Fig. 2e) retained their respective ﬂuorescent dyes after 4 weeks of chondrogenic culture in type I collagen matrix scaffolds. This aspect of the study was performed only under static conditions. Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA Simulated microgravity increases DNA content in MC and cocultures Simulated microgravity increases DNA content in MC and cocultures RESULTS Colony forming characteristics and immuno-phenotype of adipose stem cells (ASC) A proportion of the seeded adipose tissue-derived mononu- cleated cells (MNCs) formed plastic adherent cell populations with distinct colonies. The developed colonies stained with crystal violet (Fig. 1a). Microscopic visuals of the colonies revealed cells with ﬁbroblastic morphologies characteristics of plastic adherent let (Fig. 1a). Microscopic visuals of the colonies revealed cells h ﬁbroblastic morphologies characteristics of plastic adherent (6.40 ± 2.40), CD73 (8.63 ± 3.79), CD90 (113.62 ± 14.90), CD105 (1.87 ± 0.05), and CD151 (8.57 ± 0.16)—Fig. 1e. The proportion (%) 1 Colony-forming unit ﬁbroblastic characteristic and surface marker phenotype of fat pad-derived cells following tissue culture plastic herent culture expansion. a A digital photograph of a CFU-F assay Petri dish after staining with crystal violet. b Representative otomicrographs of adherent cells from a 40-year-old male at passage (P0). c Proportion (%) of total fat pad-derived MNCs seeded forming l colonies (% clonogenicity). d Surface marker phenotype of fat pad-derived adherent cells following culture expansion until passage 2 (P2). each CD marker tested, solid histograms show negative isotype control; open histograms show CD marker expression. e Represents the an ﬂuorescence intensity (MFI) of two donors. f Proportion of positive (%) cells expressing tested CD markers Microgravity (2017) 28 Published in cooperation with the Biodesign Institute at Arizona State University with the support of NASA Fig. 1 Colony-forming unit ﬁbroblastic characteristic and surface marker phenotype of fat pad-derived cells following tissue culture plastic adherent culture expansion. a A digital photograph of a CFU-F assay Petri dish after staining with crystal violet. b Representative photomicrographs of adherent cells from a 40-year-old male at passage (P0). c Proportion (%) of total fat pad-derived MNCs seeded forming cell colonies (% clonogenicity). d Surface marker phenotype of fat pad-derived adherent cells following culture expansion until passage 2 (P2). For each CD marker tested, solid histograms show negative isotype control; open histograms show CD marker expression. e Represents the mean ﬂuorescence intensity (MFI) of two donors. f Proportion of positive (%) cells expressing tested CD markers Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2017) 28 Simulated microgravity enhances chondro-induction WM Weiss et al. Simulated microgravity increases DNA content in MC and cocultures q SMG enhanced the mRNA expression of ACAN by 1.9-fold in pure MCs (p = 0.0002; Fig. 5a). SMG increased the mRNA expression of ACAN by 2.2-fold in pure ASC (p = 0.036; Fig. 5a). SMG enhanced the expression of ACAN by 2.4-fold in cocultures of 25% MC and 75% ASC (p = 0.0001; Fig. 5a). Similarly, SMG enhanced the mRNA expression of ACAN by 3.6-fold in 50% MC and 50% ASC cocultures (Fig. 5a). In contrast, the mRNA expression of COL1A2 remained unperturbed between static and SMG cultured groups; no fold changes in COL1A2 expression were Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2017) 28 Simulated microgravity enhances chondro-induction WM Weiss et al. Fig. 2 Fluorescence photomicrographs of PKH26 and PKH67 labeled cells in monolayer and in three-dimensional (3D) porou Representative ﬂuorescence photomicrograph of passage 2 (P2) fat pad-derived plastic adherent cells labeled with PKH26 (red) ce dye (male, 19 years old). Cells were labeled in suspension and imaged after 30 min of plating in monolayer. b Representative photomicrograph of primary human meniscus cells labeled with PKH67 (green) cell membrane dye (male, 56 years old). Cells we suspension and imaged after 30 min of plating in monolayer. c Representative ﬂuorescence photomicrograph of passage 2 derived plastic adherent cells (ASC) labeled with PKH26 (red) cell membrane dye (male, 19 years old) after 3 weeks of chondroge porous type I collagen scaffold. Cells were visualized after sectioning at 7 μm and additional staining with DAPI for cell nuclei ide Representative ﬂuorescence photomicrograph of primary human meniscus cells (MC) labeled with PKH67 (green) cell membrane years old) after 3 weeks of chondrogenic culture in porous type I collagen scaffold under static condition. Cells were visualized aft at 7 μm and additional staining with DAPI for cell nuclei identiﬁcation. e Representative ﬂuorescence photomicrograph of cocult h man menisc s cells (MC) labeled ith PKH67 (green) and passage 2 (P2) fat pad deri ed plastic adherent cells (ASC) labeled 4 4 Fig. 2 Fluorescence photomicrographs of PKH26 and PKH67 labeled cells in monolayer and in three-dimensional (3D) porous scaffolds. a Representative ﬂuorescence photomicrograph of passage 2 (P2) fat pad-derived plastic adherent cells labeled with PKH26 (red) cell membrane dye (male, 19 years old). Cells were labeled in suspension and imaged after 30 min of plating in monolayer. Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA Simulated microgravity increases DNA content in MC and cocultures Simulated microgravity enhances chondro-induction WM Weiss et al. 5 Fig. 3 Histological Safranin O staining after chondrogenic stimulation and differentiation of primary human meniscus cells and fat pad- derived adipose stem cells in porous type I collagen scaffolds. Representative safranin O staining of cell-scaffold constructs after 4 weeks of culture in a deﬁned serum-free chondrogenic medium containing transforming growth factor-β3 (TGFβ3) and dexamethasone (DEX) under static a–d and simulated microgravity e–h conditions: a, e pure primary human meniscus cells (MCs; male, 56 years); b, f Passage 2 (P2) fat pad- derived adipose stem cells (ASC; male, 19 years); c, g Coculture of 75% (P2) fat pad-derived adipose stem cells (ASC; male, 19 years) with 25% primary human meniscus cells (MCs; male, 56 years); d, h Coculture of 50% (P2) fat pad-derived adipose stem cells (ASC; male, 19 years) with 50% primary human meniscus cells (MCs; male, 56 years). Scale bar 100 μm 5 Fig. 3 Histological Safranin O staining after chondrogenic stimulation and differentiation of primary human meniscus cells and fat pad- derived adipose stem cells in porous type I collagen scaffolds. Representative safranin O staining of cell-scaffold constructs after 4 weeks of culture in a deﬁned serum-free chondrogenic medium containing transforming growth factor-β3 (TGFβ3) and dexamethasone (DEX) under static a–d and simulated microgravity e–h conditions: a, e pure primary human meniscus cells (MCs; male, 56 years); b, f Passage 2 (P2) fat pad- derived adipose stem cells (ASC; male, 19 years); c, g Coculture of 75% (P2) fat pad-derived adipose stem cells (ASC; male, 19 years) with 25% primary human meniscus cells (MCs; male, 56 years); d, h Coculture of 50% (P2) fat pad-derived adipose stem cells (ASC; male, 19 years) with 50% primary human meniscus cells (MCs; male, 56 years). Scale bar 100 μm culture conditions. All constructs treated with secondary anti- bodies only proved negative for the type I and II collagen (data not shown). SMG signiﬁcantly enhanced the mRNA expression of the hypertrophic differentiation marker, COL10A1 (Fig. 5e). The increase was two-fold in pure MC (p = 0.008), 7.8-fold in pure ASC (p = 0.03), 4.6-fold in cocultures of 25% MC and 75% ASC (p = 0.0009), and 2.7-fold in cocultures of 50% MC and 50% ASC (p = 0.01). Simulated microgravity increases DNA content in MC and cocultures b Representative ﬂuorescence photomicrograph of primary human meniscus cells labeled with PKH67 (green) cell membrane dye (male, 56 years old). Cells were labeled in suspension and imaged after 30 min of plating in monolayer. c Representative ﬂuorescence photomicrograph of passage 2 (P2) fat pad- derived plastic adherent cells (ASC) labeled with PKH26 (red) cell membrane dye (male, 19 years old) after 3 weeks of chondrogenic culture in porous type I collagen scaffold. Cells were visualized after sectioning at 7 μm and additional staining with DAPI for cell nuclei identiﬁcation. d Representative ﬂuorescence photomicrograph of primary human meniscus cells (MC) labeled with PKH67 (green) cell membrane dye (male, 56 years old) after 3 weeks of chondrogenic culture in porous type I collagen scaffold under static condition. Cells were visualized after sectioning at 7 μm and additional staining with DAPI for cell nuclei identiﬁcation. e Representative ﬂuorescence photomicrograph of cocultured primary human meniscus cells (MC) labeled with PKH67 (green) and passage 2 (P2) fat pad-derived plastic adherent cells (ASC) labeled with PKH26 (red) after 3 weeks of chondrogenic culture in porous type I collagen scaffold under static conditions. The cells were premixed at a cell ratio of 25% MC and 75% ASC prior to seeding a total of 250,000 cells per type I collagen scaffold. Cells were visualized after sectioning at 7 μm and additional staining with DAPI for cell nuclei identiﬁcation. Scale bars: A and B (200 μm) and C–E (100 μm) observed. The insigniﬁcant differences in the treatment groups were supported by p-values > 0.05 (Fig. 5b). SMG increased the expression of COL2A1 in pure MC, pure ASC, and in the cocultures of these cells. However, the fold increase observed were not statistically signiﬁcant; SMG increased COL2A1 mRNA expression by: 1.2-fold in pure MC (p = 0.28), 1.7-fold in pure ASC (p = 0.16), 1.4-fold in cocultures of 25% MC and 75% ASC (p = 0.06), and 1.2- fold in cocultures of 50% MC and 50% ASC (p = 0.21). Qualitative immunoﬂuorescence detection conﬁrmed the translation of these collagen genes in the constructs (Fig. 6). All groups were positive for type I and II collagen. No notable differences were evident in the deposition of these collagens between static culture and SMG Published in cooperation with the Biodesign Institute at Arizona State University, with the support of N npj Microgravity (2017) 28 Simulated microgravity enhances chondro-induction WM Weiss et al. Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA Simulated microgravity increases DNA content in MC and cocultures Expected GAG values are indicated with an “Exp.” preﬁx. The following symbols: , ≠and ¥ indicates statistically signiﬁcant differences between expected and measured GAG values of cocultured group MC/ASC. a Exp. SMG 25%/75% vs. SMG 25%/75%, ≠Exp. SMG 50%/50% vs. SMG 50%/50%, ¥Exp. Static 25%/75% vs. Static 25%/75%. b Interaction index values were determined from the ratio of measured and expected GAG as per the deﬁnition in section “Materials and methods”. Symbols and ≠indicate Wilcoxon signed rank test determined signiﬁcant differences between the interaction indices of static and simulated microgravity (SMG) cocultured groups; SMG 25%/75% vs. Static 25%/75% and ≠SMG 50%/50% vs. Static 50%/50%. c DNA contents of all groups after static and simulated microgravity culture conditions. Symbols * and ¥ indicate a paired one-tailed Student’s t-test (with unequal variance) statistically signiﬁcant difference between the DNA contents of static and simulated microgravity (SMG) groups: SMG 100% MC vs. static 100% MC, and ¥SMG 50%/50% vs. static 50%/50% Fig. 4 The interaction between primary human meniscus cells and fat pad-derived adipose stem cells on type I collagen scaffold after chondrogenic stimulation under static and simulate microgravity (SMG) conditions. Values are mean ± SD of four independent experiments. Expected GAG values are indicated with an “Exp.” preﬁx. The following symbols: , ≠and ¥ indicates statistically signiﬁcant differences between expected and measured GAG values of cocultured group MC/ASC. a Exp. SMG 25%/75% vs. SMG 25%/75%, ≠Exp. SMG 50%/50% vs. SMG 50%/50%, ¥Exp. Static 25%/75% vs. Static 25%/75%. b Interaction index values were determined from the ratio of measured and expected GAG as per the deﬁnition in section “Materials and methods”. Symbols and ≠indicate Wilcoxon signed rank test determined signiﬁcant differences between the interaction indices of static and simulated microgravity (SMG) cocultured groups; SMG 25%/75% vs. Static 25%/75% and ≠SMG 50%/50% vs. Static 50%/50%. c DNA contents of all groups after static and simulated microgravity culture conditions. Symbols and ¥ indicate a paired one-tailed Student’s t-test (with unequal variance) statistically signiﬁcant difference between the DNA contents of static and simulated microgravity (SMG) groups: SMG 100% MC vs. static 100% MC, and ¥SMG 50%/50% vs. static 50%/50% when MC and ASC cocultures are at a ratio of 25–75%, respectively. Simulated microgravity increases DNA content in MC and cocultures However, it is interesting to note that the presence of type X collagen epitope around the lacunae-like structures appear to be less evident in SMG cocultures of 50% MC and 50% ASC, but more so in the extracellular space (Fig. 7h) compared to their static cocultured counterparts (Fig. 7d). All constructs treated with secondary antibodies only proved negative for the type I and II collagen (data not shown). expression by 3.9-fold in cocultures of 25% MC and 75% ASC (p = 0.009), and by 12.4-fold in 50% MC and 50% ASC cocultures (p = 0.0001). Moreover, the mRNA expression of COL10A1 and GREM1 inversely correlated as assessed by Pearson correlation coefﬁcient (−0.166). The correlation was not statistically signiﬁcant (p = 0.100; one-tailed; Fig. 5g). In an analogous manner to collagen X, SMG increased the mRNA expression of MMP-13, another marker of hypertrophic differentiation of chondrocytes (Fig. 5h). It signiﬁ- cantly increased the expression of MMP-13: in pure MC by ﬁve-fold (p = 0.0001), in pure ASC by ﬁve-fold (p = 1.78548 × 10−6), 3.7-fold in cocultures of 25% MC and 75% ASC (p = 0.0003), and 2.1-fold in coculture of 50% MC and 50% ASC (p = 0.008). Pearson correlation demonstrated a highly signiﬁcant inverse relationship between the expression of GREM1 and MMP-13 (r = −0.305; p = 0.006; one- tailed; Fig. 5i). expression by 3.9-fold in cocultures of 25% MC and 75% ASC (p = 0.009), and by 12.4-fold in 50% MC and 50% ASC cocultures (p = 0.0001). Moreover, the mRNA expression of COL10A1 and GREM1 inversely correlated as assessed by Pearson correlation coefﬁcient (−0.166). The correlation was not statistically signiﬁcant (p = 0.100; one-tailed; Fig. 5g). In an analogous manner to collagen X, SMG increased the mRNA expression of MMP-13, another marker of hypertrophic differentiation of chondrocytes (Fig. 5h). It signiﬁ- cantly increased the expression of MMP-13: in pure MC by ﬁve-fold (p = 0.0001), in pure ASC by ﬁve-fold (p = 1.78548 × 10−6), 3.7-fold in cocultures of 25% MC and 75% ASC (p = 0.0003), and 2.1-fold in coculture of 50% MC and 50% ASC (p = 0.008). Pearson correlation demonstrated a highly signiﬁcant inverse relationship between the expression of GREM1 and MMP-13 (r = −0.305; p = 0.006; one- tailed; Fig. 5i). Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA Simulated microgravity increases DNA content in MC and cocultures We evaluated the gene expression of established inhibitor of hypertrophic differentiation, gremlin 1 (GREM1), to provide some mechanistic insight into the noted SMG-mediated upregulation of COL10A1 (Fig. 5f). SMG downregulated the mRNA level of GREM1 by two-fold (p = 0.02) in pure MC but not signiﬁcantly in pure ASC (1.4-fold; p = 0.17). SMG signiﬁcantly downregulated GREM1 Interestingly, SMG signiﬁcantly downregulated the mRNA expression of SOX9 in pure MCs by 3.3-fold (p = 0.02; Fig. 5d). Similarly, SMG downregulated the expression of SOX9 in pure ASCs and in cocultures of 25% MC and 75% ASC but not in coculture of 50% cells where a moderate increase of 1.2-fold was observed. These modulations in SOX9 gene expression were not statistically signiﬁcant with p-values >0.05. h the Biodesign Institute at Arizona State University, with the support of NASA Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2017) 28 Simulated microgravity enhances chondro-induction WM Weiss et al. Fig. 4 The interaction between primary human meniscus cells and fat pad-derived adipose stem cells on type I collagen scaffold after chondrogenic stimulation under static and simulate microgravity (SMG) conditions. Values are mean ± SD of four independent experiments. Expected GAG values are indicated with an “Exp.” preﬁx. The following symbols: , ≠and ¥ indicates statistically signiﬁcant differences between expected and measured GAG values of cocultured group MC/ASC. a Exp. SMG 25%/75% vs. SMG 25%/75%, ≠Exp. SMG 50%/50% vs. SMG 50%/50%, ¥Exp. Static 25%/75% vs. Static 25%/75%. b Interaction index values were determined from the ratio of measured and expected GAG as per the deﬁnition in section “Materials and methods”. Symbols and ≠indicate Wilcoxon signed rank test determined signiﬁcant differences between the interaction indices of static and simulated microgravity (SMG) cocultured groups; SMG 25%/75% vs. Static 25%/75% and ≠SMG 50%/50% vs. Static 50%/50%. c DNA contents of all groups after static and simulated microgravity culture conditions. Symbols and ¥ indicate a paired one-tailed Student’s t-test (with unequal variance) statistically signiﬁcant difference between the DNA contents of static and simulated microgravity (SMG) groups: SMG 100% MC vs. static 100% MC, and ¥SMG 50%/50% vs. static 50%/50% 6 Fig. 4 The interaction between primary human meniscus cells and fat pad-derived adipose stem cells on type I collagen scaffold after chondrogenic stimulation under static and simulate microgravity (SMG) conditions. Values are mean ± SD of four independent experiments. DISCUSSION The functional matrix of the knee meniscus is central to its biomechanical functionality within the joint. Generation of functional meniscus constructs, via tissue engineering and regenerative medicine strategies with a suitable cell source has the potential to revolutionize the treatment of avascular meniscus injuries, which do not heal. Avascular meniscus injuries account for most of meniscal lesions necessitating partial meniscectomies, which unfortunately predisposes the knee to early onset of osteoarthritis. Naturally, primary human MC are the optimal cell source for the generation of functional meniscal substitutes; however, limited availability from surgical specimen of partial meniscectomies is a drawback. Cell expansion strategies to increase numbers mitigate the expression of the functional matrix genes of the meniscus.18,19 To this end, direct coculture of primary human MC with adult-derived MSCs has been explored,20,21 to emulate the synergistic synthesis (i.e. chondro-induction) of the functional matrix of cartilage after coculture of primary articular or We used immunoﬂuorescence to conﬁrm the translation of collagen X in the constructs (Fig. 7). Static culture of pure MC appears to have the least deposition of collagen X in the ECM (Fig. 7a). In contrast, the ECM of the pure MC after SMG culture appears to be more positive for type X collagen epitope (Fig. 7e). Similarly, SMG culture of pure ASC (Fig. 7f) appears to be more positive for type X collagen epitope than its static culture counterpart (Fig. 7b). Interestingly, in the cocultured constructs containing MC and ASC, the epitope of type X collagen was strikingly evident around structures (indicated by white arrows) characterized by a hypertrophic phenotype due to their enlarged size and lacunae- like appearance (Fig. 7c, d, g and h). These structures appear to be larger after SMG culture (Fig. 7g) relative to static culture (Fig. 7c), Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2017) 28 Simulated microgravity enhances chondro-induction WM Weiss et al. Fig. 5 Gene expression of primary human meniscus cells, fat pad-derived adipose mesenchymal stem cells and cocultured cells under static and simulated microgravity culture conditions. Symbols , ≠, ¥ and § indicate a paired Student’s t test (one-tailed; unequal variance) statistically signiﬁcant differences between the interaction indices of static and simulated microgravity (SMG) cocultured groups; SMG 100% MC vs. static 100% MC, ≠SMG 100% ASC vs. static 100% ASC, ¥SMG 25%/75% vs. static 25%/75% and §SMG 50%/50% vs. static 50%/50%. DISCUSSION The potential of increased hypertrophic differ- entiation through GREM1 downregulation suggests a potential risk for enhanced bone formation in vivo through endochondral ossiﬁcation pathway; conditional deletion of GREM1 caused a transient increase in bone formation and bone mass.50 With that in mind, it is unclear at this point if outer and inner MC will behave differently in their capacity to modulate the expression of GREM1 under SMG. Our previous work demonstrated that outer primary human MC have a greater capacity to suppress COL10A1 and MMP-13 expression.22 However, the study was conducted under static conditions using the cell pellet model of in vitro chon- drogenesis. One may speculate that the bulk of primary human component of chondro-induction.41 An unexpected ﬁnding was the accompaniment of the microgravity enhanced chondro- induction with increased transcription of COL10A1 and MMP-13, markers for hypertrophic chondrocytes and hypertrophic differ- entiation of MSCs.43–48 Previous work had demonstrated that direct coculture of primary22 or early passage21 human MC with MSCs suppressed hypertrophic differentiation of MSCs. The mechanism underlying this unexpected ﬁnding appears to be associated with the downregulation of GREM1, a known inhibitor/ antagonist of hypertrophic differentiation.49 Parametric Pearson’s correlation coefﬁcient was used to determine the strength of the association between GREM1 and two hypertrophic markers: COL10A1 and MMP-13. Our assessment revealed an inverse correlation (r = −0.166) between COL10A1 and GREM1, albeit with a non-signiﬁcant p value. There was a highly signiﬁcant inverse correlation (r = −0.305, p = 0.006) between the expression of MMP- 13 and GREM1. The potential of increased hypertrophic differ- entiation through GREM1 downregulation suggests a potential risk for enhanced bone formation in vivo through endochondral ossiﬁcation pathway; conditional deletion of GREM1 caused a transient increase in bone formation and bone mass.50 With that in mind, it is unclear at this point if outer and inner MC will behave differently in their capacity to modulate the expression of GREM1 under SMG. Our previous work demonstrated that outer primary human MC have a greater capacity to suppress COL10A1 and MMP-13 expression.22 However, the study was conducted under static conditions using the cell pellet model of in vitro chon- drogenesis. Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA DISCUSSION a ACAN, b COL1A2, c COL2A1, d SOX9, e COL10A1 and f GREM1. g Relative gene expression of COL10A1 was plotted against the relative gene expression of GREM1. Pearson correlation analysis was used to assess the relationship between COL10A1 and GREM1. h MMP-13. i Relative gene expression of MMP-13 was plotted against the relative gene expression of GREM1. Pearson correlation analysis was used to assess the relationship between MMP-13 and GREM1 7 Fig. 5 Gene expression of primary human meniscus cells, fat pad-derived adipose mesenchymal stem cells and cocultured cells under static and simulated microgravity culture conditions. Symbols , ≠, ¥ and § indicate a paired Student’s t test (one-tailed; unequal variance) statistically signiﬁcant differences between the interaction indices of static and simulated microgravity (SMG) cocultured groups; SMG 100% MC vs. static 100% MC, ≠SMG 100% ASC vs. static 100% ASC, ¥SMG 25%/75% vs. static 25%/75% and §SMG 50%/50% vs. static 50%/50%. a ACAN, b COL1A2, c COL2A1, d SOX9, e COL10A1 and f GREM1. g Relative gene expression of COL10A1 was plotted against the relative gene expression of GREM1. Pearson correlation analysis was used to assess the relationship between COL10A1 and GREM1. h MMP-13. i Relative gene expression of MMP-13 was plotted against the relative gene expression of GREM1. Pearson correlation analysis was used to assess the relationship between MMP-13 and GREM1 component of chondro-induction.41 An unexpected ﬁnding was the accompaniment of the microgravity enhanced chondro- induction with increased transcription of COL10A1 and MMP-13, markers for hypertrophic chondrocytes and hypertrophic differ- entiation of MSCs.43–48 Previous work had demonstrated that direct coculture of primary22 or early passage21 human MC with MSCs suppressed hypertrophic differentiation of MSCs. The mechanism underlying this unexpected ﬁnding appears to be associated with the downregulation of GREM1, a known inhibitor/ antagonist of hypertrophic differentiation.49 Parametric Pearson’s correlation coefﬁcient was used to determine the strength of the association between GREM1 and two hypertrophic markers: COL10A1 and MMP-13. Our assessment revealed an inverse correlation (r = −0.166) between COL10A1 and GREM1, albeit with a non-signiﬁcant p value. There was a highly signiﬁcant inverse correlation (r = −0.305, p = 0.006) between the expression of MMP- 13 and GREM1. DISCUSSION Representative immunoﬂuorescence of cell-scaffold constructs after 4 weeks of culture in a deﬁned serum-free chondrogenic medium containing transforming growth factor-β3 (TGFβ3) and dexamethasone (DEX) under static a–d and simulated microgravity e–h conditions: a, e pure primary human meniscus cells (MCs; male, 56 years); b, f Passage 2 (P2) fat pad-derived adipose stem cells (ASC; male, 19 years); c, g coculture of 75% (P2) fat pad-derived adipose stem cells (ASC; male, 19 years) with 25% primary human meniscus cells (MCs; male, 56 years); d, h Coculture of 50% (P2) fat pad-derived adipose stem cells (ASC; male, 19 years) with 50% primary human meniscus cells (MCs; male, 56 years). Scale bar 100 μm 8 Fig. 6 Immunoﬂuorescence of types I and II collagen after chondrogenic stimulation and differentiation of primary human meniscus cells and fat pad-derived adipose stem cells in porous type I collagen scaffolds. Representative immunoﬂuorescence of cell-scaffold constructs after 4 weeks of culture in a deﬁned serum-free chondrogenic medium containing transforming growth factor-β3 (TGFβ3) and dexamethasone (DEX) under static a–d and simulated microgravity e–h conditions: a, e pure primary human meniscus cells (MCs; male, 56 years); b, f Passage 2 (P2) fat pad-derived adipose stem cells (ASC; male, 19 years); c, g coculture of 75% (P2) fat pad-derived adipose stem cells (ASC; male, 19 years) with 25% primary human meniscus cells (MCs; male, 56 years); d, h Coculture of 50% (P2) fat pad-derived adipose stem cells (ASC; male, 19 years) with 50% primary human meniscus cells (MCs; male, 56 years). Scale bar 100 μm MC in this study may have originated from the inner region of the meniscus, since the inner portion of the tissue accounts for two- thirds of the meniscus’ width. forces mediated by SMG. Thus, activation of p38 MAPK signaling may be an additional underlying mechanism contributing to the synergistic chondro-induction in our study under SMG. It is noteworthy that while Yu et al.30 reported an increase in COL2A1 and SOX9 under SMG, we did not observe a signiﬁcant increase in the expression of these genes under SMG. DISCUSSION One may speculate that the bulk of primary human nasal chondrocytes with bone marrow or adipose MSCs.41 Simulated microgravity has been shown to synergistically enhance the synthesis of the functional matrix of articular cartilage in chondrocyte seeded polymeric scaffolds.35,42 Moreover, SMG synergistically enhanced the chondrogenic differentiation of adipose MSCs in the presence of TGFβ1.30 These reported ﬁndings prompted us to ask the following question: can SMG further augment chondro-induction in direct cocultures of primary human MC and adipose MSCs? We hypothesized that SMG will further enhance chondro-induction in primary human MC and adipose MSCs in the presence of TGFβ. p p β To investigate the hypothesis, human cells: adipose-derived MSCs and MC were successfully isolated from knee fat pad and menisci, respectively. Direct coculture of these cells in a 3D porous type I collagen matrix scaffolds, as a mimic of the predominant ECM of the native meniscus, was successfully demonstrated using differential ﬂuorescent cell labeling. After 4 weeks of chondro- genic culture in the presence of TGFβ3, both pure cells and cocultured cells synthesized cartilaginous ECM under static and SMG. Our study ﬁndings supported our hypothesis with evidence of GAG and ACAN increase. Moreover, SMG increased the DNA content of constructs containing pure MCs and cocultured MCs and ASCs. This was not the case in constructs containing pure ASCs. This ﬁnding suggested that the mechanism underlying SMG enhanced chondro-induction may involve increased proliferation of MC. Increased proliferation of chondrocytes in coculture with MSCs has also been reported previously as a mechanistic npj Microgravity (2017) 28 Simulated microgravity enhances chondro-induction WM Weiss et al. Simulated microgravity enhances chondro-induction WM Weiss et al. Fig. 6 Immunoﬂuorescence of types I and II collagen after chondrogenic stimulation and differentiation of primary human meniscus cells and fat pad-derived adipose stem cells in porous type I collagen scaffolds. Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2017) 28 DISCUSSION But our ﬁndings on ACAN and COL10A1 transcriptional increase under SMG were consistent with the ﬁndings of Yu et al.30 Our ﬁnding of increased COL10A1 expression in pure adipose- derived MSCs under SMG is consistent with the reports of Yu et al.30 The authors demonstrated that SMG enhanced the in vitro chondrogenesis of adipose-derived MSCs in the presence of TGFβ1 but with increased COL10A1 mRNA expression. They also demonstrated that SB203580, a highly speciﬁc and potent inhibitor of p38 mitogen-activated protein kinases (MAPK) signaling pathway, signiﬁcantly suppressed the mRNA expression of COL10A1 during SMG culture conditions, suggesting that p38 MAPK was a signiﬁcant player in the transduction of physical In summary, the present study demonstrated that SMG enhanced the chondro-induction in cocultures of primary human MC and adipose-derived mesenchymal stem but at the cost of increased hypertrophic differentiation that is concomitant with GREM1 downregulation. npj Microgravity (2017) 28 Simulated microgravity enhances chondro-induction WM Weiss et al. 9 METHODS piperazineethanesulfonic acid (HEPES) sodium pyruvate (all from Invitro Fig. 7 Immunoﬂuorescence of type X collagen after chondrogenic stimulation and differentiation of primary human meniscus cells and fat pad-derived adipose stem cells in porous type I collagen scaffolds. Representative immunoﬂuorescence of cell-scaffold constructs after 4 weeks of culture in a deﬁned serum-free chondrogenic medium containing transforming growth factor-β3 (TGFβ3) and dexamethasone (DEX) under static a–d and simulated microgravity e–h conditions: a, e pure primary human meniscus cells (MCs; male, 56 years); b, f Passage 2 (P2) fat pad-derived adipose stem cells (ASC; male, 19 years); c, g coculture of 75% (P2) fat pad-derived adipose stem cells (ASC; male, 19 years) with 25% primary human meniscus cells (MCs; male, 56 years); d, h coculture of 50% (P2) fat pad-derived adipose stem cells (ASC; male, 19 years) with 50% primary human meniscus cells (MCs; male, 56 years). Lacunae-like structures are indicated by white arrows. Scale bar 500 μm Fig. 7 Immunoﬂuorescence of type X collagen after chondrogenic stimulation and differentiation of primary human meniscus cells and fat pad-derived adipose stem cells in porous type I collagen scaffolds. Colony-forming unit ﬁbroblastic (CFU-F) assay A CFU-F assay was performed to determine the proportion of plastic- adherent cells from adipose-derived MNCs. MNCs from each donor were plated in triplicates at 500 MNCs per 100 mm diameter sterile Petri dish (Becton Dickinson Canada Inc.) and cultured for expansion as described above at 37 °C under 95% humidity in an atmosphere of 21% O2 and 5% CO2. After the ﬁrst week, the non-adherent cell population was removed by aspiration and culture media was replenished twice for another week. After the 2 weeks of culture, the culture media was aspirated and the Petri dish was washed with sterile PBS, then ﬁxed with 10% w/v buffered formalin for 5 minutes. The formalin was aspirated and the Petri dish was washed twice with PBS, and stained with 0.25% w/v crystal violet solution (Sigma-Aldrich). Stained colonies were revealed after washing with a copious amount of distilled water. Each stained cell collection was assessed and considered to be a colony as previously described.55 The number of colonies developed from the total number of MNC seeded was used to calculate clonogenicity (%), with each colony representing a single cell- derived clone. Isolation of primary human MC Menisci were harvested from the knee joint of ﬁve donors: three males (14, 56, and 56) and two females (age 57 and 75), rinsed three times with sterile phosphate buffered saline (PBS), and ﬁnely minced. Donor information is documented in Table 2. MC were released from meniscus pieces by incubation for 16 hours at 37 °C in type II collagenase (0.15% w/v of 300 units/mg solid; Worthington, Lakewood, NJ, USA) in a standard medium, high glucose Dulbecco’s modiﬁed Eagle’s medium (DMEM) 4.5 mg/ml D- glucose supplemented with 5% v/v fetal bovine serum (FBS), 100 units/ml penicillin and 100 units/ml streptomycin, with L-glutamine (2 mM) (Invitrogen, Mississauga, Ontario, Canada), then ﬁltered through a 150- µm nylon mesh to remove cellular debris, as described previously. Isolated cells were plated in standard medium supplemented in 150 cm2 tissue culture plastic (TCP) at 37 °C and 21% O2 in a humidiﬁed incubator with 5% CO2. After 48 h of recovery in standard medium, the cells were detached with 0.15% w/v trypsin-EDTA in Hank’s buffered saline solution (HBSS; Invitrogen) and counted prior to use. METHODS piperazineethanesulfonic acid (HEPES), sodium pyruvate (all from Invitro- gen) and 5 ng/ml basic ﬁbroblast growth factor (bFGF or FGF2; from Humanzyme, Medicorp Inc., Montreal, Quebec, Canada) to maintain multipotency.51–54 The cells were cultured until passage 2 (P2) at 37 °C under 95% humidity in an atmosphere of 21% O2 and 5% CO2. DISCUSSION Representative immunoﬂuorescence of cell-scaffold constructs after 4 weeks of culture in a deﬁned serum-free chondrogenic medium containing transforming growth factor-β3 (TGFβ3) and dexamethasone (DEX) under static a–d and simulated microgravity e–h conditions: a, e pure primary human meniscus cells (MCs; male, 56 years); b, f Passage 2 (P2) fat pad-derived adipose stem cells (ASC; male, 19 years); c, g coculture of 75% (P2) fat pad-derived adipose stem cells (ASC; male, 19 years) with 25% primary human meniscus cells (MCs; male, 56 years); d, h coculture of 50% (P2) fat pad-derived adipose stem cells (ASC; male, 19 years) with 50% primary human meniscus cells (MCs; male, 56 years). Lacunae-like structures are indicated by white arrows. Scale bar 500 μm Ethics statement Approval of the Health Research Ethics Board-Biomedical Panel of the University of Alberta, Edmonton, Canada was obtained for this study, and institutional safety and ethical guidelines were followed. Ethics committee waived the need for written informed consent of patients, as specimens used in the study were intended for discard in the normal course of the surgical procedure. Extensive precautions were taken to preserve the privacy of the participants donating specimens. Flow cytometry analysis All primary monoclonal antibodies used herein were directly conjugated antibodies to ﬂuorescein isothiocynate (mAb-FITC) or to phycoerythrin (mAb- PE). Antibodies were either from BD Pharmingen or Invitrogen—see Table 1. The cells were analyzed on a FACScan ﬂow cytometer (Becton Dickinson) after detachment from culture ﬂask by 0.05% w/v trypsin-EDTA (Invitrogen). Staining buffer was prepared with PBS containing 1% w/v BSA (Sigma). The cells were re-suspended in 4 °C cold staining buffer at 5 × 106/ml. Thereafter, the cells were dispensed into sample tubes (12 × 75 mm polystyrene round- bottom tubes, Becton Dickinson) in 20 µl aliquots and incubated for 15 minutes with the antibodies at 4 °C. All incubations were implemented in 5 ml dilution tubes at room temperature in the dark and all washing steps were performed by a combination of centrifugation (400 g, 5 minutes) and aspiration of supernatant. Staining buffer (200 µl) was then added to the tubes and cells were incubated for 10 additional minutes. After removal of the supernatant by centrifugation, cells were washed with PBS and kept cold before analysis by ﬂow cytometry. Non-speciﬁc staining was assessed using relevant isotype controls. Single color immuno-ﬂuorescence analysis for the different surface markers was performed with mAb-FITC and mAb-PE. Data Type I collagen scaffold constructs Type I collagen matrix (Integra Lifesciences, PlainsBoro, NJ, USA; 10 cm × 12.5 cm; 3.5 mm total thickness collagen sponge with pore size of 115 ± 20 µm) was cut into 6 mm diameter disks using a sterile biopsy punch. To limit variability between batches of scaffolds, disks taken from the same batch of scaffolds were used for each experiment. The disks were placed in a 24- well plate for seeding with cells. Co-culture of ﬂuorescently labeled MCs and ASCs To demonstrate co-culture of MCs and ASCs, isolated MCs and ASCs were labeled with ﬂuorescent dyes in suspension according to manufacturer’s labeling protocol. Pure MCs were labeled with PKH67 (green) and pure ASCs were labeled with PKH26 (red). The labeled cells were visualized in monolayer after plating in a T25 TCP ﬂask using an Eclipse Ti–S microscope (Nikon Canada Mississauga, Canada) ﬁtted with NIS Elements (version 4.20; Nikon Canada). PKH67 and PKH26 ﬂuorescent cell linker kits were purchased from Sigma-Aldrich (Oakville, Canada). The labeled cells were seeded onto type I collagen matrix scaffolds as described above. Culture media, conditions, and duration were as described above but only static culture Isolation of human adipose stem cells The level of expression of each marker was calculated as the ratio between geometric mean ﬂuorescence intensity (MFI) of samples and that of the isotype control. collagen scaffold via a micropipette with a total of 250,000 cells suspended in 20 µl of a deﬁned serum-free chondrogenic medium consisting of high glucose DMEM containing 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 100 mM HEPES buffer, 1 mM sodium pyruvate, 100 U/ ml penicillin, 100 mg/ml streptomycin, 0.29 mg/ml L-glutamine (Invitrogen) supplemented with 0.1 mM ascorbic acid 2-phosphate, 40 mg/ml L-proline, 10−7 M dexamethasone, 1 × ITS + 1 premix (Sigma-Aldrich, Oakville, Canada), 10 ng/ml TGF-β3 (Humanzyme-Medicorp Inc.). The seeded disks were transferred to a humidiﬁed incubator at 37 °C with 21% O2 and 5% CO2 for 15 minutes to allow initial cell attachment. Thereafter, 100 µl of chondrogenic medium was gently added to the base of each well containing cell-seeded disks followed by a 30 minutes incubation period in a humidiﬁed incubator at 37 °C with 21% O2 and 5% CO2. After the incubation period, 700 µl of chondrogenic medium was added slowly to the base of each well until the entire seeded scaffolds were covered. The seeded scaffolds were then incubated at 37 °C with 21% O2 and 5% CO2 for 7 days. Chondrogenic media exchange was performed twice per week. Isolation of human adipose stem cells collagen scaffold via a micropipette with a total of 250,000 cells suspended in 20 µl of a deﬁned serum-free chondrogenic medium consisting of high glucose DMEM containing 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 100 mM HEPES buffer, 1 mM sodium pyruvate, 100 U/ ml penicillin, 100 mg/ml streptomycin, 0.29 mg/ml L-glutamine (Invitrogen) supplemented with 0.1 mM ascorbic acid 2-phosphate, 40 mg/ml L-proline, 10−7 M dexamethasone, 1 × ITS + 1 premix (Sigma-Aldrich, Oakville, Canada), 10 ng/ml TGF-β3 (Humanzyme-Medicorp Inc.). The seeded disks were transferred to a humidiﬁed incubator at 37 °C with 21% O2 and 5% CO2 for 15 minutes to allow initial cell attachment. Thereafter, 100 µl of chondrogenic medium was gently added to the base of each well containing cell-seeded disks followed by a 30 minutes incubation period in a humidiﬁed incubator at 37 °C with 21% O2 and 5% CO2. After the incubation period, 700 µl of chondrogenic medium was added slowly to the base of each well until the entire seeded scaffolds were covered The Table 1. Antibodies used to characterize infrapatellar fat pad mesenchymal stem cells Speciﬁcity Isotype Cat.#/Flurochrome Source CD13 (aminopeptidase-n) mIgG1 sc-70529/PE Santa Cruz Biotechnology CD29 mIgG1 CD2901/FITC Invitrogen CD34 mIgG1 sc-19621/FITC Santa Cruz Biotechnology CD44 (Pgp-1, H-CAM, Ly 24) mIgG2b 560977/FITC BD Pharmingen CD45 (2B11) mIgG1 Sc-20056/PE Santa Cruz Biotechnology CD73 mIgG1 550257/PE BD Pharmingen CD90 (Thy-1) mIgG1 55596/PE BD Pharmingen CD105 (endoglin) mIgG1 sc-71043/PE Santa Cruz Biotechnology CD151 (PETA-3) mIgG1 556057/PE BD Pharmingen CD184 (CXCR4) mIgG2a 560937/PE-CyTM5 BD Pharmingen Not speciﬁed (Isotype control) mIgG1 sc-2855/FITC Santa Cruz Biotechnology Not speciﬁed (Isotype control) mIgG1 sc-2866/PE Santa Cruz Biotechnology m, mouse; r, rat Table 2. Donor information of meniscal (MEN/ NMEN) and patellar fat pad (ASC) specimen Donor Age (years) Gender (M/F) Weight (kg) Height (cm) Smoker? (Y/N) Medical history/surgery reason MEN155 56 M 140 186 N Osteoarthritis MEN159 75 F 93 155 N No anticancer drug use/osteoarthritis MEN161 56 M 66 168 Y No anticancer drug use/osteoarthritis MEN172 57 F 96 151 N Osteoarthritis NMEN190 14 M 65 175 N Partial meniscectomy ASC64 20 F – – – Mitral valve prolapse/knee injury ASC68 19 M 69.5 173 – Healthy/meniscal & ACL repair ASC67 17 M 79.5 185 N Knee joint injury ASC70 21 F 71 161 Y Allergy to nickel, asthma Knee repair ASC97 40 M 88.9 180 Y Multiple ligament & knee injury Table 1. Isolation of human adipose stem cells Patellar fat pads were harvested from the knee joint of ﬁve donors undergoing routine orthopedic procedure; three males (age 17, 19 and 40) and two females (age 20 and 21). Donor information is documented in Table 2. The adipose tissue was rinsed three times with PBS, and ﬁnely minced before enzymatic digestion. Tissue was digested with type II collagenase (0.15% w/v of 300 units/mg solid; Worthington) in PBS at 37 °C using a water bath shaker at 150 rpm for 45 minutes. Nucleated cells were re-suspended in alpha modiﬁed Eagle’s medium (αMEM) supplemented with 10% v/v FBS, and 1% w/v antibiotic/antimycotic solution ﬁltered through a 100 μm nylon mesh to remove cellular debris. The number of nucleated cells in the aspirates was determined by crystal violet nuclei staining with cell counting using a hemocytometer. Thereafter, 15 million mono-nucleated cells (MNCs) were seeded per 150 cm2 TCP. The culture medium was αMEM supplemen- ted with 10% v/v FBS, penicillin–streptomycin, 4-(2-hydroxyethyl)-1- npj Microgravity (2017) 28 ublished in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NAS Simulated microgravity enhances chondro-induction WM Weiss et al. 10 acquisition was performed with Cellquest software (Becton Dickinson). FITC emission was measured at FL1 and PE at FL3. For each sample a region for live cells was deﬁned, according to the Forward Scatter (FSC) and Side Scatter (SSC) signals, which excluded aggregated cells from the analysis. Data analysis was performed with Cyﬂogic version 1.2.1, Perthu Terho & CyFlo Ltd., Finland. For each surface marker analyzed, percentage of positive cells and the level of marker expression were calculated. Percentage of positive cells was calculated as the percentage of cells having a measured ﬂuorescence greater than that of 99.5% of the cells stained with each associated isotype control. Cells were considered positive for a surface marker when the percentage of positive cells for that surface marker was ≥6%. The level of expression of each marker was calculated as the ratio between geometric mean ﬂuorescence intensity (MFI) of samples and that of the isotype control. Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA Isolation of human adipose stem cells (Y/N) Medical history/surgery reason MEN155 56 M 140 186 N Osteoarthritis MEN159 75 F 93 155 N No anticancer drug use/osteoarthritis MEN161 56 M 66 168 Y No anticancer drug use/osteoarthritis MEN172 57 F 96 151 N Osteoarthritis NMEN190 14 M 65 175 N Partial meniscectomy ASC64 20 F – – – Mitral valve prolapse/knee injury ASC68 19 M 69.5 173 – Healthy/meniscal & ACL repair ASC67 17 M 79.5 185 N Knee joint injury ASC70 21 F 71 161 Y Allergy to nickel, asthma Knee repair ASC97 40 M 88.9 180 Y Multiple ligament & knee injury Table 2. Donor information of meniscal (MEN/ NMEN) and patellar fat pad (ASC) specimen acquisition was performed with Cellquest software (Becton Dickinson). FITC emission was measured at FL1 and PE at FL3. For each sample a region for live cells was deﬁned, according to the Forward Scatter (FSC) and Side Scatter (SSC) signals, which excluded aggregated cells from the analysis. Data analysis was performed with Cyﬂogic version 1.2.1, Perthu Terho & CyFlo Ltd., Finland. For each surface marker analyzed, percentage of positive cells and the level of marker expression were calculated. Percentage of positive cells was calculated as the percentage of cells having a measured ﬂuorescence greater than that of 99.5% of the cells stained with each associated isotype control. Cells were considered positive for a surface marker when the percentage of positive cells for that surface marker was ≥6%. The level of expression of each marker was calculated as the ratio between geometric mean ﬂuorescence intensity (MFI) of samples and that of the isotype control. acquisition was performed with Cellquest software (Becton Dickinson). FITC emission was measured at FL1 and PE at FL3. For each sample a region for live cells was deﬁned, according to the Forward Scatter (FSC) and Side Scatter (SSC) signals, which excluded aggregated cells from the analysis. Data analysis was performed with Cyﬂogic version 1.2.1, Perthu Terho & CyFlo Ltd., Finland. For each surface marker analyzed, percentage of positive cells and the level of marker expression were calculated. Percentage of positive cells was calculated as the percentage of cells having a measured ﬂuorescence greater than that of 99.5% of the cells stained with each associated isotype control. Cells were considered positive for a surface marker when the percentage of positive cells for that surface marker was ≥6%. Isolation of human adipose stem cells Antibodies used to characterize infrapatellar fat pad mesenchymal stem cells Speciﬁcity Isotype Cat.#/Flurochrome Source CD13 (aminopeptidase-n) mIgG1 sc-70529/PE Santa Cruz Biotechnology CD29 mIgG1 CD2901/FITC Invitrogen CD34 mIgG1 sc-19621/FITC Santa Cruz Biotechnology CD44 (Pgp-1, H-CAM, Ly 24) mIgG2b 560977/FITC BD Pharmingen CD45 (2B11) mIgG1 Sc-20056/PE Santa Cruz Biotechnology CD73 mIgG1 550257/PE BD Pharmingen CD90 (Thy-1) mIgG1 55596/PE BD Pharmingen CD105 (endoglin) mIgG1 sc-71043/PE Santa Cruz Biotechnology CD151 (PETA-3) mIgG1 556057/PE BD Pharmingen CD184 (CXCR4) mIgG2a 560937/PE-CyTM5 BD Pharmingen Not speciﬁed (Isotype control) mIgG1 sc-2855/FITC Santa Cruz Biotechnology Not speciﬁed (Isotype control) mIgG1 sc-2866/PE Santa Cruz Biotechnology *m, mouse; r, rat Table 1. Antibodies used to characterize infrapatellar fat pad mesenchymal stem cells Table 2. Donor information of meniscal (MEN/ NMEN) and patellar fat pad (ASC) specimen Donor Age (years) Gender (M/F) Weight (kg) Height (cm) Smoker? (Y/N) Medical history/surgery reason MEN155 56 M 140 186 N Osteoarthritis MEN159 75 F 93 155 N No anticancer drug use/osteoarthritis MEN161 56 M 66 168 Y No anticancer drug use/osteoarthritis MEN172 57 F 96 151 N Osteoarthritis NMEN190 14 M 65 175 N Partial meniscectomy ASC64 20 F – – – Mitral valve prolapse/knee injury ASC68 19 M 69.5 173 – Healthy/meniscal & ACL repair ASC67 17 M 79.5 185 N Knee joint injury ASC70 21 F 71 161 Y Allergy to nickel, asthma Knee repair ASC97 40 M 88.9 180 Y Multiple ligament & knee injury acquisition was performed with Cellquest software (Becton Dickinson). FITC emission was measured at FL1 and PE at FL3. For each sample a region for live cells was deﬁned, according to the Forward Scatter (FSC) and Side Scatter (SSC) signals, which excluded aggregated cells from the analysis. Data analysis was performed with Cyﬂogic version 1.2.1, Perthu Terho & CyFlo Ltd., Finland. For each surface marker analyzed, percentage of positive cells and the level of marker expression were calculated. Percentage of positive cells was calculated as the percentage of cells having a measured ﬂuorescence greater than that of 99.5% of the cells stained with each associated isotype control. Cells were considered positive for a surface marker when the percentage of positive cells for that surface marker was ≥6%. The level of expression of each marker was calculated as the ratio between geometric mean ﬂuorescence intensity (MFI) of samples and that of the isotype control. Isolation of human adipose stem cells Type I collagen scaffold constructs Type I collagen matrix (Integra Lifesciences, PlainsBoro, NJ, USA; 10 cm × 12.5 cm; 3.5 mm total thickness collagen sponge with pore size of 115 ± 20 µm) was cut into 6 mm diameter disks using a sterile biopsy punch. To limit variability between batches of scaffolds, disks taken from the same batch f ff ld d f h i t Th di k l d i 24 collagen scaffold via a micropipette with a total of 250,000 cells suspended in 20 µl of a deﬁned serum-free chondrogenic medium consisting of high glucose DMEM containing 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 100 mM HEPES buffer, 1 mM sodium pyruvate, 100 U/ ml penicillin, 100 mg/ml streptomycin, 0.29 mg/ml L-glutamine (Invitrogen) supplemented with 0.1 mM ascorbic acid 2-phosphate, 40 mg/ml L-proline, 10−7 M dexamethasone, 1 × ITS + 1 premix (Sigma-Aldrich, Oakville, Canada), 10 ng/ml TGF-β3 (Humanzyme-Medicorp Inc.). The seeded disks were transferred to a humidiﬁed incubator at 37 °C with 21% O2 and 5% CO2 for 15 minutes to allow initial cell attachment. Thereafter, 100 µl of chondrogenic medium was gently added to the base of each well containing cell-seeded disks followed by a 30 minutes incubation period in a humidiﬁed incubator at 37 °C with 21% O2 and 5% CO2. After the incubation period, 700 µl of chondrogenic medium was added slowly to the base of each well until the entire seeded scaffolds were covered. The seeded scaffolds were then incubated at 37 °C with 21% O2 and 5% CO2 for 7 days. Chondrogenic media exchange was performed twice per week. Co-culture of ﬂuorescently labeled MCs and ASCs To demonstrate co-culture of MCs and ASCs, isolated MCs and ASCs were labeled with ﬂuorescent dyes in suspension according to manufacturer’s Table 2. Donor information of meniscal (MEN/ NMEN) and patellar fat pad (ASC) specimen Donor Age (years) Gender (M/F) Weight (kg) Height (cm) Smoker? Gene expression analysis was implemented to demonstrate co-culture of labeled cells. Cells were mixed at a ratio of 25% MC and 75% ASC prior to seeding on type I collagen matrix scaffold. Cell-scaffold constructs were cultured in chondrogenic medium as before for 28 days. Thereafter, the cultured constructs were embedded in a VWR clear frozen section compound (VWR, Mississauga, ON, Canada). Engineered tissue sections were cut on a Leica Biosystems cryostat at 7 µm. Images were captured as before and assembled in Image J version 1.51k software (NIH, Bethesda, Maryland, USA). was implemented to demonstrate co-culture of labeled cells. Cells were mixed at a ratio of 25% MC and 75% ASC prior to seeding on type I collagen matrix scaffold. Cell-scaffold constructs were cultured in chondrogenic medium as before for 28 days. Thereafter, the cultured constructs were embedded in a VWR clear frozen section compound (VWR, Mississauga, ON, Canada). Engineered tissue sections were cut on a Leica Biosystems cryostat at 7 µm. Images were captured as before and assembled in Image J version 1.51k software (NIH, Bethesda, Maryland, USA). p y Total RNA was extracted from MCs, ASCs, and scaffold cultures using Trizol (Invitrogen) after grinding with Molecular Grinding Resin (Geno Technol- ogy Inc., St Louis, USA) in combination with the use of RNeasy mini-kit (Qiagen, Mississauga, ON, Canada) and after removal of contaminating genomic DNA from the preparations with DNase treatment. Total RNA (100 ng) in a 40 µl reaction was reverse transcribed to cDNA using GoScript reverse transcriptase (Fisher Scientiﬁc, Whitby, Ontario, Canada) primed with random primer oligonucleotides. qPCR was performed either in a DNA Engine OpticonIContinuous Fluorescence Detection System (Bio-Rad) using hot start Taq and SYBR Green detection (Eurogentec North America Inc, San Diego, CA, USA) or in a Bio-Rad CFX connect real-time system (Bio-Rad Laboratories, Mississauga, ON, Canada). Primer sequences were either designed by Primer Express 3.0.1 (Applied Biosystems, ThermoFisher, ON, Canada) or taken from previously published work. All primers were obtained from Invitrogen, Mississauga, ON, Canada. Gene (mRNA) expression levels for each primer set were normalized to the expression level of human βactin, by the 2-ΔΔct method. ACKNOWLEDGEMENTS Financial support was provided by Canadian Institutes for Health Research (CIHR MOP 125921), Canadian Foundation for Innovation (CFI 33786), University of Alberta Hospital Foundation (RES0028185) and Edmonton Orthopedic Research Committee (GS22000020). In-kind donation of type I collagen scaffolds was provided by Integra LifeSciences Inc., Plainsboro, NJ, USA. 11 Culture in SMG After 7 days of culture, one set of cell co-culture on type I collagen scaffolds (MC alone, ASC alone, 1:3 MC to ASC, and 1:1 MC to ASC) were transferred to 10 mL vessels and inserted into a rotary bioreactor RCCS (Synthecon, Inc., Houston, TX). An identical set of co-cultures was used as a control, and continued to be incubated under static conditions. The rotary bioreactor was housed within the same incubator as the static controls, and both continued to be cultured in a humidiﬁed incubator at 37 °C with 21% O2 and 5% CO2. The rotation speed of the RCCS increased from 20 to 30 rpm over the course of 21 days of culture. This increase in rotation speed was required to keep the increasing mass of the scaffolds suspended under microgravity conditions. After 21 days of culture in the rotary bioreactor (28 days total from seeding of the scaffolds), the culture scaffolds were harvested and analyzed. Scaffolds were processed biochemically for GAG and DNA content, histologically and immunoﬂuor- escence for cartilage-speciﬁc matrix proteins, and at the molecular level by real time qPCR for gene expression analysis. Data availability All relevant data are available from the corresponding author. Gene expression analysis Primer sequence are as follows: ACAN19 (Accession# M55172) forward: AGG GCG AGT GGA ATG ATG TT; ACAN19 reverse: GGT GGC TGT GCC CTT TTT AC; ACTB19 (Accession# NM_001101) forward AAG CCA CCC CAC TTC TCT CTA A; ACTB19 reverse AAT GCT ATC ACC TCC CCT GTG T; COL1A219 (NM_000089) forward: TTG CCC AAA GTT GTC CTC TTC T; COL1A219 reverse: AGC TTC TGT GGA ACC ATG GAA; COL2A119 (Accession# NM_033150) forward CTG CAA AAT AAA ATC TCG GTG TTC T; COL2A1 reverse: GGG CAT TTG ACT CAC ACC AGT; COL10A1 (Accession# X60382) forward: GAA GTT ATA ATT TAC ACT GAG GGT TTC AAA; COL10A1 reverse: GAG GCA CAG CTT AAA AGT TTT AAA CA; GREM-1 (Accession# NM_001191322.1) forward: CAT GTG ACG GAG CGC AAA TA; GREM-1 reverse: GCT TAA GCG GCT GGG TTT T; MMP13 (Accession# NM_002427) forward: CATCCAAAAACGCCAGACAA; MMP13 reverse: CGGAGACTGGTAATGGCATCA; SOX919 (Accession# Z46629) for- ward: CTT TGG TTT GTG TTC GTG TTT TG; SOX919 reverse: AGA GAA AGA AAA AGG GAA AGG TAA GTT T. Histology and immuno-histochemistry Tissues generated from the scaffold-cell cultures were ﬁxed in 4% v/v phosphate buffered formalin, processed into parafﬁn wax, sectioned at 5 µm and stained with safranin O and counterstained with fast green, to reveal sulfated proteoglycan (GAG) matrix depositions. Other sections were probed with antibodies raised against collagen types I and II. The distribution of types I and II collagen were evaluated using immunoﬂuor- escence imaging. Sections were deparafﬁnized and rehydrated. Antigen retrieval was conducted with Protease XXV (Thermo Scientiﬁc) for 30 minutes at room temperature and hyaluronidase (H6254, Sigma) for 30 min at 37 °C. Sections were then blocked with 5% w/v bovine serum albumin in PBS for 30 minutes at room temperature. Collagen I, II and X labeling was completed using rabbit anti-collagen I, 1:200 dilution (CL50111AP-1, Cedarlane Labs, Burlington, ON, Canada), mouse anti-collagen II, 1:200 dilution (II-II6B3, Developmental Studies Hybridoma Bank, University of Iowa, USA) and rabbit anti-collagen X (ab58632, Abcam, UK) using a 1:100 dilution, respectively, and incubated overnight at 4 °C. Secondary labeling using goat anti-mouse IgG, Alexa Fluor 488, 1:200 (ab150117, Abcam) and goat anti-rabbit IgG, Alexa Fluor 594, 1:200 (ab150080, Abcam) incubated at room temperature for 1 hour. Slides were mounted using Everbrite Hardset Mounting Medium with DAPI (Biotium) and imaged using a Nikon Ti–S Microscope ﬁtted with DS-U3/Fi2 Color CCD camera using FITC and Texas Red ﬁlters (Nikon Canada Mississauga, Canada) with NIS Elements software (version 4.20; Nikon Canada) and assembled in Image J version 1.51k software (NIH, Bethesda, Maryland, USA). Statistical analysis A total of ﬁve independent experiments were performed with ﬁve donor specimens for each cell type. Unless stated otherwise, numerical data distribution represents data from ﬁve donors and is presented as a boxplot of the minimum, ﬁrst quartile, median, third quartile, and maximum values. Statistical analyses were performed using SPSS (version 23; IBM Canada Ltd, ON, Canada). Outliers were deﬁned as values 1.5 interquartile ranges below the ﬁrst quartile or above the third quartile. Data were assessed for normality using the Shapiro–Wilk test. Levene’s test was used to assess homogeneity of error variances. Statistical test choice was dependent on outcome of normality and homogeneity test. Paired comparisons between static and SMG groups was analyzed using either Wilcoxon signed rank test or paired Student’s t test depending on outcome of normality test. Pearson or Spearman correlation coefﬁcient was used to assess correlation between genes. Statistical signiﬁcance was considered when p < 0.05. AGexpected ¼ GAG100% MC þ GAG100% MC GAG100% ASC ð Þ ´ % M The interaction index was then calculated as the ratio of the GAG measured in the cocultured cell-seeded scaffolds (GAGmeasured) to the GAGexpected. When the interaction index is higher than 1, then chondro- induction is considered to have occurred.20,41 Histology and immuno-histochemistry Biochemical analysis After culture, scaffolds were rinsed in PBS (Invitrogen), and frozen at −80 ° C. For analysis, samples were digested in proteinase K (1 mg/ml in 50 mM Tris with 1 mM EDTA, 1 mM iodoacetamide and 10 mg/ml pepstatin A—all from Sigma-Aldrich) for 16 h at 56 °C. The sulphated GAG content was measured by 1,9 dimethylmethylene blue (DMB) binding (Sigma-Aldrich) using chondroitin sulfate (Sigma-Aldrich) as standard. The DNA content was determined using the CyQuant cell proliferation assay kit (Invitrogen) with supplied bacteriophage λ DNA as standard. Chondro-induction is deﬁned as previously described.41 Based on experimentally measured GAG contents of pure MC seeded scaffolds (GAG100% MCs) and pure ASC seeded scaffolds (GAG100% ASC), the expected total GAG (GAGexpected) in the coculture cell-seeded scaffolds was calculated as a linear function of the proportion (%) of MC using the following equation: Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA Co-culture of MCs and ASCs Cells were co-cultured in 25%:75% and 50%:50% ratios of MCs to ASCs, in addition to controls of each cell type alone. MCs and ASCs were sex matched, but obtained from different donors. After counting, cells were combined in the required ratios, and were carefully seeded on the Type I npj Microgravity (2017) 28 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA Simulated microgravity enhances chondro-induction WM Weiss et al. Simulated microgravity enhances chondro-induction WM Weiss et al. Simulated microgravity enhances chondro-induction WM Weiss et al. REFERENCES Hypertrophy in mesenchymal stem cell chondrogenesis: effect of TGF-beta isoforms and chondrogenic conditioning. Cells Tissues Organs 192, 158–166 (2010). 17. Sanchez-Adams, J. & Athanasiou, K. A. Biomechanics of meniscus cells: regional variation and comparison to articular chondrocytes and ligament cells. Biomech. Model. Mechanobiol. 11, 1047–1056 (2012). 44. Steinert, A. et al. Hypertrophy is induced during the in vitro chondrogenic dif- ferentiation of human mesenchymal stem cells by bone morphogenetic protein- 2 and bone morphogenetic protein-4 gene transfer. Arthritis Res. Ther. 11, R148 (2009). 18. Nakata, K. et al. Human meniscus cell: characterization of the primary culture and use for tissue engineering. Clin. Orthop. Relat. Res. 391, S208–S218 (2001). 19. Adesida, A. B., Grady, L. M., Khan, W. S. & Hardingham, T. E. The matrix-forming phenotype of cultured human meniscus cells is enhanced after culture with ﬁbroblast growth factor 2 and is further stimulated by hypoxia. Arthritis Res. Ther. 8, R61 (2006). 45. Mueller, M. B. & Tuan, R. S. Functional characterization of hypertrophy in chon- drogenesis of human mesenchymal stem cells. Arthritis Rheum. 58, 1377–1388 (2008). 20. Matthies, N. F., Mulet-Sierra, A., Jomha, N. M. & Adesida, A. B. Matrix formation is enhanced in co-cultures of human meniscus cells with bone marrow stromal cells. J. Tissue Eng. Regen. Med. 7, 965–973 (2013). 46. von der Mark, K. et al. Type X collagen synthesis in human osteoarthritic cartilage. Indication of chondrocyte hypertrophy. Arthritis Rheum. 35, 806–811 (1992). 47. van der Kraan, P. M. & van den Berg, W. B. Chondrocyte hypertrophy and osteoarthritis: role in initiation and progression of cartilage degeneration? Osteoarthritis Cartil. 20, 223–232 (2012). 21. Cui, X., Hasegawa, A., Lotz, M. & D’Lima, D. Structured three-dimensional co- culture of mesenchymal stem cells with meniscus cells promotes meniscal phenotype without hypertrophy. Biotechnol. Bioeng. 109, 2369–2380 (2012). 48. D’Angelo, M. et al. MMP-13 is induced during chondrocyte hypertrophy. J. Cell Biochem. 77, 678–693 (2000). 22. Saliken, D. J., Mulet-Sierra, A., Jomha, N. M. & Adesida, A. B. Decreased hyper- trophic differentiation accompanies enhanced matrix formation in co-cultures of outer meniscus cells with bone marrow mesenchymal stromal cells. Arthritis Res. Ther. 14, R153 (2012). 49. Leijten, J. C. et al. Gremlin 1, frizzled-related protein, and Dkk-1 are key regulators of human articular cartilage homeostasis. Arthritis Rheum. 64, 3302–3312 (2012). 23. Aung, A., Gupta, G., Majid, G. & Varghese, S. Osteoarthritic chondrocyte-secreted morphogens induce chondrogenic differentiation of human mesenschymal stem cells. Arthritis Rheum. REFERENCES 14, 1056–1065 (2006). 36. Freed, L. E. & Vunjak-Novakovic, G. Cultivation of cell-polymer tissue constructs in simulated microgravity. Biotechnol. Bioeng. 46, 306–313 (1995). 9. Kon, E. et al. Tissue engineering for total meniscal substitution: animal study in sheep model. Tissue Eng. Part A 14, 1067–1080 (2008). sheep model. Tissue Eng. Part A 14, 1067–1080 (2008) 37. Marsano, A. et al. Use of hydrodynamic forces to engineer cartilaginous tissues resembling the non-uniform structure and function of meniscus. Biomaterials 27, 5927–5934 (2006). 10. Stapleton, T. W. et al. Development and characterization of an acellular porcine medial meniscus for use in tissue engineering. Tissue Eng. Part A 14, 505–518 (2008). 38. Dominici, M. et al. Minimal criteria for deﬁning multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cyto- therapy 8, 315–317 (2006). 11. Baker, B. M., Nathan, A. S., Huffman, G. R. & Mauck, R. L. Tissue engineering with meniscus cells derived from surgical debris. Osteoarthritis Cartil. 17, 336–345 (2009). 39. Horwitz, E. M. et al. Clariﬁcation of the nomenclature for MSC: The International Society for Cellular Therapy position statement. Cytotherapy 7, 393–395 (2005). 12. Zellner, J. et al. Role of mesenchymal stem cells in tissue engineering of meniscus. J. Biomed. Mater. Res. A 94, 1150–1161 (2010). 40. Krawetz, R. J. et al. Synovial ﬂuid progenitors expressing CD90+ from normal but not osteoarthritic joints undergo chondrogenic differentiation without micro- mass culture. PLoS One 7, e43616 (2012). 13. Kon, E. et al. Tissue engineering for total meniscal substitution: animal study in sheep model-results at 12 months. Tissue Eng. Part A 18, 1573–1582 (2012). 14. Cucchiarini, M. et al. Advances in combining gene therapy with cell and tissue engineering-based approaches to enhance healing of the meniscus. Osteoar- thritis Cartil. 24, 1330–1339 (2016). 41. Acharya, C. et al. Enhanced chondrocyte proliferation and mesenchymal stromal cells chondrogenesis in coculture pellets mediate improved cartilage formation. J. Cell. Physiol. 227, 88–97 (2012). 15. W. Niu, et al. Cell-based strategies for meniscus tissue engineering. Stem Cells Int. 2016, 4717184 (2016). 42. Freed, L. E., Langer, R., Martin, I., Pellis, N. R. & Vunjak-Novakovic, G. Tissue engineering of cartilage in space. Proc. Natl. Acad. Sci. USA 94, 13885–13890 (1997). 16. Adams, M. E., Hukins, D. W. L. in Knee Meniscus: Basic and Clinical Foundations (eds Mow, V. C., Arnoczky, S. P., Jackson, D. W.) 15–28 (Raven Press Ltd, New York, 1992). 43. Mueller, M. B. et al. 12 ADDITIONAL INFORMATION 26. Li, Z., Kupcsik, L., Yao, S. J., Alini, M. & Stoddart, M. J. Mechanical load modulates chondrogenesis of human mesenchymal stem cells through the TGF-beta pathway. J. Cell. Mol. Med. 14, 1338–1346 (2010). Competing interests: The authors declare no competing ﬁnancial interests. y 27. Park, S. R., Choi, B. H. & Min, B. H. Low-intensity ultrasound (LIUS) as an innovative tool for chondrogenesis of mesenchymalstem cells (MSCs). Organogenesis 3, 74–78 (2007). Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. 28. Schumann, D. et al. Treatment of human mesenchymal stem cells with pulsed low intensity ultrasound enhances the chondrogenic phenotype in vitro. Bior- heology 43, 431–443 (2006). Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA AUTHOR CONTRIBUTIONS W.M.W. was involved in study design, performed cell culture, experimental assays, data acquisition, and initial manuscript writing. A.M.S. performed cell isolation, cell culture, experimental assays and data acquisition. M.K. performed gene expression assays and associated data analysis. N.M.J. was responsible for specimen procure- ment and was involved in manuscript writing. A.B.A. conceived the study, supervised the study, performed data and statistical analysis and was responsible for writing of the ﬁnal manuscript. All authors read and approved the ﬁnal manuscript. npj Microgravity (2017) 28 ublished in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA Simulated microgravity enhances chondro-induction WM Weiss et al. Simulated microgravity enhances chondro-induction WM Weiss et al. REFERENCES 1. Makris, E. A., Hadidi, P. & Athanasiou, K. A. The knee meniscus: structure-function, pathophysiology, current repair techniques, and prospects for regeneration. Biomaterials 32, 7411–7431 (2011). 29. Verdonk, P., V., R., Almqvist, F., Veys, E. M. & Verbruggen, G. Fibrochondrogenic differentiation of human mesenchymal stem cells. J. Orthop. 1, 61–71 (2009). 30. Yu, B. et al. Simulated microgravity using a rotary cell culture system promotes chondrogenesis of human adipose-derived mesenchymal stem cells via the p38 30. Yu, B. et al. Simulated microgravity using a rotary cell culture system promotes chondrogenesis of human adipose-derived mesenchymal stem cells via the p38 MAPK pathway. Biochem. Biophys. Res. Commun. 414, 412–418 (2011). 2. Andrews, S. H., Adesida, A. B., Abusara, Z. & Shrive, N. G. Current concepts on structure-function relationships in the menisci. Connect. Tissue Res.. https://doi. org/10.1080/03008207.2017.1303489 (2017). structure-function relationships in the menisci. Connect. Tissue Res.. https://doi. org/10.1080/03008207.2017.1303489 (2017). 31. Choi, J. W. et al. Mechanical stimulation by ultrasound enhances chondrogenic differentiation of mesenchymal stem cells in a ﬁbrin-hyaluronic acid hydrogel. Artif. Organs 37, 648–655 (2013). 3. Roos, H. et al. Knee osteoarthritis after meniscectomy: Prevalence of radiographic changes after twenty-one years, compared with matched controls. Arthritis Rheum. 41, 687–693 (1998). 32. Sakao, K. et al. Induction of chondrogenic phenotype in synovium-derived pro- genitor cells by intermittent hydrostatic pressure. Osteoarthritis Cartil. 16 805–814 (2008). 4. McDermott, I. D. & Amis, A. A. The consequences of meniscectomy. J. Bone Jt. Surg. Br. 88, 1549–1556 (2006). 5. Sweigart, M. A. & Athanasiou, K. A. Toward tissue engineering of the knee meniscus. Tissue Eng. JID - 9505538 7, 111–129 (2001). 33. Marsano, A. et al. Spontaneous in vivo chondrogenesis of bone marrow-derived mesenchymal progenitor cells by blocking vascular endothelial growth factor signaling. Stem Cells Transl. Med. https://doi.org/10.5966/sctm.2015-0321 (2016). 6. Buma, P., Ramrattan, N. N., van Tienen, T. G. & Veth, R. P. Tissue engineering of the meniscus. Biomaterials 25, 1523–1532 (2004). 34. Unsworth, B. R. & Lelkes, P. I. Growing tissues in microgravity. Nat. Med. 4, 901–907 (1998). 7. Adams, S. B. Jr, Randolph, M. A. & Gill, T. J. Tissue engineering for meniscus repair. J. Knee Surg. 18, 25–30 (2005). 35. Freed, L. E. & Vunjak-Novakovic, G. Microgravity tissue engineering. In Vitro Cell. Dev. Biol. Anim. 33, 381–385 (1997). 8. Chiari, C. et al. A tissue engineering approach to meniscus regeneration in a sheep model. Osteoarthritis Cartil. 14, 1056–1065 (2006). sheep model. Osteoarthritis Cartil. © The Author(s) 2017 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons. org/licenses/by/4.0/. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, REFERENCES 63, 148–158 (2011). 50. Gazzerro, E. et al. Conditional deletion of gremlin causes a transient increase in bone formation and bone mass. J. Biol. Chem. 282, 31549–31557 (2007). 51. Martin, I., Muraglia, A., Campanile, G., Cancedda, R. & Quarto, R. Fibroblast growth factor-2 supports ex vivo expansion and maintenance of osteogenic precursors from human bone marrow. Endocrinology 138, 4456–4462 (1997). 24. Johnstone, B., Hering, T. M., Caplan, A. I., Goldberg, V. M. & Yoo, J. U. In vitro chondrogenesis of bone marrow-derived mesenchymal progenitor cells. Exp. Cell Res. 238, 265–272 (1998). 52. Bianchi, G. et al. Ex vivo enrichment of mesenchymal cell progenitors by ﬁbro- blast growth factor 2. Exp. Cell Res. 287, 98–105 (2003). 25. Adesida, A. B., Mulet-Sierra, A. & Jomha, N. M. Hypoxia mediated isolation and expansion enhances the chondrogenic capacity of bone marrow mesenchymal stromal cells. Stem Cell Res. Ther. 3, 9 (2012). 53. Solchaga, L. A., Penick, K., Goldberg, V. M., Caplan, A. I. & Welter, J. F. Fibroblast growth factor-2 enhances proliferation and delays loss of chondrogenic potential npj Microgravity (2017) 28 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA Simulated microgravity enhances chondro-induction WM Weiss et al. 13 adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons. org/licenses/by/4.0/. in human adult bone-marrow-derived mesenchymal stem cells. Tissue Eng. Part A 16, 1009–1019 (2010). 54. Di Maggio, N. et al. Fibroblast growth factor-2 maintains a niche-dependent population of self-renewing highly potent non-adherent mesenchymal pro- genitors through FGFR2c. Stem Cells 30, 1455–1464 (2012). 55. Bornes, T., Jomha, N., Mulet-Sierra, A. & Adesida, A. Hypoxic culture of bone marrow-derived mesenchymal stromal stem cells differentially enhances in vitro chondrogenesis within cell-seeded collagen and hyaluronic acid porous scaf- folds. Stem Cell Res. Ther. 6, 84 (2015). Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2017) 28 npj Microgravity (2017) 28
https://openalex.org/W2977617580	https://europepmc.org/articles/pmc6791899?pdf=render	English	null	The role of ant nests in European ground squirrel’s (Spermophilus citellus) post-reintroduction adaptation in two Bulgarian mountains	Biodiversity data jurnal	2,019	cc-by	5,340	The role of ant nests in European ground squirrel’s (Spermophilus citellus) post-reintroduction adaptation in two Bulgarian mountains Maria Kachamakova , Vera Antonova , Yordan Koshev ‡ ‡ ‡ Corresponding author: Maria Kachamakova (maria.n.kachamakova@gmail.com) Academic editor: Ricardo Moratelli Received: 16 Jul 2019 \| Accepted: 09 Sep 2019 \| Published: 07 Oct 2019 Citation: Kachamakova M, Antonova V, Koshev Y (2019) The role of ant nests in European ground squirrel’s (Spermophilus citellus) post-reintroduction adaptation in two Bulgarian mountains. Biodiversity Data Journal 7: e38292. https://doi.org/10.3897/BDJ.7.e38292 Corresponding author: Maria Kachamakova (maria.n.kachamakova@gmail.com) Academic editor: Ricardo Moratelli Received: 16 Jul 2019 \| Accepted: 09 Sep 2019 \| Published: 07 Oct 2019 Citation: Kachamakova M, Antonova V, Koshev Y (2019) The role of ant nests in European ground squirrel’s (Spermophilus citellus) post-reintroduction adaptation in two Bulgarian mountains. Biodiversity Data Journal 7: e38292. https://doi.org/10.3897/BDJ.7.e38292 Corresponding author: Maria Kachamakova (maria.n.kachamakova@gmail.com) Academic editor: Ricardo Moratelli Received: 16 Jul 2019 \| Accepted: 09 Sep 2019 \| Published: 07 Oct 2019 Citation: Kachamakova M, Antonova V, Koshev Y (2019) The role of ant nests in European ground squirrel’s (Spermophilus citellus) post-reintroduction adaptation in two Bulgarian mountains. Biodiversity Data Journal 7: e38292. https://doi.org/10.3897/BDJ.7.e38292 © Kachamakova M et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Biodiversity Data Journal 7: e38292 doi: 10.3897/BDJ.7.e38292 Short Communications The role of ant nests in European ground squirrel’s (Spermophilus citellus) post-reintroduction adaptation in two Bulgarian mountains Maria Kachamakova , Vera Antonova , Yordan Koshev ‡ Institute of Biodiversity and Ecosystem Research at Bulgarian Academy of Sciences, Sofia, Bulgaria Corresponding author: Maria Kachamakova (maria.n.kachamakova@gmail.com) Academic editor: Ricardo Moratelli Received: 16 Jul 2019 \| Accepted: 09 Sep 2019 \| Published: 07 Oct 2019 Citation: Kachamakova M, Antonova V, Koshev Y (2019) The role of ant nests in European ground squirrel’s (Spermophilus citellus) post-reintroduction adaptation in two Bulgarian mountains. Biodiversity Data Journal 7: e38292. https://doi.org/10.3897/BDJ.7.e38292 ‡ ‡ ‡ Biodiversity Data Journal 7: e38292 doi: 10.3897/BDJ.7.e38292 Short Communications Biodiversity Data Journal 7: e38292 doi: 10.3897/BDJ.7.e38292 Short Communications Short Communications Introduction The European ground squirrel or the souslik (Spermophilus citellus), is a rodent inhabiting pastures and meadows in Central and South-Eastern Europe. It constructs complex burrows up to 2 m deep (Brinkmann 1951, Ruzič 1978) where it hides, rests, reproduces and hibernates. During the last decades, the souslik's populations throughout its entire range are declining mainly because of habitat destruction and degradation (Coroiu et al. 2008). Currently it is listed as “Vulnerable” in the IUCN Red List and conservation measures have been taken all over its range. They often include conservation reintroductions – reestablishing the species in places where it was extinct. Most of the ground squirrel reintroductions in Bulgaria were organised by nature parks directorates and implemented in the mountains (Koshev et al. 2019). Playing a key role in the ecosystem, the souslik lives in close interaction with several invertebrate species inhabiting its burrows - scarabid beetles (Carpaneto et al. 2011) and ectoparasites such as ticks (Honzakova et al. 1980) and fleas (Ryba et al. 1980). Some of these relationships are highly specific and not always reappear after reintroduction (Lindtner et al. 2019). Shabanova et al. (2014) also reported ants, as well as digger wasps and beetles, using sousliks’ burrows. The possible symbiosis with ants is especially interesting as they are also key ecosystem species, soil engineers and dominate in almost every terrestrial habitat. They may change the landscape by creating networks of soil macropores and building stationary, perennial mounds with altered physico-chemical structure and change the availability of resources for plants and animals (Alonso 2000, Ehrle et al. 2017). The nest’s structure (tunnels and chambers) allows water and gas circulation (Folgarait 1998). All these characteristics could be beneficial for the ground squirrels and possibly be linked to the conservation practice. Hapl et al. (2006) mentioned that the mounds of Lasius flavus could be used for initial hiding places in Slovakia for disorientated reintroduced sousliks, but the authors did not provide any references or further explanations. Interactions between ants and small mammals, such as rodents and shrews, have rarеly been documented (Panteleeva et al. 2016, Gilev and Nakonechny 2010, Scherba 1965, Mironov 1986). Often they concern the competition between ants and rodents for seeds (e.g. Brown et al. 1979). Vygonyailova (2011) reports the superficial and surrounding mound’s material of red wood ants is attractive food for rodents, possibly because of the higher organic components. Keywords anthills, Lasius flavus, mountain, reintroduction, Spermophilus citellus, symbiosis anthills, Lasius flavus, mountain, reintroduction, Spermophilus citellus, symbiosis Abstract The European ground squirrel (Spermophilus citellus) is a vulnerable species, whose populations are declining throughout its entire range in Central and South-Eastern Europe. To a great extent, its conservation depends on habitat restoration, maintenance and protection. In order to improve the conservation status of the species, reintroductions are increasingly applied. Therefore, researchers focus their attention on factors that facilitate these activities and contribute to their success. In addition to the well-known factors like grass height and exposition, others, related to the underground characteristics, are more difficult to evaluate. The presence of other digging species could help this evaluation. Here, we present two reintroduced ground squirrel colonies, where the vast majority of the burrows are located in the base of anthills, mainly of yellow meadow ant (Lasius flavus). This interspecies relationship offers numerous advantages for the ground squirrel and is mostly neutral for the ants. The benefits for the ground squirrel, including reduced energy demand for digging, as well as additional surveillance and hiding places available, could greatly enhance the post-reintroduction adaptation process. © Kachamakova M et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Kachamakova M et al Kachamakova M et al 2 Materials and methods Reintroduced colonies of ground squirrel in Bulgaria are regularly monitored. In 2017, it was observed that the majority of the holes were located in the base of anthills in two of these colonies (20.06.2017 in Bulgarka Nature Park and 30.06.2017 in Vrachanski Balkan Nature Park). The observations were confirmed in June 2018. On 11.06.2019 (Bulgarka Nature Park) and on 29.05.2019 (Vrachanski Balkan Nature Parka), the colonies were investigated in detail – multiple transects were designed in order to count and map the burrows and determine what percentage of them is associated with ant nests. The transects were chosen randomly, aiming to cover the entire area of the colonies (Suppl. materials 1, 2). When no more burrows were found, the transect was finished. Both holes of S. citellus and Microtus sp. were observed (identification based on holes' size and size and the surrounding excrement) but only the first type was considered in the current study. Each ant nest with a burrow was investigated - a small hole was made at the nest’s top and 3 to 5 ants were taken, fixed in 75% alcohol and identified under a microscope. The walls of the burrow at the entry hole were scraped, using a small garden shovel in order to check if the ants were present in the burrow’s interior. The behaviour of the ground squirrels was observed with binoculars for several minutes before the start of the transects. Located on the northern slopes of Central Stara Planina mountain, Bulgarka Nature Park occupies 236.9 km with an average altitude of 870 m. Most of the park area (89%) is covered by deciduous forests and only the highest parts, close to the park's borders, are meadows. Ground squirrel colonies have been previously documented there (V. Popov – personal data for 2003, Koshev 2013). In 2013, the park’s directorate started a reintroduction programme in one such locality - Karamandra (42.7410N; 25.2510E, 1410 m a.s.l.). For the next three years, 149 animals were translocated there (Koshev et al. 2019). 2 The Vrachanski Balkan Nature Park is located in western Bulgaria, in the Stara Planina mountain. Its area is 288.03 km and the average altitude is 700 m (Bechev and Georgiev 2016). The species disappeared at the end of the 1950s due to a ban on transhumance (G. Stoyanov - unpubl. record). Introduction Taking into account these scare data, our goal is to report a confirmed case of an interspecific relationship between souslik and ants and to discuss its implications in the conservation reintroduction practice. The role of ant nests in European ground squirrel’s (Spermophilus citellus) p ... 3 Materials and methods In 2013, park's authorities started a ground squirrel reintroduction project near Parshevitsa hut (43.1379 N; 23.4855 E, 1420 m a.s.l.) where there were historical data of its presence in the past. Until 2016, 132 individuals in total were translocated in order to form a new colony (Koshev et al. 2019). 2 The habitat in both locations is similar - mesophyte mountain meadows with diverse vegetation including both dicotyledonous and monocotyledonous plants. It is on a southern slope with a limestone base. A common species covering most of the ant nests is the Thymus sp. A moderate level of livestock grazing occurs (cattle in Bulgarka and horses in Vrachanski Balkan). Kachamakova M et al Kachamakova M et al 4 Results The number of burrows were from one (97%) to three per nest (Table 1). a b c d Figure 1. L. flavus nests in two reintroduced S. citellus colony. a: Nests of L. flavus in the colony in Bulgarka Nature Park b: Nest of L. flavus with burrow in the base in Bulgarka Nature Park c: The reintroduced colony in Vrachanski Balkan Nature Park - a general view d: Nest of L. flavus with burrow in Vrachanski Balkan Nature Park The role of ant nests in European ground squirrel’s (Spermophilus citellus) p ... 5 The role of ant nests in European ground squirrel’s (Spermophilus citellus) p ... 5 b a a a c d d d gu e . L. flavus nests in two reintroduced S. citellus colony. a: Nests of L. flavus in the colony in Bulgarka Nature Park b: Nest of L. flavus with burrow in the base in Bulgarka Nature Park c: The reintroduced colony in Vrachanski Balkan Nature Park - a general view d: Nest of L. flavus with burrow in Vrachanski Balkan Nature Park In the Vrachanski Balkan colony, 84 active burrows were counted, 59 of them (70.2%) being in the bаse of anthills (Fig. 1c, d). In some nests, Myrmica scabrinodis or Tetramorium sp. were co-existing with the yellow meadow ant (Table 1). There were cases when abandoned L. flavus' nests were entirely occupied by other ant species. Burrows were also found near nests of the dominant protected ant species Formica pratensis, but not in it. The number of burrows were from one (97%) to three per nest (Table 1). In the Vrachanski Balkan colony, 84 active burrows were counted, 59 of them (70.2%) being in the bаse of anthills (Fig. 1c, d). In some nests, Myrmica scabrinodis or Tetramorium sp. were co-existing with the yellow meadow ant (Table 1). There were cases when abandoned L. flavus' nests were entirely occupied by other ant species. Burrows were also found near nests of the dominant protected ant species Formica pratensis, but not in it. The number of burrows were from one (97%) to three per nest (Table 1). At both locations, the active nests of Lasius flavus were covered with vegetation, mainly Thymus sp. (in accordance with Bernard 1968). The mounds were with diameter between 30 cm and 150 cm (average 89 cm) and height between 8 cm and 50 cm (average 25 cm). Results In Bulgarka Nature Park, 82.3% of all 132 burrows mapped were at the base of ant mounds (Fig. 1a, b). The most abundant ant species was Lasius flavus (65% active L. flavus nests of all nests with burrows) (Table 1). The rest of the nests with burrows were probably also built by the yellow meadow ant but currently abandoned - empty or occupied by other ant species. There was usually one (95%) and rarely two holes at one nest (Table 1). Nests of Formica pratensis were also observed, but there were no souslik burrows in the immediate vicinity. The area where the colony was detected in 2017 is 750 m distant from the release site and is densely covered with Lasius flavus nests. On the release site, only two ant nests were found. Bulgarka Nature Park Vrachanski Balkan Nature Park Total number of burrows mapped 132 84 Transect length 5043 m 4398 m Percentage of burrows in ant nests by ant species: Lasius flavus nests 64.7% 57.1% Formica cunicularia nests - 1.2% Formica rufibarbis nests - 1.2% Abandoned or very old nests (more than 10 years) of L. flavus occupied by Lasius alienus - 6% Abandoned or very old nests (more than 10 years) of L. flavus occupied by Formica fusca - 3.5% Abandoned or very old nests (more than 10 years) of L. flavus occupied by Myrmica scabrinodis 2.9% - Abandoned or very old nests (more than 10 years) of L. flavus occupied by Tetramorium sp. 2.9% - Nests of Lasius flavus where it was found coexisting with Myrmica scabrinodis 1.5% - Nests of Lasius flavus where it was found coexisting with Tetramorium sp. 1.5% - Empty nests 8.8% 1.2% Total percentage of burrows in ant nests 82.3% 70.2% Ant nests with one hole 95% 97% Ant nests with two or three holes 5% 3% Table 1. Types of ant nests with burrows mapped in 2019. In the Vrachanski Balkan colony, 84 active burrows were counted, 59 of them (70.2%) being in the bаse of anthills (Fig. 1c, d). In some nests, Myrmica scabrinodis or Tetramorium sp. were co-existing with the yellow meadow ant (Table 1). There were cases when abandoned L. flavus' nests were entirely occupied by other ant species. Burrows were also found near nests of the dominant protected ant species Formica pratensis, but not in it. Discussion As the reintroduced animals were taken from the plain - 480 m a.s.l. for Bulgarka and 100 m a.s.l. for Vrachanski Balkan (Koshev et al. 2019), the new habitat presented multiple difficulties for their adaptation. Artificial burrows (at least 5 per released individual) were prepared but the entire burrow system had to be re-established. In these conditions, each facilitating factor was crucial. The presence of the anthills should be considered as such a factor. The soil into and under the nest is more friable and drained and the vegetation on the nest is not so dense and tough. These factors greatly reduce the digging effort for initiating the tunnel. Not only the physical characteristics in and around the mound are specific, but also the chemical constituents (Bierbaß et al. 2015) - pH is frequently higher (King and Woodell 1975) and the microbiological - bacteria, fungi, actinomycetes and micro-arthropod communities differ (Sharma and Sumbali 2013). The last may have an indirect impact creating a cleaner micro-environment. We also know that the soil in the mounds, constructed by the ground squirrels themselves during excavation activities, has a higher pH and reduced microbiological acivity, diversity and heterogenity (Lindtner et al. 2019b). On the other hand, the formic acid, an organic substance of ants (subfamily Formicinae, including Lasius genus) mainly used for defence, trail marking and predation (Hölldobler and Wilson 1990), has repellent properties. Some authors reported about vertebrates which use formic acid for repellent (Falótico et al. 2007). The animals rub their fur or plumage with ants (so-called “anting” process) to avoid ticks (Acari) and other ectoparasites. In addition, as part of the micro-relief of the habitat, the ant nests provide two additional benefits for the souslik. They are up to 50 cm tall and, when digging in their base, the animals are partly protected from predators, especially from raptors. Gedeon et al. 2012 showed that, in the new environment, the newly reintroduced sousliks prefer patches with higher grass cover that potentially also have a shelter role. On the other hand, the tops of the mounds of L. flavus are thick, covered with vegetation and there are no ants on the surface. In consequence, the S. citellus use them as watchtowers. In both locations, surveillance behaviour was observed on the top of the ant nests during the observation session. Results Ants were not detected in the burrow’s interior after scraping. Surveillance behaviour of S. citellus (including juveniles) was observed at the top of the ant nests at each observation session done before the start of the transects. Kachamakova M et al Kachamakova M et al 6 Discussion Such features are especially important during the summer when, despite livestock grazing, the grass could become as tall as 50 - 60 cm and the visibility for the ground squirrels drops. Considering all these positive effects, we could at least partly explain the settling of the Bulgarka colony 750 m distant from the release site with searched cohabitation with ants. The average nests’ dimensions showed that they are more than 10 years old - the nest’s diameter increases by 7 cm/year and the height by 2.2 cm/year (Waloff and Blackith 1962). Therefore, they were present well before the reintroduction activities started. This was also confirmed by the park’s authorities. These multiple benefits are not associated with conflict with the ants. Potential conflict seems probable with the territorial ants Formica pratensis, but not with the peaceful L. flavus. Scherba (1965) reports a case when the ants do not avoid and do not attack the voles and both live in a “myrmecocole” interaction. The author considers the microhabitat formed in ants nests is used by the voles only when the most suitable habitats are already The role of ant nests in European ground squirrel’s (Spermophilus citellus) p ... 7 7 occupied. Trophic competition may arise with the red wood ants for small invertebrates (snails, beetles, worms etc.) and with the harvester ants for seeds (O'Dowd and Hay 1980, Brown et al. 1979, Abramsky 1983, Davidson et al. 1984). According to Panteleeva et al. (2016), the red wood ants may disturb the digging activity of rodents and may compete for small invertebrates. The case with yellow meadow ant is different. L. flavus is feeding underground with aphids, excretions of root-living aphids and coccids (King and Woodell 1975). Their nests are about 15 - 20 cm deep (Steinmeyer et al. 2012) so the souslik tunnel system is developed below it. As there were no ants present even in the upper part of the tunnel, we suggest that the ants’ structures, destroyed during the initial digging, are quickly sealed and the direct interactions between the two species are limited. There are scarce data on the presence of ants in the S. citellus' diet - Formicidae species (Tetramorium caespidum, Lasius alienus, Tapinoma erraticum, Solenopsis fugax, div. Puppen, Myrmica scabrinodis, M. Discussion rugulosa) have been identified in gut contents and droppings, but their share is not considerable - 11% of all insects found in the diet (Herzig- Straschil 1976). Nevertheless, we did not find any trace of scratching and digging on the nest tops - the vegetation was intact and the nests continue to be viable. It is possible that the ants have even some indirect benefits from the ground squirrel presence, related to general improvement of the ecosystem functioning and diversity (Dundas et al. 2018). Based on the observations described, we would conclude that the relationship between the two species (Spermphilus citellus and Lasius flavus) could be considered as commensalism (see Dickman 1992). We suggest that the souslik has numerous benefits from cohabitation with the ants and, for the ants, the interaction is neutral. Panteleeva et al. (2016) assumed that there is “coadaptation” (symbiosis, commensalism type) between the ants and the small rodents, that were using the same microhabitats. In the investigated case, it is also possible that the interactions have been enforced by the reintroduction event, as we have not observed it in the natural colonies until now. With regard to the conservation status of the S. citellus, the mentioned benefits could have a role in helping the conservation efforts. Hapl et al. (2006) describe a hard-release method when additional cover of branches was put around the Lasuis flavus’ nests so that the sousliks could easily and safely dig their first holes after translocation. Nevertheless, nothing is reported about the scientific reasons on which this method is based, neither about the percentage of successful results. Therefore, our study provides a robust proof for the suitability of this practice. In addition, according to our observations, the yellow meadow ant and the souslik have common microhabitat preferences - in the investigated cases, the areas with anthills coincided with the souslik colony. The ant nests of Lasius flavus are also long-lasting structures - they can be active for between 22 and 150 years (Dostal 2005, Steinmeyer et al. 2012) and are densely situated - up to 2500 nests/ha (Elmes 1991). This means that the presence of Lasuis flavus mounds could serve as additional confirmation for the habitat suitability for S. citellus reintroduction. In conclusion, we state that the reported ecological findings could be beneficial for planning and implementation of future ground squirrel reintroduction projects. Kachamakova M et al Kachamakova M et al 8 Acknowledgements The study was supported by the Program for the Support of Young Researchers and PhD Students at the Bulgarian Academy of Sciences (Grant no 17-110/2017). The activities implemented in the NPs were funded by OP Environment 2007–2013. We warmly thank to Daniela Borisova (NP Vrachanski Balkan), Yasen Mutafchiev, V.R. Popov and Dimitar Ragyov for the precious support during the field work. References • Abramsky Z (1983) Experiments on seed predation by rodents and ants in the Israeli desert. Oecologia 57 (3): 328‑332. • Alonso LE (2000) Ants as indicators of diversity. In: Agosti D, Majer JD, Alonso LE, Schultz T (Eds) Ants: standard methods for measuring and monitoring biodiversity. Biological diversity handbook series. Smithsonian Institution Press, Washington. • Alonso LE (2000) Ants as indicators of diversity. In: Agosti D, Majer JD, Alonso LE, Schultz T (Eds) Ants: standard methods for measuring and monitoring biodiversity. Biological diversity handbook series. Smithsonian Institution Press, Washington. • Bechev D, Georgiev D (2016) Geographic features of Vrachanska Planina Mountains. ZooNotes, Suppl 3: 13‑16. URL: http://web.uni-plovdiv.bg/bechev/Bechev_pdfs/Geographic %20Features_Vrachanski%20Balkan.pdf • Bechev D, Georgiev D (2016) Geographic features of Vrachanska Planina Mountains. ZooNotes, Suppl 3: 13‑16. URL: http://web.uni-plovdiv.bg/bechev/Bechev_pdfs/Geographic %20Features_Vrachanski%20Balkan.pdf • Bierbaß P, Gutknecht JL, Michalzik B (2015) Nest-mounds of the yellow meadow ant ( Lasius flavus) at the “Alter Gleisberg”, Central Germany: Hot or cold spots in nutrient cycling? Soil Biology and Biochemistry 80: 209‑217. • Bierbaß P, Gutknecht JL, Michalzik B (2015) Nest-mounds of the yellow meadow ant ( Lasius flavus) at the “Alter Gleisberg”, Central Germany: Hot or cold spots in nutrient cycling? Soil Biology and Biochemistry 80: 209‑217. • Brinkmann M (1951) Uber die Zieselkolonien in Oberschlesien. Bonner Zoologische Beiträge 3-4: 191‑216. • Brinkmann M (1951) Uber die Zieselkolonien in Oberschlesien. Bonner Zoologische Beiträge 3-4: 191‑216. • Brown JH, Davidson DW, Reichman OJ (1979) An experimental study of competition between seed-eating desert rodents and ants. American Zoologist 19 (4): 1129‑1143. Brown JH, Davidson DW, Reichman OJ (1979) An experimental study of competition between seed-eating desert rodents and ants. American Zoologist 19 (4): 1129‑1143. Carpaneto GM, Mazziotta A, Pittino R, Luiselli L (2011) Exploring co-extinction correlates: The effects of habitat, biogeography and anthropogenic factors on ground squirrels-dung beetles associations. Biodiversity and Conservation 20 (13): 3059‑3076. https:// • Carpaneto GM, Mazziotta A, Pittino R, Luiselli L (2011) Exploring co-extinction correlates: The effects of habitat, biogeography and anthropogenic factors on ground squirrels-dung beetles associations. Biodiversity and Conservation 20 (13): 3059‑3076. https:// doi.org/10.1007/s10531-011-0162-5 • Coroiu C, Kryštufek B, Vohralík V, Zagorodnyuk I (2008) Spermophilus citellus. The IUCNRed list of threatened species 2008: e.T20472A9204055. https://www.iucnredlist.org/ species/ 20472/9204055 • Davidson DW, Inouye RS, Brown JH (1984) Granivory in a desert ecosystem: experimental evidence for indirect facilitation of ants by rodents. Ecology 65 (6): 1780‑1786. References • Dickman CR (1992) Commensal and mutualistic interactions among terrestrial vertebrates. Trends in Ecology & Evolution 7 (6): 194‑197. • Dostal P (2005) Effect of three mound-building ant species on the formation of soil seed bank in mountain grassland. Flora-Morphology, Distribution. Functional Ecology of Plants 200 (2): 148‑158. • Dostal P (2005) Effect of three mound-building ant species on the formation of soil seed bank in mountain grassland. Flora-Morphology, Distribution. Functional Ecology of Plants 200 (2): 148‑158. • Dundas SJ, Hopkins AJ, Ruthrof KX, Tay NE, Burgess TI, Hardy GESJ, Fleming PA (2018) Digging mammals contribute to rhizosphere fungal community composition and seedling growth. Biodiversity and Conservation 27 (12): 3071‑3086. https://doi.org/10.1007/ s10531-018-1575-1 • Ehrle A, Andersen AN, Levick SR, Schumacher J, Trumbore SE, Michalzik B (2017) Yellow-meadow ant (Lasius flavus) mound development determines soil properties and • Ehrle A, Andersen AN, Levick SR, Schumacher J, Trumbore SE, Michalzik B (2017) Yellow-meadow ant (Lasius flavus) mound development determines soil properties and The role of ant nests in European ground squirrel’s (Spermophilus citellus) p ... 9 growth responses of different plant functional types. European Journal of Soil Biology 81: 83‑93. • Elmes GW (1991) Ant colonies and Environmental disturbance. In: Meadows P, Meadows A (Eds) The Environmental Impact of Burrowing Animals and Animal Burrows. Symposium growth responses of different plant functional types. European Journal of Soil Biology 81: 83‑93. • Elmes GW (1991) Ant colonies and Environmental disturbance In: Meadows P Meadows • Elmes GW (1991) Ant colonies and Environmental disturbance. In: Meadows P, Meadows A (Eds) The Environmental Impact of Burrowing Animals and Animal Burrows. Symposium 63 Zoological Society of London. Clarendon Press, Oxford, 15-32 pp. • Elmes GW (1991) Ant colonies and Environmental disturbance. In: Meadows P, Meadows A (Eds) The Environmental Impact of Burrowing Animals and Animal Burrows. Symposium 63 Zoological Society of London. Clarendon Press, Oxford, 15-32 pp. • Falótico T, Labruna MB, Verderane MP, De Resende BD, Izar P, Ottoni EB (2007) Repellent efficacy of formic acid and the abdominal secretion of carpenter ants (Hymenoptera: Formicidae) against Amblyomma ticks. Ixodidae). Journal of medical 44 (4): 718‑721. • Folgarait PJ (1998) Ant biodiversity and its relationship to ecosystem functioning: a review. Biodiversity & Conservation 7 (9): 1221‑1244. • Gedeon CI, Boross G, Németh A, Altbäcker V (2012) Release site manipulation to favour European ground squirrel Spermophilus citellus translocations: translocation and habitat manipulation. Wildlife Biology 18 (1): 97‑104. References Journal of Animal Ecology 421‑437. • Waloff N, Blackith RE (1962) The growth and distribution of the mounds of Lasius flavus (Fabricius) (Hym: Formicidae) in Silwood Park, Berkshire. Journal of Animal Ecology 421‑437. References https://doi.org/10.2981/10-124 • Gilev AV, Nakonechny NV (2010) Ants of the ordinary mole burrow complex in the Western Sibiria forest zone. The Bulletin of KrasGAU 6: 85‑89. [In Russian]. • Hapl E, Ambros M, Oleksak M, Adamec M (2006) Suslik (Spermophilus citellus) reintroduction in Slovakia. Guidelines. State Nature Conservancy of the Slovak Republic, Banská Bystrica, 28 pp. • Herzig-Straschil B (1976) Nahrung und Nahrungserwerb des Ziesels. Acta Theriologica 21: 131‑139. • Hölldobler B, Wilson EO (1990) The ants. Springer-Verlag, Berlin, 732 pp. • Honzakova E, Cerný V, Daniel M, Dusbabek F (1980) Development of the tick Ixodes laguri Ol. in the nests of the European suslik Citellus citellus (L.). Folia Parasitologica 27 (1): 71‑75. • King TJ, Woodell SR (1975) The use of the mounds of Lasius fiavus in teaching some principles of ecological investigation. Journal of Biological Education 9 (3-4): 109‑113. • Koshev Y (2013) Reports on distribution and determining conservation status of European ground squirrel (Spermophilus citellus) in Nature 2000 sites BG0000113 “Vitosha”, BG0000166 “Vrachanski Balkan”, BG0000399 “Bulgarka”, BG0000117 “Kotlenska Planina”, BG0000164 “Sinite Kamani”, BG0001389 ”Sredna gora”, BG0000219 “Derventski vazvishenia 2”. Project "Mapping and determining conservation status of mammals in NATURA 2000 network in Bulgaria 2011-2013". Founded by MOEW-Bulgaria and Operational Programme Environment 2007–2013. URL: http:// nat ra2000 moe go ernment bg/Home/Nat ra2000ProtectedSites • Koshev Y, Kachamakova M, Arangelov S, Ragyov D (2019) Translocations of European ground squirrel (Spermophilus citellus) along altitudinal gradient in Bulgaria – an overview. Nature Conservation 35: 63‑95. https://doi.org/10.3897/natureconservation.35.30911 • Koshev Y, Kachamakova M, Arangelov S, Ragyov D (2019) Translocations of European ground squirrel (Spermophilus citellus) along altitudinal gradient in Bulgaria – an overview. Nature Conservation 35: 63‑95. https://doi.org/10.3897/natureconservation.35.30911 • Lindtner P, Šoltís M, Kubovčík V (2019a) Translocation of keystone species may not mean translocation of keystone effect. European Journal of Wldlife Research 65 (2): 24. • Lindtner P, Gömöryová E, Gömöry D, Stašiov S, Kubovčík V (2019b) Development of physico-chemical and biological soil properties on the European ground squirrel mounds. Geoderma 339: 85‑93. https://doi.org/10.1016/j.geoderma.2018.12.043. Mironov AD (1986) The influence of forest ants on the spatial distribution of voles. The Bulletin of Leningrad State University 1: 95‑96. [In Russian]. O'Dowd DJ, Hay ME (1980) Multalism between harvester ants and a desert ephemeral: seed escape from rodents. Ecology 61 (3): 531‑540. 10 Kachamakova M et al • Panteleeva SN, ZhI R, Sinkova OB (2016) Spatio-ethologicals apects of interactions between small mammals and wood ants. Suppl. material 1: Transect and burrows in Vrachanski Balkan Nature Park Authors: Maria kachamakova Data type: map Download file (145.49 kb) Supplementary materials Suppl. material 1: Transect and burrows in Vrachanski Balkan Nature Park Authors: Maria kachamakova Data type: map Download file (145.49 kb) Suppl. material 2: Transect and burrows in Bulgarka Nature park Authors: Maria Kachamakova Data type: map Download file (159.32 kb) Suppl. material 1: Transect and burrows in Vrachanski Balkan Nature Park References Journal of General Biology 77 (5): 346‑358. [In Russian]. • Ruzič A (1978) Citellus citellus (Linnaeus, 1766)—der Oder das Europäische Ziesel. In: Niethammer J, Krap F (Eds) Handbuch der Säugetiere Europas\|. Bd. 1, Nagetiere I (Sciuridae, Castoridae, Gliridae, Muridae). Akademische Verlagsgesellschaft Wiesbaden, Wiesbaden. • Ryba J, Simonova V, Daniel M, Cerny V (1980) Changes in the flea fauna in the nests of the European suslik (Citellus citellus L.) under conditions of field experiment. Folia Parasitologica 27 (3): 281‑287. • Ryba J, Simonova V, Daniel M, Cerny V (1980) Changes in the flea fauna in the nests of the European suslik (Citellus citellus L.) under conditions of field experiment. Folia Parasitologica 27 (3): 281‑287. • Scherba G (1965) Observations on Microtus nesting in ant mounds. Psyche: A Journal of Entomology 72 (2): 127‑132. Scherba G (1965) Observations on Microtus nesting in ant mounds. Psyche: A Journal of Entomology 72 (2): 127‑132. • Shabanova NP, Verba ML, Bykov AV (2014) The effect of digging activity of little souslik on soils of the first terrace of Khaki Sor in the Botkul’sk-Khaki depression. Eurasian Soil Science 47 (3): 141‑152. https://doi.org/10.1134/s1064229314030065 Shabanova NP, Verba ML, Bykov AV (2014) The effect of digging activity of little souslik on soils of the first terrace of Khaki Sor in the Botkul’sk-Khaki depression. Eurasian Soil • Shabanova NP, Verba ML, Bykov AV (2014) The effect of digging activity of little souslik on soils of the first terrace of Khaki Sor in the Botkul’sk-Khaki depression. Eurasian Soil Science 47 (3): 141‑152. https://doi.org/10.1134/s1064229314030065 Science 47 (3): 141‑152. https://doi.org/10.1134/s1064229314030065 • Sharma V, Sumbali G (2013) An overview of the symbiotic interaction between ants, fungi and other living organisms in ant-hill soils. International Journal of Environmental Sciences 4 (3): 432‑443. • Sharma V, Sumbali G (2013) An overview of the symbiotic interaction between ants, fungi and other living organisms in ant-hill soils. International Journal of Environmental Sciences 4 (3): 432‑443. • Steinmeyer C, Pennings PS, Foitzik S (2012) Multicolonial population structure and nestmate recognition in an extremely dense population of the European ant Lasius flavus . Insectes Sociaux 59 (4): 499‑510. • Vygonyailova OB (2011) Geophagy and preference for ant-hill materials in rodents. Izvestia Penzenskogo Gosudarstvennogo Pedagogicheskogo Universiteta Imeni V.G. Belinskogo, Natural Sciences 25: 330‑336. [In Russian]. g • Waloff N, Blackith RE (1962) The growth and distribution of the mounds of Lasius flavus (Fabricius) (Hym: Formicidae) in Silwood Park, Berkshire. Suppl. material 2: Transect and burrows in Bulgarka Nature park Authors: Maria Kachamakova Data type: map Download file (159.32 kb) Authors: Maria Kachamakova Data type: map Download file (159.32 kb)
https://openalex.org/W2053898782	https://europepmc.org/articles/pmc3891621?pdf=render	English	null	Radiobiology of Radiosurgery for the Central Nervous System	BioMed research international	2,013	cc-by	8,080	Antonio Santacroce,1 Marcel A. Kamp,2 Wilfried Budach,3 and Daniel Hänggi2 1 Departments of Radiation Oncology and Neurosurgery, Medical Faculty, Heinrich Heine University, Moorenstraße 5, 40225 D¨usseldorf, Germany 2 f y 2 Department of Neurosurgery, Medical Faculty, Heinrich Heine University, Moorenstraße 5, 40225 D¨usseldorf, Germany 3 Department of Radiation Oncology, Medical Faculty, Heinrich Heine University, Moorenstraße 5, 40225 D¨usseldorf, Germany Correspondence should be addressed to Daniel H¨anggi; daniel.haenggi@uni-duesseldorf.de Received 10 May 2013; Accepted 19 September 2013 Received 10 May 2013; Accepted 19 September 2013 Academic Editor: Ana Maria Tari Academic Editor: Ana Maria Tari Copyright © 2013 Antonio Santacroce 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. According to Leksell radiosurgery is defined as “the delivery of a single, high dose of irradiation to a small and critically located intracranial volume through the intact skull.” Before its birth in the early 60s and its introduction in clinical therapeutic protocols in late the 80s dose application in radiation therapy of the brain for benign and malignant lesions was based on the administration of cumulative dose into a variable number of fractions. The rationale of dose fractionation is to lessen the risk of injury of normal tissue surrounding the target volume. Radiobiological studies of cell culture lines of malignant tumors and clinical experience with patients treated with conventional fractionated radiotherapy helped establishing this radiobiological principle. Radiosurgery provides a single high dose of radiation which translates into a specific toxic radiobiological response. Radiobiological investigations to study the effect of high dose focused radiation on the central nervous system began in late the 50s. It is well known currently that radiobiological principles applied for dose fractionation are not reproducible when single high dose of ionizing radiation is delivered. A review of the literature about radiobiology of radiosurgery for the central nervous system is presented. Hindawi Publishing Corporation BioMed Research International Volume 2013, Article ID 362761, 9 pages http://dx.doi.org/10.1155/2013/362761 Hindawi Publishing Corporation BioMed Research International Volume 2013, Article ID 362761, 9 pages http://dx.doi.org/10.1155/2013/362761 Hindawi Publishing Corporation BioMed Research International Volume 2013, Article ID 362761, 9 pages http://dx.doi.org/10.1155/2013/362761 2. Radiosurgery and Fractionated Stereotactic Radiotherapy (1) repair capacity of cells after sublethal damage radia- tion induced, The use of “stereotactic coordinates” implies the support of three-dimensional mapping techniques to perform a medical procedure which might be applied to radiation therapy and surgery in particular of the central nervous system. Stereotac- tic radiation is a highly precise technique to deliver conformal radiation to a small target volume, either neoplastic or non- neoplastic sparing surrounding tissue by radiation exposure. This radiation if applied as high single dose fraction, in most of cases of photons, is defined as radiosurgery (RS), also named by many authors as stereotactic radiosurgery (SRS). If this radiation dose is delivered using more than one dose fraction, always with support of stereotactic coordinates, it is defined as fractionated stereotactic radiotherapy (FSRT). Stereotactic fractionated radiotherapy should not be con- fused with intensity modulated radiation therapy (IMRT). In this case a cumulative radiation dose is, like for 3D con- formal radiation, applied to a given target volume with dose fractionation but without supply of stereotactic coordinates. Furthermore individual treatment beams irradiate only part of the target at the time: it assigns nonuniform intensities (i.e., weights) to tiny subdivision of irradiation beams defined as “beamlets.”h (2) repopulation of surviving tumor stem cells during fractionated radiotherapy, (3) redistribution of cells between the cell cycle after radi- ation injury in equally distributed radiation sensitive and resistant subpopulations, (4) reoxygenation of hypoxic tumor cells after repeated radiation exposure. The radiosensitivity of cells is inversely proportional to the hypoxic cell rate. The application of a dose fraction produces death of oxygenated tumor cells followed by oxygenation of hypoxic cells now more sensitive to the following dose fraction. These radiobiological principles are based on “in vitro” experiments applying a dose of ionizing radiation, usually photons, to cell in vitro cultivated, for example, fibroblasts [14, 15]. The results of these experimental models together with clinical experience with three-dimensional conformal radiotherapy brought the physicians to conclude that dose fractionation lessens the risk of injury of normal tissues and thus the rate of side effects [16]. f It has been reported that the relationship between radi- ation dose to desired outcome and undesired effect can be represented by a sigmoid dose-response curve [17]. 3. Radiobiological Principles Together with radiosurgery as neurosurgical tool, newly developed high precision radiotherapy techniques, that is, stereotactic fractionated radiation and intensity modulated radiotherapy (IMRT), are now steadily establishing their role in definitive cancer therapy. While advantages of the new radiation techniques are evident upon physical grounds a few radiobiological issues remain unresolved regarding the evaluation of radiation doses employed in these treatment modalities [6]. 3.1. 4Rs of Radiobiology. The goal of any medical intervention is to reach the highest rate of clinical success in terms of desired effect with the minimum rate of side effects treatment related.hf The radiobiological (toxic) effect obtained with radiation therapy aims to achieve a high tumor control rate together with low complications rate treatment related. Historically radiation was delivered considering variable safety margin to include microscopic tumor infiltration in normal tissue. First aim of this paper is to review the studies currently published about radiobiological principles for radiosurgery, clarifying definitions and terminology used for stereotactic radiation techniques. Dose application, in particular for malignant brain lesions, consisted in the application of cumulative dose into a variable number of fractions, usually not more than 2 Gy for standard fraction more than 2 Gy for hypofractionation either in curative or in palliative setting. The cumulative dose was currently applied with 3D imaging simulation obtained with CT scan and MRI support. The relationship between time of radiation, dose, and number of fractions to influence biological effect to a given tissue is based on four basic principles of radiobiology defined as the “4Rs” of ionizing radiation [14, 15]: Second aim is to review and compare the reliability of the mathematical formalisms used in clinical dose fractionated radiotherapy when applied to radiosurgery. Third aim is a brief summary of the clinical indications and experimental lines of evidence of radiosurgery for the central nervous system. 1. Introduction Therefore it is not surprising that CNS may react differ- ently when exposed to ionizing radiation compared to other organs of the human body. This makes the CNS somehow “unique” in the field of radiation oncology, radiobiology, and radioresistance. Over the last century radiation therapy for the management of human cancer had a tremendous evolution. Radiation delivery is based in most cases on photon irradiation. It might be applied with different techniques devices and dose schedules. More recently in particular at the beginning of this century experiments were performed with heavier ions radiation such as protons or carbon ions; in more recent times this highly biological effective radiation was also intro- duced in clinical routine thus with encouraging results but deserving longer term followup to draw better conclusions and therefore to establish clinical therapeutic indications.h Any physician is aware of the eloquence of specific nervous regions, like speech areas, optic pathways, brainstem, limbic lobe, and the “respect” they deserve. Radiation therapy is not an exception to this issue. Assuming these presumptions Larsson et al. began radio- biological investigations to study the effect of high dose focused radiation on the central nervous system more than 5 decades ago. The introduction of highly focused single ses- sion radiation delivered with the help of stereotactic framed based coordinates within the brain led to the definition of radiosurgery [3, 4]. The central nervous system (CNS), in particular the brain parenchyma, deserves some separate considerations [1]. First the TNM classification cannot be applied. Furthermore secondary metastases of primary brain tumors even if malig- nant are a truly rare event, described in the literature, but nevertheless uncommon [2]. Today it is not surprising, assuming these considerations, that the radiobiological principles used for dose fractionation 2 BioMed Research International 2 are not applicable for single session radiation most particu- larly for the CNS. These controversies come out either from experimental data or from clinical experience [1, 5]. disorders such as trigeminal neuralgia and pharmacological resistant epilepsies. Goals of Radiosurgery are as follows: (1) exposure of a target volume to a single high dose of ionizing radiation which ultimately translates into a specific (toxic) radiobiological response [17], (1) exposure of a target volume to a single high dose of ionizing radiation which ultimately translates into a specific (toxic) radiobiological response [17], In dose fractionated radiotherapy the effect of different fractionation schedules could have substantial relevance in clinical practice.f (2) precise destruction of a chosen target containing healthy and/or pathological cells, without significant concomitant or late radiation damage to adjacent tissue [3]. According to the LQ formalism isoeffect curves calcu- lating the effect of changing the dose fraction schedule and thus to compare two treatment regimens expressing an equal biological effect have been proposed [19]: BioMed Research International The linear quadratic formula is presently the standard way to mathematically represent the effect of radiotherapy to account for the effects of different fractionation schedules [5].h The principle underlying radiosurgery is that by reducing the volume target and the safety margin applied with a high conformal radiation delivery the complication rate might be drastically lower if compared to larger target volumes.i ff The clinical consequence of this model is that normal tissues can be classified in early responding tissue (high 𝛼/𝛽ratio) and late responding tissue (low 𝛼/𝛽ratio). For neoplastic tissues this classification reports relatively high 𝛼/𝛽ratios for malignant tumors and low 𝛼/𝛽ratios for slow growing benign tumors. According to this presumptions the following definitions of radiosurgery are available: (1) “the delivery of a single high dose of irradiation to a small and critically located intracranial volume through the intact skull” [3], Rationale of dose fractionation is to reach a compromise between desired effect (tumors cure/target destruction) and undesired effects (injury of normal tissue/complications): by delivering a cumulative dose in several fractions it is possible to spare normal tissue from severe damage and to repair from sublethal damage. Moreover the reoxygenation of hypoxic tumor cells after repeated radiation exposure reduces the radioresistance of the tumor tissue thus increasing the rate of cell death by increasing the rate of reoxygenation of hypoxic cells.h (2) “stereotactic radiosurgery: stereotactically guided delivery of focused radiation to a defined target volume in single session” [17], (3) “discipline that uses externally generated ionizing radiation delivered in single session to eradicate or inactivate a target defined by high resolution stereotactic imaging” (ASTRO SRS Model Coverage Policy), (4) “technique designed to deliver a high dose of focused radiation to a defined target volume to elicit a decide radiobiological response” [18]. The late toxicity of a radiation treatment can be well described with the linear quadratic model applying dose fraction from 1 untill 8 Gy given intervals between dose applications longer than 6 hours. On the other hand for acute toxicity in early responding tissues as well as for neoplastic tissue this model can be applied only for the same total treatment time.ff 2. Radiosurgery and Fractionated Stereotactic Radiotherapy The logical consequence of this sigmoid curve representation is to define a therapeutic window through which the better desired outcome can be achieved (i.e., imaging control of the target volume) without increasing the rate of undesired outcomes (complication rate). Reducing the volume of tissue irradiated shifts dose-response curve for complications increasing the separation between cure and complication probability [17]. The current radiosurgery concept is that damage to tissue within the target volume (either normal or neoplastic) is the desired effect. Historically developed by Larsson et al. [3], the number of clinical indications of radiosurgery has increased greatly. Nowadays radiosurgery has a well-established role for the treatment of small volume brain lesions like AVMs [7, 8], vestibular schwannomas [9], small remnants recurrent WHO Gr. I meningiomas, and imaging defined meningiomas [10– 13] and in more recent times also in the field of functional 3 3.2. Mathematical Formalisms and Models Few months later, a reply to this contribution proposed a more detailed classification for the application of radio- surgery to the central nervous system with respect to target and surrounding tissues [23]: (b) The biological effect produced by ionizing radiation allows us to classify normal tissue in acute and early responding through defined 𝛼/𝛽ratios expressed in Grays. (1) late responding target embedded within late respond- ing tissue: AVM, (2) late responding target surrounded by late responding tissue: meningioma/schwannoma, (c) The central nervous system is classified as late reacting tissue with different 𝛼/𝛽ratios with regard to brain parenchyma (𝛼/𝛽= 2) and spinal cord (cervical 2- 3—lumbar 4-5). (3) early responding target embedded within late re- sponding tissue: low gr. Glioma, (4) early responding target surrounded by late respond- ing tissue: glioblastomas/metastases. Formulas (3), (4), and (6) reviewed allow to calculate the response of a given tissue and relative 𝛼/𝛽ratio to a different fractionation schedule.if According to this oversimplified classification the indication to radiosurgery appears relatively simple to give. Nevertheless some variables must be considered: the different anatomic parts of central nervous system do not have the same uniform tolerance to radiation and 2nd cranial nerve optic chiasm and brainstem represent a dose limiting factor. The target volume plays a role to give a proper indication to radiosurgery. Finally the use of a highly conformal radiation dose is applicable with different techniques and the devices whose differences must be considered before applying radiosurgery. Formula (5) defines the probabilistic effect of cell response when exposed to fractionated radiation.h This mathematic extrapolations derived by the LQ for- mula can be considered reliable when applied to dose frac- tionation. Nevertheless some remarks have to be made: first the LQ response formulas are not reliably applicable at any dose level [20, 21]. Furthermore some estimation of 𝛼/𝛽ratios obtained with cell in vitro cultivated exposed to ionizing radiation was uncertain in particular of late responding tissue. Besides the LQ model does not take into consideration the time of radiation. To conclude uncertainty is reported in the literature about the 𝛼/𝛽ratios of many tumors [17]. Reliability of the LQ Formalisms in Radiosurgery. As previ- ously reported in [24] the LQ model is simple and convenient, and by far it has been the most useful means for isodose calculation in treating tumors with conventional fractionated radiation therapy [24–27]. 3.3. Controversies about Radiobiology of Radiosurgery for the Central Nervous System. 3.2. Mathematical Formalisms and Models (2) The delivery of a high focused radiation dose to a target implies a high conformality to produce a toxic effect limited to the target volume defined with a high steep dose falloff to avoid damage of the surrounding tissue. the respective linear and quadratic coefficients. The value of the 𝛼-coefficient divided by the 𝛽-coefficient (the 𝛼/𝛽ratio) characterizes how a tissue or tumor is affected by different fractionation schemes.h The same group [17] proposed the following formula to equate the effect of a course of fractionated radiotherapy administered with dose 𝑋per fraction in terms of an equivalent dose for treatment with dose 𝑌per fractions: (3) The rationale of dose fractionation for radiation deliv- ery to the CNS is to avoid the clinical consequences that a radionecrosis implies. (3) The rationale of dose fractionation for radiation deliv- ery to the CNS is to avoid the clinical consequences that a radionecrosis implies. [total dose (𝑥)] × [1 + 𝑋 (𝛼/𝛽)] = [total dose (𝑦)] × [1 + 𝑌 (𝛼/𝛽)] , (6) As previously reported in the literature [16] there was a consensus with the use of radiosurgery for benign brain lesions such as arteriovenous malformations (AVMs) and benign tumors, but its role for the management of malignant tumors is questioned. An improved ratio would be expected from fractionation for malignant lesions. The argument was based on the concept that hypoxic cells could reestablish their oxygenated state and become more sensitive to irradiation when treated with multiple fractions. Since AVMs and benign tumors are late-responding tissues, nothing was felt to be gained by fractionation. (6) where total dose(𝑦) is the total dose given at 𝑌Gy per fraction and is equal to the number of fractions times 𝑌. q Formulas (1) and (2) reviewed allow drawing the follow- ing conclusions. (a) The biological response of cell in terms of sur- vival/death fraction exposed to a given radiation dose is expressed mathematically by a curve with linear (𝛼) and exponential quadratic (𝛽) component. (a) The biological response of cell in terms of sur- vival/death fraction exposed to a given radiation dose is expressed mathematically by a curve with linear (𝛼) and exponential quadratic (𝛽) component. (1) The CNS is classified as late responding tissue with accepted 𝛼/𝛽ratio of ca. 2. 3.2. Mathematical Formalisms and Models 𝐷ref 𝐷new = (𝛼/𝛽+ 𝑑new) (𝛼/𝛽+ 𝑑ref) ; (3) (3) The Linear Quadratic Model. The biological response of a given tissue to radiation and dose fractionation has been theorized with many models. The linear quadratic (LQ) equation formula is so far the most used: briefly it defines the biological response of tissue in terms of surviving fraction (SF) to a given dose (𝐷) according to a linear dose coefficient 𝛼for low doses and a coefficient for the square of the dose 𝛽 for high dose fraction within a dose range from 1 untill 8 Gy; biological effect is proportional to 𝛼𝐷+ 𝛽𝐷2: then 𝐷new = 𝐷ref ∗[ (𝛼/𝛽+ 𝑑ref) (𝛼/𝛽+ 𝑑new)] , (4) (4) where 𝐷new is the new total dose obtained with a new fractionation schedule (𝑑new) compared to a known total dose 𝐷ref and fractionation dose (𝑑ref) and the 𝛼/𝛽ratio of the given tissue irradiated. SF = 𝑒−(𝑎∗𝐷+𝛽∗𝐷2); (1) (1) As proposed by Niranjan and coworkers [17] for single fraction irradiation the linear quadratic formula represents the probability of a desired outcome or undesired response (cure of a tumor or normal tissue injury) by the following probabilistic double exponential equation: then ln SF = −𝛼𝐷−𝛽𝐷2. (2) (2) The extrapolated cellular survival curve related to the dose applied allows us to define a linear component of cell killingh The extrapolated cellular survival curve related to the dose applied allows us to define a linear component of cell killing 𝛼and a quadratic component of cell killing 𝛽. The ratio of these two variables defines 𝛼/𝛽ratio of a tissue and expresses the point where the linear component 𝛼and the exponential (curvier) component of the survival curve 𝛽are equal or, in other words, it express the dose at which the two components of cell killing are equal. 𝑃(response) = 𝐸−[𝑘∗𝐸−(𝛼∗dose+ 𝛽∗dose2)], (5) (5) where 𝑃(response) is the probability of cure or complications, EXP represents the number 𝑒(2.7183, commonly used in natural logarithms) risen exponentially to the power of the terms that follow, and 𝑘represents the number of clonogens in the target tissue, while 𝛼and 𝛽are coefficients for 4 BioMed Research International BioMed Research International (2) The delivery of a high focused radiation dose to a target implies a high conformality to produce a toxic effect limited to the target volume defined with a high steep dose falloff to avoid damage of the surrounding tissue. 3.2. Mathematical Formalisms and Models The model is based on the incidence of possible interactions of radiation of direct interactions of radiation with specific cellular targets (i.e., DNA strands). Because the LQ survival curve continu- ously bends downward with increasing radiation dose, some authors state that the LQ formula might overestimate cell death caused by high dose-per-fraction radiation therapy. The reliability of the LQ formalisms for high dose radi- ation is nowadays still strong matter of debate [28, 29]. A growing number of contributions analyzing laboratory and clinical show the risk of misestimating the equivalent dose by applying the LQ models for radiosurgery or high dose radiation fractions [6]. In his recent review Shibamoto and coworkers emphasize that many clinicians have used the LQ formalisms to convert hypofractionated doses to single doses [30–32]. Furthermore it is also reported that together with the introduction of the biological effective dose model (BED = 𝐷(1 + 𝑑/[𝛼/𝛽]) [19] the issue has become even more complicated thus not making the entire matter easier to understand. Conversely clinical results have shown that the LQ model actually underestimates tumor control by stereotactic radi- ation therapy or radiosurgery [29]. Therefore other mecha- nisms together with DNA strand breaks and/or chromosome aberrations may be involved in response of tumors to stereo- tactic radiation therapy or radiosurgery. A number of experimental models have studied the effects of radiosurgery on the central nervous system [1, 5, 17]. The magnitude of radiosurgical effects remains poorly under- stood, especially when described in terms of conventional radiation therapy doses. Drawing outcomes from either laboratory data [33–39] and normal tissue data [25, 40–43] on of the author shows the reliability of LQ formalism if applied to isoeffects within a range of dose from 1–8 Gy. Nevertheless other authors proposed the validity of the LQ formalism and isoeffects curves also for doses higher than 8 Gy [28, 44]. Conversely to these theoretical extrapolations the same author reports further experimental data [45] which show contradictory results, in particular reliability of the LQ model for spinal cord as late reacting tissue with a fractionation dose up to 10 Gy. Benign Tumors. 3.2. Mathematical Formalisms and Models Since its birth radiosurgery was a matter of debate among physicians. A number of contribu- tions have been published since the early 90s describing all the possible issues that a conformal stereotactic single session radiation implies [16, 22, 23]. Despite the current use in clinical fractionated radiother- apy the LQ formalism has some limitations if applied to radio- surgery. First the LQ model cannot be applied with a dose of 8 Gy or more. Second the validity of the linear quadratic model has not been sufficiently investigated for very small target volumes (major diameter < 2 cm) so far. This implies that increasing the fractionation for slow growing tumors or nonneoplastic lesion may not bring a biological better response of the tumor if compared to a single dose if we accept Several points have to be mentioned. (1) The CNS is classified as late responding tissue with accepted 𝛼/𝛽ratio of ca. 2. 5 BioMed Research International a low 𝛼/𝛽ratio for benign or slow growing tumors according to the most recent clinical data. Furthermore highly confor- mal radiosurgery spares dose exposure to surrounding tissue; therefore the compromise of dose fractionation to balance between imaging tumor control and normal surrounding tissue reaction might not be a consideration, depending on dose delivered, target volume, and eloquence of surrounding structures. To conclude many efforts to extrapolate a survival curve applying high doses to a benign lesions (>10 Gy) gave improbable values for which the risk of missing estimates of 𝛼/𝛽ratios is too high, thus confirming the usefulness of such a model in radiosurgery [17, 24]. As previously published in the literature [17, 23] estimations of the equivalent dose for fractionated radiotherapy with 2 Gy fractions and single fraction radiosurgery doses using 𝛼/𝛽 = 2 commonly accepted for the brain as late reacting tissue are proposed. (2) Mathematical models of radiation injury probability need to take into account that the target/tumor tissue’s radiation response may affect the reaction of the surrounding normal tissue. (3) The predominant radiation response of a radiosur- gical target is mediated through the target or tumor vasculature. 3.4. Experimental Lines of Evidence of Radiosurgery for Central Nervous System. The validity of the linear quadratic model for calculating isoeffect doses in radiation therapy has been intensively described [24, 26, 27, 50]. 3.2. Mathematical Formalisms and Models When tumor volume shrinks due to death of parenchymal cells after irradiation, the tumor vascular beds may become further disorganized, aggregated, and fragmented.h The Issue of Vascular Damage in Radiosurgery. It is well known that the radiobiological effect of radiosurgery is based on direct cytotoxic effect after low dose radiation therapy [58]. On the other hand it is well accepted that intratumor microenvironment greatly influences the radiosensitivity of tumor cells and the intratumor microenvironment is closely related to the functional status of tumor microvasculature [57]. The authors concluded that the radiation-induced vascu- lar damage and the resulting indirect death of tumor cells play important roles in the response of tumors to high dose hypofractionated radiotherapy and radiosurgery. In addition, enhanced immune reactions and increased eradiation of cancer stem cells might be involved in the response of tumors to stereotactic fractionated radiotherapy and radiosurgery [57]. Available information from AVM radiosurgery or menin- gioma radiosurgery has shown that normal vessels rarely decrease in size or occlude after radiosurgery and therefore they conclude that the abnormal vessels of neoplasms or vascular malformations have a relative sensitivity to radio- surgery in comparison to normal surrounding vessels since no occurrence of perforator occlusion leading to an infarct has been identified [5]. On the other hand it must also be said that chance to produce a damage of normal capillary vessels is directly proportional to the dose increasing [59]. Toxicity of Radiosurgery. These emerging applications of radiosurgery open as the last point the discussion about the different dose tolerance of brain structures after radiosurgery. With regard to radiobiological properties the CNS is a “late reacting tissue.” Despite this not all brain regions including cranial nerves have the same eloquence. In a recent review of the literature [57] an analysis of the studies published about vascular damage in tumors after stereotactic high dose hypofractionated radiation ther- apy and radiosurgery was performed. The authors indicate that the functional vascularity in human tumors remains unchanged or improves slightly during the early period of conventional fractionated radiotherapy with 1.5–2.0 Gy daily doses but gradually diminishes during the latter part of treatment. By delivering radiation doses higher than 10 Gy in a single fraction or 20–60 Gy in limited numbers of fractions severe vascular damage leading to the deterioration of the intratumor microenvironment and indirect death of tumor cells is observed. 3.2. Mathematical Formalisms and Models An emerging field of radiosurgery is the treatment of functional neurological disorders which BioMed Research International 6 6 were interestingly the first clinical application evaluated once radiosurgery was introduced in the late 50s. After half a century we can describe an arising number of indications in the literature. cells. The authors strictly distinguished between endothelial cells derived from normal and tumor tissue classifying them as radioresistant and radiosensitive, respectively, in accor- dance with other experimental lines of evidence [60] demon- strating that developing vessels are more radiosensitive than mature vessels.i The first experiments about this issue were conducted with dose delivery of 150 Gy to small volumes producing tissue necrosis of the target (3 × 5 mm diameter within 1 month not changing over the ensuing years as reported by Kondziolka and coworkers) [3, 5, 54, 55]. Better outcomes in terms of desired targeting were observed with the use of 4 mm conic collimator. The doses applied were different with respect to the target volume and varied from 130 Gy for thalamotomies to 80 Gy for trigeminal neuralgia. As reported by Kondziolka and coworkers histological features of the target volumes after radiosurgery consisted in necrosis of the target volume a surrounding gliotic rim and normalisation of the parenchyma within 2 mm [56]. Even the higher doses used in functional radiosurgery do not appear to cause vascular injury to surrounding tissue with proper targeting, in accordance with the findings of more recent studies [57]. Most specifically as reported by the authors [57] the death of endothelial cells after direct radiation damage would cause focal microscopic or macroscopic vascular damage and collapse of the affected capillary-like vessels. Soon after vascular permeability in tumors increases rapidly after irradiation due to damage in the endothelial cells followed by widening of the gaps between endothelial cells. Further extravasation of plasma due to vascular permeability might increase the erythrocyte concentration within the narrow capillaries, thereby leading to retardation or stasis of blood perfusion. In addition, the increased permeability of capil- laries may increase the extravascular or interstitial plasma protein concentrations, thereby elevating interstitial fluid pressure. The elevation of interstitial fluid pressure above the intravascular blood pressure will cause vascular collapse. Therefore, it is probable that the early decline in functional vascularity after irradiation in tumors may be caused at least in part by collapse of blood vessels as a result of elevation of interstitial fluid pressure. 3.2. Mathematical Formalisms and Models If we consider the application of radiosurgery for benign intracranial lesions like meningiomas and schwan- nomas, it has been observed that the radiobiological effect on meningiomas and other benign neoplasms is a combination of both cytotoxic and radiation induced vascular damage as already reported [5].t After both vestibular schwannoma and meningioma radiosurgery a doubling of the number of apoptotic cells after radiosurgery when compared to controls, within the first 48 h after irradiation, was observed. g y Due to this uncertainty further mathematical models are also quoted: particular emphasis is given to the recently pro- posed survival curve model [46], the linear quadratic-linear model (LQL) [47, 48], and the generalized linear quadratic model (gLQ) [49]. Each of these models was developed modifying or existing model trying to draw conclusions which could better fit the isoeffect curves at high dose range; the experience with these models is somehow very limited and deserves further clinical application and laboratory lines of evidence to be used in clinical routine. Metastases. Some investigators have reported that apoptosis may play a significant role in the early effects of radio- surgery also for malignant tumors [51]. With regard to brain metastases some issues have to be considered. As previously reported [17] a RTOG dose escalation study (RTOG 90- 05) [52, 53] showed results about dose tolerance of brain tissue after radiosurgery of recurrent brain metastases. A treatment protocol was defined applying initial dosis of 18, 15, and 12 Gy for diameters <20 mm, 21–30 mm, and 31–40 mm, respectively. A prescription dose escalation in 3 Gy steps was applied till toxicity rate was seen in over 30% of the sample population. The final recommendation was 24, 18, and 15 Gy for lesion of diameter <20, 21–30, and 31–40 mm, respectively. Furthermore it was reported that no difference in terms of progression free survival rate and tumor control was seen by stratifying the sample per histology of the brain metastases. These contradictory findings are confirmed also by a previous report of the group from Pittsburgh [17] drawing outcomes from radiobiological analysis of clinical data. (1) The linear quadratic equation cannot reliably repre- sent equivalent radiation effects when extrapolating from conventional fractionation (1.5–4 Gy per frac- tion) to high dose (12–25 Gy) single fractions for radiosurgery. Functional Radiosurgery. References [1] A. Niranjan, G. T. Gobbel, D. Kondziolka, J. C. Flickinger, and L. D. Lunsford, “Experimental radiobiological investigations into radiosurgery: present understanding and future directions,” Neurosurgery, vol. 55, no. 3, pp. 495–504, 2004. Furthermore the clinical experience with AVM radio- surgery allowed also defining specific dose tolerance within the brain parenchyma. Results of an AVM radiosurgery study group tolerance showed no difference in the likelihood of injury imaging changes after radiosurgery for AVMs with respect to brain location. Conversely relevant differences were seen in the development of clinical side effects being then observed at the frontal region as the most radioresistant followed by parietal region, temporal lobe, cerebellar and brainstem and thalamus/basal ganglia as the most sensitive [61, 63, 64]. [2] A. E. Romero-Rojas, J. A. Diaz-Perez, D. Amaro, A. Lozano- Castillo, and S. I. Chinchilla-Olaya, “Glioblastoma metastasis to parotid gland and neck lymph nodes: fine-needle aspiration cytology with histopathologic correlation,” Head and Neck Pathology, 2013. [3] B. Larsson, L. Leksell, B. Rexed, P. Sourander, W. Mair, and B. Andersson, “The high-energy proton beam as a neurosurgical tool,” Nature, vol. 182, no. 4644, pp. 1222–1223, 1958. [4] L. Steiner, D. Forster, and L. Leksell, “Gammathalamotomy in intractable pain,” Acta Neurochirurgica, vol. 52, no. 3-4, pp. 173– 184, 1980. The Issue of Volume Dose Effect. A major issue in radiosurgery for CNS is not only the highly conformal dosis to deliver in order to achieve in one session the same radiobiological effect of a dose fractionation schedule but also the effect of the target volume. According to experimental data [65] the tolerance of the spinal cord of rats increases from 20 Gy to 80 Gy delivered in single session if the volume of spinal cord irradiated is reduced from 20 mm to 2 mm. [5] D. Kondziolka, A. Niranjan, L. D. Lunsford, and J. C. Flickinger, “Radiobiology of radiosurgery,” Progress in Neuro- logical Surgery, vol. 20, pp. 16–27, 2007. [6] Y. Shibamoto, S. Otsuka, H. Iwata, C. Sugie, H. Ogino, and N. Tomita, “Radiobiological evaluation of the radiation dose as used inhigh-precision radiotherapy: effect of prolonged deliverytime and applicability of the linear-quadratic model,” Journal of Radiation Research, vol. 53, no. 1, pp. 1–9, 2012. For target volumes with diameter more than 3 cm the dosis falloff at the target margin is progressively flatter thus increasing the volume of normal tissue irradiated and then the toxicity rate. 3.2. Mathematical Formalisms and Models Of note experimental data about radiation induced vascular damage shows that high dose delivery in single session produces decrease of vascular volume and increase of vascular permeability. It is also observed that radiation induced changes in blood perfusion, functional intravascular volume, and vascular permeability are directly related to the functional integrity and activity of endothelial The incidence of cranial neuropathies after radiosurgery is well described in the literature. Variables to consider are the dose delivered and its distribution (isodose line), the volume of the tissue irradiated, the sensitivity of the tissue affected, and history of any prior irradiation. The clinical manifesta- tion may vary greatly according to the volume and location of the target [13]. As previously reported [61], a prior history of fractionated radiation therapy to the same region of interest appears to have limited effects on the risk of developing postradiosurgery parenchymal edema with exception to the optic nerve. Each cranial nerve has a specific tolerance to dose radiation. According to clinical experience with radiosurgery and conventional fractionated radiotherapy sensory nerves appear to be the most sensitive, followed by somatic sensory nerves and motor nerves [61]. The anterior optic pathways are the most dose sensitive structures, thus implying that the 7 BioMed Research International dose applying the second cranial nerve (optic nerve) should be always a consideration. Many contributions report a dose maximum tolerance of the optic nerve after radiosurgery to be at 8 Gy. Nevertheless more recent studies [62] report that the maximal dose tolerance at 10 Gy may be related to better imaging technique available. The correlation of data with dose fractionation to conclude brought physicians to assume an 𝛼/𝛽ratio for the optic nerve of ca. 1 as reference value to calculate dose equivalent between fractionation and radiosurgery dose protocols. system tissue is crucial in particular to define mathematical models reliable to this special radiation technique. Particular emphasis should be given to the mechanisms of vascular damage after high dose radiation and the tolerance doses of CNS structures for radiosurgery. References Better appreciation of the tolerance of CNS structures to high radiation dose schedules is still a matter of investigation and requires further studies. [7] H. Kano, D. Kondziolka, J. C. Flickinger et al., “Stereotactic radiosurgery after embolization for arteriovenous malforma- tions,” Progress in Neurological Surgery, vol. 27, pp. 89–96, 2013. [8] L. D. Lunsford, A. Niranjan, H. Kano, and D. Kondziolka, “The technical evolution of gamma knife radiosurgery for arteriovenous malformations,” Progress in Neurological Surgery, vol. 27, pp. 22–34, 2013. 4. Conclusions 4, pp. 556–593, 1960. [32] M. T. Milano, A. W. Katz, M. C. Schell, A. Philip, and P. Okunieff, “Descriptive analysis of oligometastatic lesions treated with curative-intent stereotactic body radiotherapy,” International Journal of Radiation Oncology Biology Physics, vol. 72, no. 5, pp. 1516–1522, 2008. [16] E. J. Hall and D. J. Brenner, “The radiobiology of radiosurgery: rationale for different treatment regimes for AVMs and malig- nancies,” International Journal of Radiation Oncology Biology Physics, vol. 25, no. 2, pp. 381–385, 1993. [33] S. Otsuka, Y. Shibamoto, H. Iwata et al., “Compatibility of the linear-quadratic formalism and biologically effective dose concept to high-dose-per-fraction irradiation in a murine tumor,” International Journal of Radiation Oncology Biology Physics, vol. 81, no. 5, pp. 1538–1543, 2011. [17] A. Niranjan and J. C. Flickinger, “Radiobiology, principle and technique of radiosurgery,” Progress in Neurological Surgery, vol. 21, pp. 32–42, 2008. [34] Y. Shibamoto, Y. Kitakabu, R. Murata et al., “Reoxygenation in the SCCVII tumor after KU-2285 sensitization plus single or fractionated irradiation,” International Journal of Radiation Oncology Biology Physics, vol. 29, no. 3, pp. 583–586, 1994. [18] A. Niranjan and L. D. Lunsford, “Radiosurgery: where we were, are, and may be in the third millennium,” Neurosurgery, vol. 46, no. 3, pp. 531–543, 2000. [19] H. R. Withers, H. D. Thames Jr., and L. J. Peters, “A new isoeffect curve for change in dose per fraction,” Radiotherapy and Oncology, vol. 1, no. 2, pp. 187–191, 1983. [35] R. Murata, Y. Shibamoto, K. Sasai et al., “Reoxygenation after single irradiation in rodent tumors of different types and sizes,” International Journal of Radiation Oncology Biology Physics, vol. 34, no. 4, pp. 859–865, 1996. [20] K. K. Ang, G. Jiang, Y. Feng, L. C. Stephens, S. L. Tucker, and R. E. Price, “Extent and kinetics of recovery of occult spinal cord injury,” International Journal of Radiation Oncology Biology Physics, vol. 50, no. 4, pp. 1013–1020, 2001. [36] H. Iwata, Y. Shibamoto, R. Murata et al., “Estimation of errors associated with use of linear-quadratic formalism for evaluation of biologic equivalence between single and hypofractionated radiation doses: an in vitro study,” International Journal of Radiation Oncology Biology Physics, vol. 75, no. 2, pp. 482–488, 2009. [21] K. K. Ang, A. J. van der Kogel, and E. van der Schueren, “Lack of evidence for increased tolerance of rat spinal cord with decreasing fraction doses below 2 Gy,” International Journal of Radiation Oncology Biology Physics, vol. 4. Conclusions [9] J. Regis, R. Carron, S. Moucharrafien et al., “Role of radio- surgery and stereotactic radiotherapy in the management of vestibular schwannomas,” Cancer/Radioth´erapie, vol. 16, pp. S70–S78, 2012. The radiobiology of radiosurgery in particular for the central nervous system plays a crucial role for the clinical indication and the application of a therapeutic radiation dose in single session. The current mathematical models applied for dose fractionation allow us to predict the rate of late neurological side effects but are not applicable for a dose over 8 Gy. There- fore in order to avoid late complications a highly conformal radiation delivery with high dose gradient is required. Target location and volume are crucial to achieve the conformality required. The current indications to radiosurgery concern small rest or recurrent benign lesions and metastases and in the last years also for small imaging defined benign lesions without histological confirmation. Radiosurgery for functional neurological diseases is an emerging field and the results are encouraging. As previously reported [33] the effects of radiosurgery on the brain tumor microenvironment are still under investigation. The pathogenesis of biological effects after radiosurgery may be unique. The need for basic research concerning the radiobiological effects of high dose, single fraction, and ionizing radiation on nervous [10] A. E. Elia, H. A. Shih, and J. S. Loeffler, “Stereotactic radiation treatment for benign meningiomas,” Neurosurgical Focus, vol. 23, no. 4, p. E5, 2007. [11] D. Kondziolka, D. Mathieu, L. D. Lunsford et al., “Radiosurgery as definitive management of intracranial meningiomas,” Neuro- surgery, vol. 62, no. 1, pp. 53–60, 2008. [12] A. Kollova, R. Liscak, J. Novotny et al., “Gamma Knife surgery for benign meningioma,” Journal of Neurosurgery, vol. 107, no. 2, pp. 325–336, 2007. [13] A. Santacroce, M. Walier, J. R´egis et al., “Long-term tumor control of benign intracranial meningiomas after radiosurgery in a series of 4565 patients,” Neurosurgery, vol. 70, no. 1, pp. 32– 39, 2012. [14] M. M. Elkind, H. Sutton-Gilbert, W. B. Moses, T. Alescio, and R. W. Swain, “Radiation response of mammalian cells grown in culture. V. Temperature dependence of the repair of X-ray damage in surviving cells (Aerobic and Hypoxic),” Radiation Research, vol. 25, no. 2, pp. 359–376, 1965. BioMed Research International 8 8 [15] M. M. Elkind and H. Sutton, “Radiation response of mam- malian cells grown in culture. 1. Repair of X-ray damage in surviving Chinese hamster cells.,” Radiation Research, vol. 13, no. 4. Conclusions 11, no. 1, pp. 105–110, 1985. [37] T. T. Puck and P. I. Marcus, “Action of x-rays on mammalian cells,” Journal of Experimental Medicine, vol. 103, no. 5, pp. 653– 666, 1956. [22] D. A. Larson and M. W. McDermott, “Radiosurgery,” Western Journal of Medicine, vol. 165, no. 1-2, pp. 59–60, 1996.flh [38] L. M. Garcia, J. Leblanc, D. Wilkins, and G. P. Raaphorst, “Fitting the linear-quadratic model to detailed data sets for different dose ranges,” Physics in Medicine and Biology, vol. 51, no. 11, pp. 2813–2823, 2006. [23] D. A. Larson, J. C. Flickinger, and J. S. Loeffler, “The radio- biology of radiosurgery,” International Journal of Radiation Oncology Biology Physics, vol. 25, no. 3, pp. 557–561, 1993.fi [24] C. W. Song, L. C. Cho, J. Yuan, K. E. Dusenbery, R. J. Griffin, and S. H. Levitt, “Radiobiology of stereotactic body radiation ther- apy/stereotactic radiosurgery and the linear-quadratic model,” International Journal of Radiation Oncology ‘Biology’ Physics, vol. 87, no. 1, pp. 18–19, 2013.h [39] N. Tomita, Y. Shibamoto, M. Ito et al., “Biological effect of intermittent radiation exposure in vivo: recovery from sublethal damage versus reoxygenation,” Radiotherapy and Oncology, vol. 86, no. 3, pp. 369–374, 2008. [40] B. G. Douglas and J. F. Fowler, “The effect of multiple small doses of X rays on skin reactions in the mouse and a basic interpretation,” Radiation Research, vol. 66, no. 2, pp. 401–426, 1976. [25] J. F. Fowler, “The linear-quadratic formula and progress in fractionated radiotherapy,” British Journal of Radiology, vol. 62, no. 740, pp. 679–694, 1989.h [26] J. D. Chapman and C. J. Gillespie, “The power of radiation biophysics-let’s use it,” International Journal of Radiation Oncol- ogy ‘Biology’ Physics, vol. 84, no. 2, pp. 309–311, 2012. [41] J. F. Fowler, “Review: total doses in fractionated radiotherapy— implications of new radiobiological data,” International Journal of Radiation Biology, vol. 46, no. 2, pp. 103–120, 1984. [27] D. J. Brenner, R. K. Sachs, L. J. Peters, H. R. Withers, and E. J. Hall, “We forget at our peril the lessons built into the alpha/beta model,” International Journal of Radiation Oncology Biology Physics, vol. 82, no. 4, pp. 1312–1314, 2012.h [42] A. J. van der Kogel, “Chronic effects of neutrons and charged particles on spinal cord, lung, and rectum,” Radiation Research, vol. 8, pp. S208–S216, 1985. [43] J. W. Peck and F. A. 4. Conclusions Gibbs, “Mechanical assay of consequential and primary late radiation effects in murine small intestine: alpha/beta analysis,” Radiation Research, vol. 138, no. 2, pp. 272– 281, 1994. [28] D. J. Brenner, “The linear-quadratic model is an appropriate methodology for determining isoeffective doses at large doses per fraction,” Seminars in Radiation Oncology, vol. 18, no. 4, pp. 234–239, 2008. [44] J. F. Fowler, W. A. Tom´e, J. D. Fenwick, and M. P. Mehta, “A challenge to traditional radiation oncology,” International Journal of Radiation Oncology Biology Physics, vol. 60, no. 4, pp. 1241–1256, 2004. [29] J. P. Kirkpatrick, J. J. Meyer, and L. B. Marks, “The linear- quadratic model is inappropriate to model high dose per frac- tion effects in radiosurgery,” Seminars in Radiation Oncology, vol. 18, no. 4, pp. 240–243, 2008. [45] M. Astrahan, “Some implications of linear-quadratic-linear radiation dose-response with regard to hypofractionation,” Medical Physics, vol. 35, no. 9, pp. 4161–4172, 2008. [30] J. Wulf, K. Baier, G. Mueller, and M. P. Flentje, “Dose-response in stereotactic irradiation of lung tumors,” Radiotherapy and Oncology, vol. 77, no. 1, pp. 83–87, 2005.l [46] C. Park, L. Papiez, S. Zhang, M. Story, and R. D. Timmerman, “Universal survival curve and single fraction equivalent dose: useful tools in understanding potency of ablative radiotherapy,” International Journal of Radiation Oncology ‘Biology’ Physics, vol. 70, no. 3, pp. 847–852, 2008. [31] U. Haedinger, T. Krieger, M. Flentje, and J. Wulf, “Influ- ence of calculation model on dose distribution in stereotactic radiotherapy for pulmonary targets,” International Journal of Radiation Oncology Biology Physics, vol. 61, no. 1, pp. 239–249, 2005. BioMed Research International 9 Radiation Oncology, vol. 15, pp. 378–388, Lippincott Williams & Wilkins, Philadelphia, Pa, USA, 2008. [47] M. Guerrero and X. A. Li, “Extending the linear-quadratic model for large fraction doses pertinent to stereotactic radio- therapy,” Physics in Medicine and Biology, vol. 49, no. 20, pp. 4825–4835, 2004. [62] S. L. Stafford, B. E. Pollock, J. A. Leavitt et al., “A study on the radiation tolerance of the optic nerves and chiasm after stereotactic radiosurgery,” International Journal of Radiation Oncology∗Biology∗Physics, vol. 55, no. 5, pp. 1177–1181, 2003. [48] M. Guerrero and M. Carlone, “Mechanistic formulation of a lineal-quadratic-linear (LQL) model: split-dose experiments and exponentially decaying sources,” Medical Physics, vol. 37, no. 8, pp. 4173–4181, 2010. [63] J. C. Flickinger, D. Kondziolka, B. E. Pollock, A. H. Maitz, and L. D. 4. Conclusions Lunsford, “Complications fromarteriovenous malformation radiosurgery:multivariate analysis and riskmodeling,” Interna- tional Journal of Radiation Oncology Biology Physics, vol. 38, no. 3, pp. 485–490, 1997. [49] J. Z. Wang, Z. Huang, S. S. Lo, W. T. C. Yuh, and N. A. Mayr, “A generalized linear-quadratic model for radiosurgery, stereotac- tic body radiation therapy, and high-dose rate brachytherapy,” Science Translational Medicine, vol. 2, no. 39, Article ID 39ra48, pp. 39–48, 2010. [64] J. C. Flickinger, D. Kondziolka, L. D. Lunsford et al., “Devel- opment of a model to predict permanent symptomatic postra- diosurgery injury for arteriovenous malformation patients,” International Journal of Radiation Oncology Biology Physics, vol. 46, no. 5, pp. 1143–1148, 2000. [50] E. Glatstein, “The omega on alpha and beta,” International Journal of Radiation Oncology Biology Physics, vol. 81, no. 2, pp. 319–320, 2011. [65] H. P. Bijl, P. Van Luijk, R. P. Coppes, J. M. Schippers, A. W. T. Konings, and A. J. Van der Kogel, “Dose-volume effects in the rat cervical spinal cord after proton irradiation,” International Journal of Radiation Oncology Biology Physics, vol. 52, no. 1, pp. 205–211, 2002. [51] T. F. Witham, H. Okada, W. Fellows et al., “The characterization of tumor apoptosis after experimental radiosurgery,” Stereotac- tic and Functional Neurosurgery, vol. 83, no. 1, pp. 17–24, 2005. [52] E. Shaw, C. Scott, L. Souhami et al., “Radiosurgery for the treatment of previously irradiated recurrent primary brain tumors and brain metastases: initial report of Radiation Ther- apy Oncology Group protocol 90-05,” International Journal of Radiation Oncology Biology Physics, vol. 34, no. 3, pp. 647–654, 1996. [53] E. Shaw, C. Scott, L. Souhami et al., “Single dose radiosurgical treatment of recurrent previously irradiated primary brain tumors and brain metastases: final report of RTOG protocol 90- 05,” International Journal of Radiation Oncology Biology Physics, vol. 47, no. 2, pp. 291–298, 2000. [54] B. Andersson, B. Larsson, L. Leksell et al., “Histopathology of late local radiolesions in the goat brain,” Acta Radiologica, vol. 9, no. 5, pp. 385–394, 1970. [55] L. Kihlstr¨om, T. Hindmarsh, I. Lax, B. Lippitz, P. Mindus, and C. Lindquist, “Radiosurgical lesions in the normal human brain 17 years after gamma knife capsulotomy,” Neurosurgery, vol. 41, no. 2, pp. 396–402, 1997. [56] D. Kondziolka, L. D. Lunsford, D. Claassen, A. H. Maitz, J. C. Flickinger, and P. H. Gutin, “Radiobiology of radiosurgery: part I. The normal rat brain model,” Neurosurgery, vol. 31, no. 2, pp. 271–279, 1992. 4. Conclusions [57] H. J. Park, R. J. Griffin, S. Hui, S. H. Levitt, and C. W. Song, “Radiation-induced vascular damage in tumors: implications of vascular damage in ablative hypofractionated radiotherapy (SBRT and SRS),” Radiation Research, vol. 177, no. 3, pp. 311–327, 2012. [58] T. Tsuzuki, S. Tsunoda, T. Sakaki et al., “Tumor cell proliferation and apoptosis associated with the Gamma Knife effect,” Stereo- tactic and Functional Neurosurgery, vol. 66, supplement 1, pp. 39–48, 1997. [59] M. Yamamoto, M. Jimbo, M. Kobayashi et al., “Long-term results of radiosurgery for arteriovenous malformation: neuro- diagnostic imaging and histological studies of angiographically confirmed nidus obliteration,” Surgical Neurology, vol. 37, no. 3, pp. 219–230, 1992. [60] P. Grabham, B. Hu, P. Sharma, and C. Geard, “Effects of ionizing radiation on three-dimensional human vessel models: differential effects according to radiation quality and cellular development,” Radiation Research, vol. 175, no. 1, pp. 21–28, 2011. [61] J. C. Flickinger and A. Niranjan, “Stereotactic radiosurgery and radiotherapy,” in Perez Brady’s Principles and Practice of
https://openalex.org/W2972008116	https://europepmc.org/articles/pmc6789636?pdf=render	English	null	A Cellular Model of Infection with Brucella melitensis in Ovine Macrophages: Novel Insights for Intracellular Bacterial Detection	Veterinary sciences	2,019	cc-by	8,135	Received: 2 June 2019; Accepted: 22 August 2019; Published: 3 September 2019 Abstract: Intracellular bacteria provoking zoonoses, such as those of the genus Brucella, present a host cell tropism mostly limited to the monocyte/macrophage lineage, leading to chronic inﬂammatory reactions, diﬃcult-to-eradicate-infections, and widespread prevalence among ruminants. Eradication of brucellosis has been based on programs that translate into a substantial ﬁnancial burden for both the authorities and stockbreeders, if not strictly followed. To this end, we sought to create an in vitro cell model that could be utilized as future reference for adequately measuring the number of engulfed brucellae/cell, using peripheral blood-derived sheep macrophages infected with B. melitensis at decimal multiplicities of infection (MOI = 5000-5), to simulate the host cell/microorganism interaction and monitor bacterial loads up to 6 days post-infection. We show that the MOI = 5000 leads to high numbers of engulfed bacteria without aﬀecting macrophages’ viability and that the minimum detection limit of our Real-Time PCR assay was 3.97 ± 5.58 brucellae/cell. Moreover, we observed a time-associated, signiﬁcant gradual reduction in bacterial loads from Day 2 to Day 6 post-infection (p = 0.0013), as part of the natural bactericidal properties of macrophages. Overall, the work presented here constitutes a reliable in vitro cell model of Brucella melitensis for research purposes that can be utilized to adequately measure the number of engulfed brucellae/cell and provides insights towards future utilization of molecular biology-based methods for detection of Brucella. Keywords: brucellosis; macrophages; ruminant animals; infectious diseases A Cellular Model of Infection with Brucella in Ovine Macrophages: Novel Insights for Intracellular Bacterial Detection Garyfalia Karponi 1,, Spyridon K. Kritas 1, Eleni Papanikolaou 2,† and Evanthia Petridou 1,† 1 Department of Microbiology and Infectious Diseases, School of Veterinary Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece 2 Laboratory of Biology, School of Medicine, National and Kapodistrian University of Athens, 11527 Athens, Greece Garyfalia Karponi 1,, Spyridon K. Kritas 1, Eleni Papanikolaou 2,† and Evanthia Petridou 1,† 1 Department of Microbiology and Infectious Diseases, School of Veterinary Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece 2 Laboratory of Biology, School of Medicine, National and Kapodistrian University of Athens, 11527 Athens, Greece * Correspondence: gkarponi@vet.auth.gr † These authors contributed equally to this work. ceived: 2 June 2019; Accepted: 22 August 2019; Published: 3 September 2019 Vet. Sci. 2019, 6, 71; doi:10.3390/vetsci6030071 veterinary sciences veterinary sciences veterinary sciences veterinary sciences veterinary sciences www.mdpi.com/journal/vetsci 2.1. Bacteria and Ovine Macrophages B. melitensis 16 M strain (ATCC 23456) was purchased from Culture Collections Public Health England (Salisbury, UK). To store-purchased B. melitensis long-term, a standard procedure was followed, as per the manufacturer’s instructions, in order to open the freeze-dried vial containing the 16 M strain upon arrival. Then the strain was cultured aerobically both on Brucella agar (Oxoid, Hampshire, UK) and Columbia agar sheep blood plates (Oxoid, Hampshire, UK) for 3 days at 37 ◦C. From the initial culture, ten vials containing 108 CFU/mL B. melitensis in brain heart broth (Oxoid, Hampshire, UK) with 15% glycerol (Sigma-Aldrich, St. Louis, MO, USA) were primarily screened for contamination and then stored at −80 ◦C, under the label “master seed”. Working seed was prepared by spreading 10 µL of a master seed vial on Columbia agar sheep blood plates. After 3 days of incubation, ten vials of working seed were prepared following the same protocol for the master seed. Also, initial titration of bacteria, was conducted utilizing a suspension turbidity detector (Lioﬁlchem, Teramo, Italy). Purity of culture to B. melitensis was assessed by spreading working seed brucellae on Columbia agar sheep blood plates. To infect macrophages at particular MOIs, serial dilutions of cultured bacteria were plated on Columbia agar sheep blood plates (Oxoid, Hampshire, UK), and bacterial colony-forming units (CFU) were determined after 3 days to estimate bacterial concentrations per mL. Ovine macrophages were obtained by culturing the mononuclear cell fraction of sheep peripheral blood (n = 6) after ﬁcoll density gradient centrifugation. Characterization of the extracted and cultured primary macrophages was extensively described with ﬂow cytometry and a phagocytosis assay as previously described [9]. 1. Introduction Intracellular infectious agents, such as bacteria of the genus Brucella, provoke abortions and infertility in ruminant animals [1] and present a host cell tropism mostly limited to the monocyte/macrophage lineage [2]. Thus, brucellosis is diﬃcult to eradicate, since the microorganism may eﬀectively evade antibiotic treatment. Currently, vaccination programs in livestock, monitoring, and slaughtering of seropositive carriers constitute the only available means for eradicating the disease [3]. However, the available animal vaccine [4] is sometimes ineﬀective [5] and even infectious for veterinarians [5]. Moreover, the disease cannot be prevented in humans, since no vaccine currently exists and antibiotic therapy is long-lasting, potentially leading to relapse or re-infection after de novo exposure to the microorganism [6]. Consequently, Brucella infection in livestock not only poses severe public health issues, but also translates into a substantial ﬁnancial burden for stockbreeders [7]. Therefore the need to adequately monitor Brucella infection in ﬂocks is necessary. Current methods for Brucella detection involve classic microbiological assays, i.e., colony-forming units (CFU) detection Vet. Sci. 2019, 6, 71; doi:10.3390/vetsci6030071 www.mdpi.com/journal/vetsci Vet. Sci. 2019, 6, 71 2 of 9 on agar plates as well as several serological assays based on immune response [8]. Surprisingly, molecular-based methods such as Real-Time PCR and classic end-point PCR are not considered eﬀective in terms of surveillance of the prevalence in herds or ﬂocks [8]. In an eﬀort to understand the potential reasons that hamper the wider use of Real-Time PCR to detect Brucella infection, we aimed to generate an ovine macrophage infection model, using decimally diluted B. melitensis, to imitate the host cell/microorganism interaction in vitro and monitor the number of incorporated brucellae/cell by simultaneously comparing the classic CFU detection method with a molecular-based Real-Time PCR detection method. Our results indicate that the Real-Time PCR method has a lower sensitivity level of detection and suggest that potential future diagnosis for Brucella infection by Real-Time PCR can be reliable only if large numbers of infected cells are present. 2 3 PCR Analysis of Brucella in Macrophages 2.3. PCR Analysis of Brucella in Macrophages 2.3. PCR Analysis of Brucella in Macrophages Genomic DNA from cultured cells was extracted using the High Pure PCR template preparation kit (Roche, Basel, Switzerland) as per the manufacturer’s instructions. Furthermore, to exclude the presence of PCR inhibitors at a high concentration, we spiked an exogenous source of DNA template to our samples (Primerdesign, Chandler’s Ford, UK), which, after co-purification with our target DNA d i i l f h i i h R l Ti PCR Genomic DNA from cultured cells was extracted using the High Pure PCR template preparation kit (Roche, Basel, Switzerland) as per the manufacturer’s instructions. Furthermore, to exclude the presence of PCR inhibitors at a high concentration, we spiked an exogenous source of DNA template to our samples (Primerdesign, Chandler’s Ford, UK), which, after co-puriﬁcation with our target DNA, was used as a positive control for the extraction process with Real-Time PCR. DNA, was used as a positive control for the extraction process with Real-Time PCR. Quantification of internalized brucellae, was conducted with the Brucella genus Genesig advanced kit (Primerdesign, Chandler’s Ford, UK). From initial experimentation, we realized that utilization of the kit’s standards leads to different quantification of brucellae. Therefore, we generated in-house standards based on various amounts of B. melitensis 16 M genomic DNA deriving from lysed brucellae cells cultivated in our laboratory for the detection of the L-glutamine: 2-deoxy-scylloinosose aminotransferase gene as indicated by the commercial kit. Based on these results the number of brucellae that can be quantified ranges from 2.5 × 103 to 2.5 × 108 copies (R2 = 0.991). To determine the ratio of incorporated brucellae/cell, the absolute quantification method was employed, with the implementation of two single-plex reactions, one for the detection of the L-glutamine: 2-deoxy- scylloinosose aminotransferase gene as indicated by the commercial kit and a second one for the wild type ovine genomic DNA for the detection of the endogenous ovine prion protein (ovPrp) single- copy chromosomal gene [11,12] as previously described [9] (range 1–1 × 105 copies, R2 = 0.992). The primers for the ovPrp gene (ovPrpF 5′-GCCAAAAACCAACATGAAGCAT-3′ and ovPrpR 5′- TGCTCATGGCACTTCCCAG-3′) yielded a 95-bp amplicon. The standard curves produced were then used to extrapolate the absolute number of brucellae and ovPrp copies per reaction. 2.2. Infection of Macrophages with B. melitensis Fifty thousand ovine macrophages were seeded in 24-well tissue culture plates at a ﬁnal volume of 1 mL (Day −1). 24 h later (Day 0), the cells were counted by trypan blue exclusion, washed with phosphate-buﬀered saline (PBS) (Lonza, Basel, Switzerland), and exposed to Brucella at an MOI of 5000, 500, 50, or 5. The macrophage-speciﬁc medium (Macrophage-SFM, Invitrogen, Carlsbad, CA, USA, supplemented with heat-inactivated fetal bovine serum, Invitrogen, Carlsbad, CA, USA) used for the duration of the whole cell culture, did not contain any antibiotics at this point. The next day (Day 1), cells were washed with PBS and cultivated in macrophage-speciﬁc culture medium supplemented with 5% penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA) and 0.5 mg/mL lysozyme (Roche, Basel, Switzerland) to kill extracellular bacteria overnight. At the next day of the procedure (Day 2), cells were either washed with PBS to remove lysozyme, counted, and left in culture up to 4 more days (Day 3 or Day 6) in the macrophage-speciﬁc culture medium, or washed with PBS, detached from the culture plates with 0.05% trypsin-EDTA (Invitrogen, Carlsbad, CA, USA) and pelleted at 1400 rpm for 10 min, to exclude precipitating any extracellular brucellae that may had survived exposure to antibiotics and lysozyme. The pellets were resuspended in PBS and underwent 3 freezing-thawing cycles. Before DNA extraction, all samples were additionally incubated with 0.25 mg/mL lysozyme 3 of 9 e that S and Vet. Sci. 2019, 6, 71 USA) and pelle may had surviv for 1 h at 37 ◦C. Quantiﬁcation of internalized brucellae was conducted with quantitative Real-time PCR and CFU assay. Uninfected cells served as control at all times. Viability of macrophages was maintained by changing the cell culture medium and assessed by trypan blue exclusion at all time points of the experimental procedure. Especially for the time gap between Days 3 and 6, the wells were supplemented with the appropriate fresh medium volume in order to maintain the culture supernatant at 1 mL. The study is graphically outlined in Figure 1. incubated with 0.25 mg/mL lysozyme for 1 h at 37 °C. Quantification of internalized brucellae was conducted with quantitative Real-time PCR and CFU assay. Uninfected cells served as control at all times. Viability of macrophages was maintained by changing the cell culture medium and assessed by trypan blue exclusion at all time points of the experimental procedure. 2.2. Infection of Macrophages with B. melitensis Especially for the time gap between Days 3 and 6, the wells were supplemented with the appropriate fresh medium volume in order to maintain the culture supernatant at 1 mL. The study is graphically outlined in Figure 1. Figure 1. Schematic diagram and timeline of the study. Figure 1. Schematic diagram and timeline of the study. Figure 1 Schematic diagram and timeline of the study Figure 1. Schematic diagram and timeline of the study. At all time points, the presence or absence of live brucellae in the culture supernatant was verified by collecting the old culture media, spinning at 14,000 rpm and seeding the resuspended pellets in Columbia agar sheep blood plates. The same procedure was also applied in the supernatant collected from the final cell wash, that was implemented to remove trypsin, after harvesting the infected cells. To assess intracellular live brucellae, 500 infected cells were isolated and processed by adding 0.1% saponin (Sigma-Aldrich, St. Louis, MO, USA), incubated at room temperature for 5 min, pelleted at 14,000 rpm, resuspended in PBS and plated in Columbia agar sheep blood plates as previously described [10] At all time points, the presence or absence of live brucellae in the culture supernatant was veriﬁed by collecting the old culture media, spinning at 14,000 rpm and seeding the resuspended pellets in Columbia agar sheep blood plates. The same procedure was also applied in the supernatant collected from the ﬁnal cell wash, that was implemented to remove trypsin, after harvesting the infected cells. To assess intracellular live brucellae, 500 infected cells were isolated and processed by adding 0.1% saponin (Sigma-Aldrich, St. Louis, MO, USA), incubated at room temperature for 5 min, pelleted at 14,000 rpm, resuspended in PBS and plated in Columbia agar sheep blood plates as previously described [10]. 2 3 PCR Analysis of Brucella in Macrophages 2.3. PCR Analysis of Brucella in Macrophages Ultimately, brucellae per cell were calculated by normalizing the absolute number of brucellae to the ovPrp Quantiﬁcation of internalized brucellae, was conducted with the Brucella genus Genesig advanced kit (Primerdesign, Chandler’s Ford, UK). From initial experimentation, we realized that utilization of the kit’s standards leads to diﬀerent quantiﬁcation of brucellae. Therefore, we generated in-house standards based on various amounts of B. melitensis 16 M genomic DNA deriving from lysed brucellae cells cultivated in our laboratory for the detection of the L-glutamine: 2-deoxy-scylloinosose aminotransferase gene as indicated by the commercial kit. Based on these results the number of brucellae that can be quantiﬁed ranges from 2.5 × 103 to 2.5 × 108 copies (R2 = 0.991). To determine the ratio of incorporated brucellae/cell, the absolute quantiﬁcation method was employed, with the implementation of two single-plex reactions, one for the detection of the L-glutamine: 2-deoxy-scylloinosose aminotransferase gene as indicated by the commercial kit and a second one for the wild type ovine genomic DNA for the detection of the endogenous ovine prion protein (ovPrp) single-copy chromosomal gene [11,12] as previously described [9] (range 1–1 × 105 copies, R2 = 0.992). The primers for the ovPrp gene (ovPrpF 5′-GCCAAAAACCAACATGAAGCAT-3′ and ovPrpR 5′-TGCTCATGGCACTTCCCAG-3′) yielded a 95-bp amplicon. The standard curves produced were then used to extrapolate the absolute number of brucellae and ovPrp copies per reaction. Ultimately, brucellae per cell were calculated by normalizing the absolute number of brucellae to the ovPrp copies, in order to adjust for equal loading of genomic DNA per reaction. All reactions were conducted in a StepOne Real-Time PCR instrument (Applied Biosystems, Foster City, CA, USA). Vet. Sci. 2019, 6, 71 conducted in a A qualita 4 of 9 SA). was also A qualitative assessment of the intracellular and extracellular presence of brucellae was also performed by conventional end-point PCR (all reagents from Invitrogen, Carlsbad, CA, USA) in a total of 50 µL containing 5 µL DNA, 1× Dream Taq buﬀer, 0.2 mM dNTPs, 0.05 U/µL Dream Taq polymerase and a 1 µM concentration of the BMEII0843f (5′-TTTACACAGGCAATCCAGCA-3′) and BMEII0844r (5′-GCGTCCAGTTGTTGTTGATG-3′) primers as described previously [13], producing a 1071 bp amplicon. The ampliﬁcation was carried out according to the following conditions: initial denaturation at 95 ◦C for 7 min and 40 cycles of 95 ◦C for 1 min, 60 ◦C for 1 min and 72 ◦C for 1 min. The amplicons were analyzed by agarose gel electrophoresis. Establishment of the Ovine Macrophage Infection In Vitro Model We utilized wild type peripheral blood macrophages i i l dil ti th t d d t MOI f 5000 We utilized wild type peripheral blood macrophages from healthy sheep to infect with Brucella in serial dilutions that corresponded to an MOI of 5000, 500, 50, or 5 and assess the minimum detection limit of our assay regarding quantiﬁcation of internalized brucellae by Real-Time PCR. As depicted in Figure 2, the number of intracellular brucellae 48 h post-infection, was proportionally related to the increasing MOIs, since, on average, half of the bacteria indicated per MOI showed successful incorporation into macrophages. The minimum detection limit of our assay was produced at the lowest macrophage: Brucella ratio (MOI = 5) and corresponded to 3.97 ± 5.58 incorporated brucellae/cell. As expected, the MOI of 5000 produced the largest numbers of engulfed bacteria into the host cytoplasm (1549.32 ± 635.85 incorporated brucellae/cell). Moreover, the Ct’s produced from the exogenous DNA extraction control fell within the range indicated by the supplier (Ct = 25–31), demonstrating that minimal or no PCR inhibitors were present in our samples. However, it should be noted that the lowest number of total brucellae detectable by Real-Time PCR was approximately 2500 brucellae. in serial dilutions that corresponded to an MOI of 5000, 500, 50, or 5 and assess the minimum detection limit of our assay regarding quantification of internalized brucellae by Real-Time PCR. As depicted in Figure 2, the number of intracellular brucellae 48 h post-infection, was proportionally related to the increasing MOIs, since, on average, half of the bacteria indicated per MOI showed successful incorporation into macrophages. The minimum detection limit of our assay was produced at the lowest macrophage: Brucella ratio (MOI = 5) and corresponded to 3.97 ± 5.58 incorporated brucellae/cell. As expected, the MOI of 5000 produced the largest numbers of engulfed bacteria into the host cytoplasm (1549.32 ± 635.85 incorporated brucellae/cell). Moreover, the Ct’s produced from the exogenous DNA extraction control fell within the range indicated by the supplier (Ct = 25–31) demonstrating that minimal or no PCR inhibitors were present in our samples. However, it should be noted that the lowest number of total brucellae detectable by Real-Time PCR was approximately 2500 brucellae. Figure 2. Comparative analysis of incorporated brucellae per cell in ovine macrophages infected with Brucella at various multiplicities of infection (MOIs), 2 days post-infection (quantitative Real-Time PCR). 2.4. Statistics Multip considered s Multiple comparisons were performed using the one-way ANOVA. Values of p < 0.05 were considered statistically signiﬁcant. All results are expressed as means ± standard deviation (SD). y g p ( ) 3. Results 2 3 PCR Analysis of Brucella in Macrophages 2.3. PCR Analysis of Brucella in Macrophages total of 50 μL containing 5 μL DNA, 1× Dream Taq buffer, 0.2 mM dNTPs, 0.05 U/μL Dream Taq polymerase and a 1 μM concentration of the BMEII0843f (5′-TTTACACAGGCAATCCAGCA-3′) and BMEII0844r (5′-GCGTCCAGTTGTTGTTGATG-3′) primers as described previously [13], producing a 1071 bp amplicon. The amplification was carried out according to the following conditions: initial denaturation at 95 °C for 7 min and 40 cycles of 95 °C for 1 min, 60 °C for 1 min and 72 °C for 1 min The amplicons were analyzed by agarose gel electrophoresis. 2.4. Statistics Establishment of the Ovine Macrophage Infection In Vitro Model We utilized wild type peripheral blood macrophages i i l dil ti th t d d t MOI f 5000 MOI: multiplicity of infection. Figure 3. Comparative analysis of incorporated brucellae per cell in ovine macrophages infected with Brucella at various MOIs, 2 days post-infection (results from colony-forming unit (CFU) assay from 500 infected macrophages seeded in blood agar dishes). Each MOI condition was repeated 6 times (six different experiments on macrophages of 6 different sheep). Data are represented as means ± Figure 3. Comparative analysis of incorporated brucellae per cell in ovine macrophages infected with Brucella at various MOIs, 2 days post-infection (results from colony-forming unit (CFU) assay from 500 infected macrophages seeded in blood agar dishes). Each MOI condition was repeated 6 times (six diﬀerent experiments on macrophages of 6 diﬀerent sheep). Data are represented as means ± standard deviation. MOI: multiplicity of infection. Figure 3. Comparative analysis of incorporated brucellae per cell in ovine macrophages infected with Brucella at various MOIs, 2 days post-infection (results from colony-forming unit (CFU) assay from 500 infected macrophages seeded in blood agar dishes). Each MOI condition was repeated 6 times (six different experiments on macrophages of 6 different sheep). Data are represented as means ± standard deviation. MOI: multiplicity of infection. standard deviation. MOI: multiplicity of infection. Figure 4. Intracellular and extracellular brucellae from one representative experiment at an MOI = 5000. Lane 1: 100–1000 base pair ladder; Lane 2: culture supernatant from macrophages before addition of lysozyme and antibiotics (Day 1); Lane 3: culture supernatant from macrophages after addition of lysozyme and antibiotics (Day 2); Lane 4: infected macrophages after addition of lysozyme and antibiotics (Day 2); Lane 5: B. melitensis 16 M positive control; Lane 6: negative PCR control; Lane 7 i f d h Figure 4. Intracellular and extracellular brucellae from one representative experiment at an MOI = 5000. Lane 1: 100–1000 base pair ladder; Lane 2: culture supernatant from macrophages before addition of lysozyme and antibiotics (Day 1); Lane 3: culture supernatant from macrophages after addition of lysozyme and antibiotics (Day 2); Lane 4: infected macrophages after addition of lysozyme and antibiotics (Day 2); Lane 5: B. melitensis 16 M positive control; Lane 6: negative PCR control; Lane 7: uninfected macrophages. Figure 4. Intracellular and extracellular brucellae from one representative experiment at an MOI = 5000. Establishment of the Ovine Macrophage Infection In Vitro Model We utilized wild type peripheral blood macrophages i i l dil ti th t d d t MOI f 5000 Lane 1: 100–1000 base pair ladder; Lane 2: culture supernatant from macrophages before addition of lysozyme and antibiotics (Day 1); Lane 3: culture supernatant from macrophages after addition of lysozyme and antibiotics (Day 2); Lane 4: infected macrophages after addition of lysozyme and antibiotics (Day 2); Lane 5: B. melitensis 16 M positive control; Lane 6: negative PCR control; Lane 7: uninfected macrophages. MOI: multiplicity of infection. Figure 4. Intracellular and extracellular brucellae from one representative experiment at an MOI = 5000. Lane 1: 100–1000 base pair ladder; Lane 2: culture supernatant from macrophages before addition of lysozyme and antibiotics (Day 1); Lane 3: culture supernatant from macrophages after addition of lysozyme and antibiotics (Day 2); Lane 4: infected macrophages after addition of lysozyme and antibiotics (Day 2); Lane 5: B. melitensis 16 M positive control; Lane 6: negative PCR control; Lane Figure 4. Intracellular and extracellular brucellae from one representative experiment at an MOI = 5000. Lane 1: 100–1000 base pair ladder; Lane 2: culture supernatant from macrophages before addition of lysozyme and antibiotics (Day 1); Lane 3: culture supernatant from macrophages after addition of lysozyme and antibiotics (Day 2); Lane 4: infected macrophages after addition of lysozyme and antibiotics (Day 2); Lane 5: B. melitensis 16 M positive control; Lane 6: negative PCR control; Lane 7: uninfected macrophages. Figure 4. Intracellular and extracellular brucellae from one representative experiment at an MOI = 5000. Lane 1: 100–1000 base pair ladder; Lane 2: culture supernatant from macrophages before addition of lysozyme and antibiotics (Day 1); Lane 3: culture supernatant from macrophages after addition of lysozyme and antibiotics (Day 2); Lane 4: infected macrophages after addition of lysozyme and antibiotics (Day 2); Lane 5: B. melitensis 16 M positive control; Lane 6: negative PCR control; Lane 7: uninfected macrophages. 7: uninfected macrophages. Furthermore, we proceeded to culturing the Brucella-infected macrophages for up to 6 days, in order to evaluate any potential fluctuations in the number of incorporated brucellae over time, under conditions of heavy bacterial loads produced by the MOI of 5000. Summative analysis of experiments conducted with Brucella-infected macrophages at MOI = 5000, demonstrated a gradual drop in the bacterial load associated with duration in culture; from Day 2 to Day 6 post-Brucella infection bacterial numbers were significantly reduced (Day 2: 1549.32 ± 635.85 vs. Day 6: 276.67 ± 138.01, p = 0.0013, one-way ANOVA) (Figure 5). Establishment of the Ovine Macrophage Infection In Vitro Model We utilized wild type peripheral blood macrophages i i l dil ti th t d d t MOI f 5000 2019, 6, 71 supernatant p p p were formed. A g them in blood agar dishes. After approximately one week, single colonies were formed xperimental groups that further corroborated our previous observations (Figure 3, Table 1). ted cells and seeding them in blood agar dishes. After approximately one week, single colonies formed in all experimental groups that further corroborated our previous observations (Figure ble 1) The aforementioned were also verified with conventional PCR (Figure 4) rmed in all experimental groups that further corroborated our previous observations (Figure e 1). The aforementioned were also verified with conventional PCR (Figure 4). seeding them in blood agar dishes. After approximately one week, single colonies were formed in all experimental groups that further corroborated our previous observations (Figure 3, Table 1). The aforementioned were also veriﬁed with conventional PCR (Figure 4). infected cells and seeding them in blood agar dishes. After approximately one week, single colonies were formed in all experimental groups that further corroborated our previous observations (Figure 3, Table 1). The aforementioned were also verified with conventional PCR (Figure 4). were formed in all experimental groups that further corroborated our previous observations (Figure 3, Table 1). The aforementioned were also verified with conventional PCR (Figure 4). Figure 3. Comparative analysis of incorporated brucellae per cell in ovine macrophages infected with Brucella at various MOIs, 2 days post-infection (results from colony-forming unit (CFU) assay from 500 infected macrophages seeded in blood agar dishes). Each MOI condition was repeated 6 times (six different experiments on macrophages of 6 different sheep). Data are represented as means ± d d d i i MOI l i li i f i f i Figure 3. Comparative analysis of incorporated brucellae per cell in ovine macrophages infected with Brucella at various MOIs, 2 days post-infection (results from colony-forming unit (CFU) assay from 500 infected macrophages seeded in blood agar dishes). Each MOI condition was repeated 6 times (six diﬀerent experiments on macrophages of 6 diﬀerent sheep). Data are represented as means ± standard deviation. MOI: multiplicity of infection. Figure 3. Comparative analysis of incorporated brucellae per cell in ovine macrophages infected with Brucella at various MOIs, 2 days post-infection (results from colony-forming unit (CFU) assay from 500 infected macrophages seeded in blood agar dishes). Each MOI condition was repeated 6 times (six different experiments on macrophages of 6 different sheep). Data are represented as means ± standard deviation. Establishment of the Ovine Macrophage Infection In Vitro Model We utilized wild type peripheral blood macrophages i i l dil ti th t d d t MOI f 5000 Each MOI condition was repeated 6 times (six different experiments on macrophages of 6 different sheep) Data are represented as means ± standard deviation MOI: multiplicity of infection Figure 2. Comparative analysis of incorporated brucellae per cell in ovine macrophages infected with Brucella at various multiplicities of infection (MOIs), 2 days post-infection (quantitative Real-Time PCR). Each MOI condition was repeated 6 times (six diﬀerent experiments on macrophages of 6 diﬀerent sheep). Data are represented as means ± standard deviation. MOI: multiplicity of infection. Figure 2. Comparative analysis of incorporated brucellae per cell in ovine macrophages infected with Brucella at various multiplicities of infection (MOIs), 2 days post-infection (quantitative Real-Time PCR). Each MOI condition was repeated 6 times (six different experiments on macrophages of 6 diff t h ) D t t d t d d d i ti MOI lti li it f i f ti Figure 2. Comparative analysis of incorporated brucellae per cell in ovine macrophages infected with Brucella at various multiplicities of infection (MOIs), 2 days post-infection (quantitative Real-Time PCR). Each MOI condition was repeated 6 times (six diﬀerent experiments on macrophages of 6 diﬀerent sheep). Data are represented as means ± standard deviation. MOI: multiplicity of infection. p p p y Our results truly reflected intracellular bacteria, since the culture supernatant of the infected cells that was collected 24 h after their exposure to antibiotics and lysozyme and seeded in blood aga dishes, did not yield any colonies at all MOIs based on the CFU assay. Moreover, to verify that al extracellular brucellae were killed before proceeding to DNA extraction, we also washed the infected Our results truly reﬂected intracellular bacteria, since the culture supernatant of the infected cells that was collected 24 h after their exposure to antibiotics and lysozyme and seeded in blood agar dishes, did not yield any colonies at all MOIs based on the CFU assay. Moreover, to verify that all extracellular brucellae were killed before proceeding to DNA extraction, we also washed the infected cells to remove trypsin, after harvesting them from the 24-well plates at Day 2, and utilized the supernatant produced from this ﬁnal cell wash to seed in blood agar dishes. Once more, no colonies were formed. Additionally, the presence of intracellular live brucellae was veriﬁed by lysing 500 infected cells and 5 of 9 colonies i 500 o ies ng 500 l i Vet. Sci. Establishment of the Ovine Macrophage Infection In Vitro Model We utilized wild type peripheral blood macrophages i i l dil ti th t d d t MOI f 5000 This is consistent with a previous report with B. abortus in an infected macrophage cell line stating that the number of intracellular brucellae usually peaks at 48 h Furthermore, we proceeded to culturing the Brucella-infected macrophages for up to 6 days, in order to evaluate any potential fluctuations in the number of incorporated brucellae over time, under conditions of heavy bacterial loads produced by the MOI of 5000. Summative analysis of experiments conducted with Brucella-infected macrophages at MOI = 5000, demonstrated a gradual drop in the bacterial load associated with duration in culture; from Day 2 to Day 6 post-Brucella infection, bacterial numbers were significantly reduced (Day 2: 1549.32 ± 635.85 vs. Day 6: 276.67 ± 138.01, p = 0.0013, one-way ANOVA) (Figure 5). This is consistent with a previous report with B. abortus in an infected macrophage cell line stating that the number of intracellular brucellae usually peaks at 48 h Furthermore, we proceeded to culturing the Brucella-infected macrophages for up to 6 days, in order to evaluate any potential ﬂuctuations in the number of incorporated brucellae over time, under conditions of heavy bacterial loads produced by the MOI of 5000. Summative analysis of experiments conducted with Brucella-infected macrophages at MOI = 5000, demonstrated a gradual drop in the bacterial load associated with duration in culture; from Day 2 to Day 6 post-Brucella infection, bacterial numbers were signiﬁcantly reduced (Day 2: 1549.32 ± 635.85 vs. Day 6: 276.67 ± 138.01, p = 0.0013, one-way ANOVA) (Figure 5). This is consistent with a previous report with B. abortus in an infected macrophage cell line stating that the number of intracellular brucellae usually peaks at 48 h post-infection and until 96 h the load is gradually dropping [10]. This may be a result of natural intracellular killing of brucellae until they reach residual numbers [6] that basically persist for life, and not of macrophage death, since viability of cells, measured by trypan blue exclusion at all time 6 of 9 Vet. Sci. 2019, 6, 71 points, remained >90%. The high variation in incorporated Brucella numbers on Day 2 most likely reﬂects each animal’s sensitivity to infection. Table 1. Establishment of the Ovine Macrophage Infection In Vitro Model We utilized wild type peripheral blood macrophages i i l dil ti th t d d t MOI f 5000 Detailed results from all the experiments conducted to determine the number of incorporated brucellae in ovine macrophages infected with Brucella at various MOIs, 2 days post-infection either from 500 infected and lysed macrophages seeded in blood agar dishes or from 50,000 infected macrophages analyzed by Real-Time PCR. Each MOI condition was repeated 6 times (six diﬀerent experiments on macrophages of 6 diﬀerent sheep). MOI: multiplicity of infection. Table 1. Detailed results from all the experiments conducted to determine the number of incorporated brucellae in ovine macrophages infected with Brucella at various MOIs, 2 days post-infection either from 500 infected and lysed macrophages seeded in blood agar dishes or from 50,000 infected macrophages analyzed by Real-Time PCR. Each MOI condition was repeated 6 times (six diﬀerent experiments on macrophages of 6 diﬀerent sheep). MOI: multiplicity of infection. Total Number of Colonies (Agar) Brucellae/Cell (Real-Time PCR) Total Number of Colonies (Agar) Brucellae/Cell (Real-Time PCR) MOI 5000 Exp 1 350 2057.46 Exp 2 232 997.86 Exp 3 201 909.56 Exp 4 327 1111.22 Exp 5 288 1771.21 Exp 6 249 2448.61 Average 274.5 1549 SD 57.44 635.8 MOI 500 Exp 1 103 163.38 Exp 2 91 131.42 Exp 3 56 206.07 Exp 4 83 269.77 Exp 5 45 150.06 Exp 6 93 372.37 Average 78.5 215.5 SD 22.87 91.34 MOI 50 Exp 1 5 28.94 Exp 2 6 24.57 Exp 3 5 15.56 Exp 4 3 13.32 Exp 5 4 20.23 Exp 6 4 11.39 Average 4.5 19.00 SD 1.05 6.832 MOI 5 Exp 1 0 0.00 Exp 2 0 0.00 Exp 3 0 0.00 Exp 4 1 6.65 Exp 5 2 14.00 Exp 6 1 3.17 Average 0.67 3.97 SD 0.82 5.58 Total Number of Colonies (Agar) Brucellae/Cell (Real-Time PCR) 7 of 9 7 of 9 Vet. Sci. 2019, 6, 71 Vet. Sci. 2019, 6, x . Sci. 2019, 6, 71 7 o Vet. Sci. 2019, 6, x FOR PEER REVIEW 7 Figure 5. Summative analysis of incorporated brucellae per cell in ovine macrophages infected with Brucella at MOI = 5000, on Days 2, 3, and 6 post-infection (quantitative Real-Time PCR). Statistical comparison was performed with the one-way ANOVA and data are expressed as means ± standard deviation. Each MOI condition was repeated 6 times (six different experiments on macrophages of 6 different sheep). p > 0.005. Figure 5. 4. Discussion No PCR inhibitors were detected, that may have led For the ﬁrst time, we created a cellular model, using macrophages from sheep peripheral blood to simulate infection with Brucella melitensis. To the best of our knowledge, all previous studies reporting B. melitensis infection models, were based either on cell lines [15,16], or wild type monocytes isolated from peripheral venous blood from healthy volunteers [17]. There are also studies reporting cellular models with wild type sheep macrophages using other Brucella species, such as Brucella ovis [18]. Here, we exposed wild type ovine macrophages to serial dilutions of the microorganism and subjected the infected cells to Real-Time PCR. Our assay warranted a minimum detectable number of as little as 3.97 internalized brucellae per cell. No PCR inhibitors were detected, that may have led to jeopardizing the integrity of our data. to jeopardizing the integrity of our data. Viability of brucellae that were added to macrophages overnight was verified at all MOIs by collecting the culture supernatant, before adding antibiotics and lysozyme to cells, and seeding it after spinning, to blood agar dishes. Moreover, by lysing 500 infected macrophages after thei exposure to antibiotics and lysozyme, we were able to culture the intracellular brucellae in blood aga dishes. It is well-known that isolation and culture of Brucella in clinical samples by routine bacteriological culture methods can be particularly challenging due to the slow growth of the microorganism. Since seemingly negative culture dishes are often discarded after a one-week incubation period, diagnosis may be missed altogether. In order to maximize the detection o brucellae, long-term incubation of cultures that may take up to 30 days, as well as blind subculturing Viability of brucellae that were added to macrophages overnight was veriﬁed at all MOIs by collecting the culture supernatant, before adding antibiotics and lysozyme to cells, and seeding it, after spinning, to blood agar dishes. Moreover, by lysing 500 infected macrophages after their exposure to antibiotics and lysozyme, we were able to culture the intracellular brucellae in blood agar dishes. It is well-known that isolation and culture of Brucella in clinical samples by routine bacteriological culture methods can be particularly challenging due to the slow growth of the microorganism. Since seemingly negative culture dishes are often discarded after a one-week incubation period, diagnosis may be missed altogether. 4. Discussion 4. Discussion The basic approach for controlling and eradicating brucellosis has always been vaccination o herds, testing animals for infection and sending infected animals to slaughter [3]. These program have failed in southern Europe and alternatives are necessary to be found [14]. For instance, in mainland Greece, the program has been ongoing since 1978, and it has been failing to decrease the rates of infection under 2%, posing safety concerns towards traditional dairy products, such as feta cheese. In addition, reimbursement costs for seropositive animals, outlines a substantial financia burden not only for the European Union, but also for animal breeders as well [7]. Moreover, the attenuated vaccine used until nowadays, is not entirely safe for veterinarians who administer it and The basic approach for controlling and eradicating brucellosis has always been vaccination of herds, testing animals for infection and sending infected animals to slaughter [3]. These programs have failed in southern Europe and alternatives are necessary to be found [14]. For instance, in mainland Greece, the program has been ongoing since 1978, and it has been failing to decrease the rates of infection under 2%, posing safety concerns towards traditional dairy products, such as feta cheese. In addition, reimbursement costs for seropositive animals, outlines a substantial ﬁnancial burden not only for the European Union, but also for animal breeders as well [7]. Moreover, the attenuated vaccine used until nowadays, is not entirely safe for veterinarians who administer it and may cause undesired side eﬀects in animals [5]. y y may cause undesired side effects in animals [5]. For the first time, we created a cellular model, using macrophages from sheep peripheral blood to simulate infection with Brucella melitensis. To the best of our knowledge, all previous studie reporting B. melitensis infection models, were based either on cell lines [15,16], or wild type monocyte isolated from peripheral venous blood from healthy volunteers [17]. There are also studies reporting cellular models with wild type sheep macrophages using other Brucella species, such as Brucella ovi [18]. Here, we exposed wild type ovine macrophages to serial dilutions of the microorganism and subjected the infected cells to Real-Time PCR. Our assay warranted a minimum detectable numbe of as little as 3.97 internalized brucellae per cell. Establishment of the Ovine Macrophage Infection In Vitro Model We utilized wild type peripheral blood macrophages i i l dil ti th t d d t MOI f 5000 Summative analysis of incorporated brucellae per cell in ovine macrophages infected with Brucella at MOI = 5000, on Days 2, 3, and 6 post-infection (quantitative Real-Time PCR). Statistical comparison was performed with the one-way ANOVA and data are expressed as means ± standard deviation. Each MOI condition was repeated 6 times (six diﬀerent experiments on macrophages of 6 diﬀerent sheep). p > 0.005. Figure 5. Summative analysis of incorporated brucellae per cell in ovine macrophages infected with Brucella at MOI = 5000, on Days 2, 3, and 6 post-infection (quantitative Real-Time PCR). Statistical comparison was performed with the one-way ANOVA and data are expressed as means ± standard deviation. Each MOI condition was repeated 6 times (six different experiments on macrophages of 6 different sheep) p > 0 005 Figure 5. Summative analysis of incorporated brucellae per cell in ovine macrophages infected with Brucella at MOI = 5000, on Days 2, 3, and 6 post-infection (quantitative Real-Time PCR). Statistical comparison was performed with the one-way ANOVA and data are expressed as means ± standard deviation. Each MOI condition was repeated 6 times (six diﬀerent experiments on macrophages of 6 diﬀerent sheep). p > 0.005. 4. Discussion In order to maximize the detection of brucellae, long-term incubation of cultures that may take up to 30 days, as well as blind subculturing have been advised [19]. However, this approach is labor-intensive, expensive and diagnosis is substantially delayed. g y p y g have been advised [19]. However, this approach is labor-intensive, expensive and diagnosis is substantially delayed. Here, we’ve employed different methods, both molecular- and culture-based, to demonstrate Here, we’ve employed diﬀerent methods, both molecular- and culture-based, to demonstrate the presence of truly engulfed bacteria into the host cytoplasm. Based on our results, the CFU assay generates lower numbers of intracellular brucellae/cell, probably because not all engulfed brucellae 8 of 9 Vet. Sci. 2019, 6, 71 can ultimately grow in culture after lysis of macrophages. Another reason for this discrepancy might be that intracellular brucellae are no longer alive but their DNA is still detectable. However, the CFU assay was a lot more sensitive compared to our Real-Time PCR assay since it can detect engulfed brucellae from only 500 infected macrophages with an average copy number of 0.67 as indicated in Table 1 and as indicated in Figure 3 it was able to detect as low as 0.005 or less brucellae/cell, compared to the calculated sensitivity of the Real-Time PCR assay which had a cutoﬀof 2,500 brucellae in total. These results indicate that in order to reliably diagnose B. melitensis by Real-Time PCR, we would need at least 2,500 cells isolated from an infected animal with an average of 1 engulfed Brucella/cell. Hence, because the level of sensitivity of the Real-Time PCR was lower compared to the CFU assay, the safe conclusion that can be drawn suggests that the Real-Time PCR could be potentially utilized to detect intracellular brucellae only if there is either a high bacterial load, or if the starting number of cells in the isolated tissue from infected animals is over 2,500. Alternatively, addition of concentration steps in clinical samples would further facilitate detection of residual brucellae by real-time PCR, since culture of Brucella in clinical samples by routine bacteriological culture methods can be particularly challenging due to the slow growth of the microorganism. In fact, we are already successfully implementing the method on samples from conﬁrmed B. melitensis cases in farms all over Greece (manuscript in preparation). 4. Discussion Although culturing is an indispensable method to provide proof of intracellular viable bacteria, we herein provide further insight on the detection of Brucella infection with an incremental approach for detecting intracellular brucellae which could accompany the culture-based techniques speciﬁcally when results need to be delivered in a short timeline. Overall, our Real-Time PCR approach could be particularly useful in the detection of persistent Brucella infections in animals, where too few bacteria are present in diﬃcult-to-lyse tissues, such as ganglia lymph nodes, and may thus not be easily traced by conventional blood agar culturing methods, given that high cell numbers are obtained from infected animals. 5. Conclusions In summary, the work presented here constitutes a reliable in vitro cell model of Brucella melitensis for research purposes that can be utilized to adequately measure the number of engulfed brucellae/cell and provides insights towards future utilization of molecular biology-based methods for detection of Brucella. Author Contributions: G.K. designed and performed the experiments and wrote the manuscript. S.K.K. coordinated the study and helped to draft the manuscript. E.P. (Eleni Papanikolaou) conceived the study, designed the experiments and helped to draft the manuscript. E.P. (Evanthia Petridou) conceived and coordinated the study and helped to draft the manuscript. All authors read and approved the ﬁnal manuscript. Funding: This post-doctoral research was conducted with a scholarship from the Greek State Scholarships Foundation, which was funded by the Operation “Support for post-doctoral researchers” from the resources of the Operational Program “Human Resources Development, Education and Lifelong Learning” with Priority Axes 6, 8 and 9 and is co-funded by the European Social Fund (ESF) and the Greek state. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. 1. Díaz Aparicio, E. Epidemiology of brucellosis in domestic animals caused by Brucella melitensis, Brucella suis and Brucella abortus. Rev. Sci. Tech. 2013, 32, 53–60. [CrossRef] 2. Cutler, S.J.; Whatmore, A.M.; Commander, N.J. Brucellosis—New aspects of an old disease. J. Appl. Microbiol. 2005, 98, 1270–1281. [CrossRef] [PubMed] 3. Avila-Calderón, E.; Lopez-Merino, A.; Sriranganathan, N.; Boyle, S.; Contreras-Rodríguez, A. A history of the development of Brucella vaccines. Biomed. Res. Int. 2013, 2013, 743509. [CrossRef] [PubMed] 4. Higgins, J.L.; Bowen, R.A.; Gonzalez-Juarrero, M. Evaluation of shedding, tissue burdens, and humoral immune response in goats after experimental challenge with the virulent Brucella melitensis strain 16M and the reduced virulence vaccine strain Rev. 1. PLoS ONE 2017, 12, e0185823. [CrossRef] References 1. Díaz Aparicio, E. Epidemiology of brucellosis in domestic animals caused by Brucella melitensis, Brucella suis and Brucella abortus. Rev. Sci. Tech. 2013, 32, 53–60. [CrossRef] 2. Cutler, S.J.; Whatmore, A.M.; Commander, N.J. Brucellosis—New aspects of an old disease. J. Appl. Microbiol. 2005, 98, 1270–1281. [CrossRef] [PubMed] 3. Avila-Calderón, E.; Lopez-Merino, A.; Sriranganathan, N.; Boyle, S.; Contreras-Rodríguez, A. A history of the development of Brucella vaccines. Biomed. Res. Int. 2013, 2013, 743509. [CrossRef] [PubMed] 4. Higgins, J.L.; Bowen, R.A.; Gonzalez-Juarrero, M. Evaluation of shedding, tissue burdens, and humoral immune response in goats after experimental challenge with the virulent Brucella melitensis strain 16M and the reduced virulence vaccine strain Rev. 1. PLoS ONE 2017, 12, e0185823. [CrossRef] 4. Higgins, J.L.; Bowen, R.A.; Gonzalez-Juarrero, M. Evaluation of shedding, tissue burdens, and humoral immune response in goats after experimental challenge with the virulent Brucella melitensis strain 16M and the reduced virulence vaccine strain Rev. 1. PLoS ONE 2017, 12, e0185823. [CrossRef] Vet. Sci. 2019, 6, 71 9 of 9 5. Blasco, J.M. A review of the use of B. melitensis Rev 1 vaccine in adult sheep and goats. Prev. Vet. Med. 1997, 31, 275–283. [CrossRef] 6. Von Bargen, K.; Gorvel, J.P.; Salcedo, S.P. Internal aﬀairs: Investigating the Brucella intracellular lifestyle. FEMS Microbiol. Rev. 2012, 36, 533–562. [CrossRef] [PubMed] 7. Donev, D.M. Brucellosis as priority public health challenge in South Eastern European countries. Croat. Med. J. 2010, 51, 283–284. [CrossRef] [PubMed] 8. World Organization for Animal Health (OIE). Brucellosis (Brucella abortus, B. melitensis and B. suis). In OIE Terrestrial Manual 2018; World Organization for Animal Health (OIE): Paris, France, 2018; Chapter 3.1.4; pp. 355–398. pp 9. Karponi, G.; Kritas, S.; Petridou, E.; Papanikolaou, E. Eﬃcient transduction and expansion of ovine macrophages for gene therapy implementations. Vet. Sci. 2018, 5, 57. [CrossRef] [PubMed] 10. Jiang, X.; Baldwin, C.L. Eﬀects of cytokines on intracellular growth of Brucella abortus. Infect. Immun. 1993, 61, 124–134. [PubMed] 11. Lee, I.Y.; Westaway, D.; Smit, A.F.A.; Wang, K.; Seto, J.; Chen, L.; Acharya, C.; Ankener, M.; Baskin, D.; Cooper, C.; et al. Complete genomic sequence and analysis of the prion protein gene region from three mammalian species. Genome Res. 1998, 8, 1022–1037. [CrossRef] [PubMed] 12. Garcia-Crespo, D.; Juste, R.A.; Hurtado, A. Selection of ovine housekeeping genes for normalisation by real-time RT-PCR; analysis of PrP gene expression and genetic susceptibility to scrapie. BMC Vet. Res. 2005, 1, 3. [CrossRef] [PubMed] 13. References García-Yoldi, D.; Marín, C.M.; De Miguel, M.J.; Muñoz, P.M.; Vizmanos, J.L.; López-Goñi, I. Multiplex PCR assay for the identiﬁcation and diﬀerentiation of all Brucella species and the vaccine strains Brucella abortus S19 and RB51 and Brucella melitensis Rev1. Clin. Chem. 2006, 52, 779–781. [CrossRef] [PubMed] 14. Pappas, G.; Papadimitriou, P.; Akritidis, N.; Christou, L.; Tsianos, E.V. The new global map of human brucellosis. Lancet Infect. Dis. 2006, 6, 91–99. [CrossRef] 15. Jiménez De Bagüés, M.P.; Terraza, A.; Gross, A.; Dornand, J. Diﬀerent responses of macrophages to smooth and rough Brucella spp.: relationship to virulence. Infect. Immun. 2004, 72, 2429–2433. [CrossRef] [PubMed] 15. Jiménez De Bagüés, M.P.; Terraza, A.; Gross, A.; Dornand, J. Diﬀerent responses of macrophages to smooth and rough Brucella spp.: relationship to virulence. Infect. Immun. 2004, 72, 2429–2433. [CrossRef] [PubMed] 16. Lecároz, C.; Blanco-Prieto, M.J.; Burrell, M.A.; Gamazo, C. Intracellular killing of Brucella melitensis in 15. Jiménez De Bagüés, M.P.; Terraza, A.; Gross, A.; Dornand, J. Diﬀerent responses of macrophages to smooth and rough Brucella spp.: relationship to virulence. Infect. Immun. 2004, 72, 2429–2433. [CrossRef] [PubMed] 16. Lecároz, C.; Blanco-Prieto, M.J.; Burrell, M.A.; Gamazo, C. Intracellular killing of Brucella melitensis in human macrophages with microsphere-encapsulated gentamicin. J. Antimicrob. Chemother. 2006, 58, 549–556. [CrossRef] [PubMed] 16. Lecároz, C.; Blanco-Prieto, M.J.; Burrell, M.A.; Gamazo, C. Intracellular killing of Brucella melitensis in human macrophages with microsphere-encapsulated gentamicin. J. Antimicrob. Chemother. 2006, 58, 549–556. [CrossRef] [PubMed] 17. Fernandez-Prada, C.M.; Zelazowska, E.B.; Nikolich, M.; Hadﬁeld, T.L.; Roop, R.M., II; Robertson, G.L.; Hoover, D.L. Interactions between Brucella melitensis and human phagocytes: Bacterial surface O-polysaccharide inhibits phagocytosis, bacterial killing, and subsequent host cell apoptosis. Infect. Immun. 2003, 71, 2110–2119. [CrossRef] [PubMed] 18. Macedo, A.A.; Silva, A.P.C.; Mol, J.P.S.; Costa, L.F.; Garcia, L.N.; Araújo, M.S.; Martins Filho, O.A.; Paixão, T.A.; Santos, R.L. The abcEDCBA-encoded ABC transporter and the virB operon-encoded type IV secretion system of brucella ovis are critical for intracellular traﬃcking and survival in ovine monocyte-derived macrophages. PLoS ONE 2015, 10, e0138131. [CrossRef] [PubMed] 9. Yagupsky, P. Detection of brucellae in blood cultures. J. Clin. Microbiol. 1999, 37, 3437–3442. [PubMed] 19. Yagupsky, P. Detection of brucellae in blood cultures. J. Clin. Microbiol. 1999, 37, 3437–3442. [PubMed] © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). © 2019 by the authors. References Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W3014892831	https://www.frontiersin.org/articles/10.3389/fphar.2020.00275/pdf	English	null	Short-Term Efficacy and Tolerability of Paroxetine Versus Placebo for Panic Disorder: A Meta-Analysis of Randomized Controlled Trials	Frontiers in pharmacology	2,020	cc-by	7,733	Short-Term Efﬁcacy and Tolerability of Paroxetine Versus Placebo for Panic Disorder: A Meta-Analysis of Randomized Controlled Trials Beilin Zhang †, Chao Wang †, Lexiang Cui †, Jiguo Gao, Chenglin Wang, Xiangyu Tan and Shaokuan Fang* Department of Neurology, Neuroscience Centre, The First Teaching Hospital of Jilin University, Changchun, China Edited by: Arjan Blokland, Maastricht University, Netherlands Objective: To explore the short-term efﬁcacy and tolerability of paroxetine in the treatment of panic disorder in adults. Methods: Multiple electronic databases were searched to ﬁnd randomized controlled trials (RCTs) on paroxetine and panic disorder. The primary efﬁcacy outcomes were: the mean change compared to the baseline in the total number of full panic attacks, Clinical Global Impression-Severity of Illness (CGI-S) score, and the proportion of participants with zero full panic attacks and with a 50% or greater reduction in the number of full panic attacks. The tolerability outcomes included withdrawal rate and the incidence of adverse events (AEs). Reviewed by: Marin Veldic, Mayo Clinic, United States Edward John Ogden, Swinburne University of Technology, Australia Correspondence: Shaokuan Fang fang20063536@sina.com †These authors have contributed equally to this work Correspondence: Shaokuan Fang fang20063536@sina.com †These authors have contributed equally to this work g fang20063536@sina.com †These authors have contributed equally to this work Results: 13RCTs were included. The pooled analyses showed patients who received paroxetine experienced greater improvements in the number of full panic attacks (total: MD=-1.96, 95%CI -3.45 to -0.47, P=0.010; ≥50% reduction: OR=1.66, 95%CI 1.08 to 2.55, P=0.02; zero full panic attacks: OR=1.70, 95%CI 1.42 to 2.03, P < 0.00001) and CGI-S (MD=-0.37, 95%CI -0.74 to -0.01, P=0.05) than placebo. There was no evident difference in the total withdrawal rate (OR=0.91, 95%CI 0.76 to 1.08, P=0.26) and withdrawal rate due to AEs (OR=1.29, 95%CI 0.97 to 1.72, P=0.07) between the two groups. Withdrawal rate due to lack of efﬁcacy or relapse (OR=0.44, 95%CI 0.31 to 0.63, P < 0.00001) and the incidence of serious AEs (OR=0.42, 95%CI 0.23 to 0.79, P=0.007) in the paroxetine group was lower than the placebo group. Meanwhile, the incidence of any treatment-emergent adverse events (TEAEs) (OR=1.32, 95%CI 1.05 to 1.64, P=0.02) in the paroxetine group was higher in comparison with the placebo. †These authors have contributed equally to this work Specialty section: This article was submitted to Neuropharmacology, a section of the journal Frontiers in Pharmacology Specialty section: This article was submitted to Neuropharmacology, a section of the journal Frontiers in Pharmacology Received: 21 November 2019 Accepted: 26 February 2020 Published: 31 March 2020 Received: 21 November 2019 Accepted: 26 February 2020 Published: 31 March 2020 Keywords: efﬁcacy, tolerability, paroxetine, panic disorder, meta-analysis SYSTEMATIC REVIEW published: 31 March 2020 doi: 10.3389/fphar.2020.00275 SYSTEMATIC REVIEW published: 31 March 2020 doi: 10.3389/fphar.2020.00275 INTRODUCTION above data. (5) randomized controlled trials (RCTs) were used as the study types. Panic disorder (PD) is a common mental health disorder with a lifetime prevalence of 1.6% to 2.2% in the general population (American Psychiatric Association, 2009), while in primary care settings, panic syndromes have been reported to have a prevalence of around 10% (King et al., 2008). PD is characterized by repeated, unexpected panic attacks, which are discrete periods of fear or anxiety that have a rapid onset, reach a peak within 10 minutes, and in which at least 4 of 13 characteristic symptoms are experienced (Bighelli et al., 2018). Many of these symptoms involve bodily systems, such as a racing heart, chest pain, sweating, shaking, dizziness, ﬂushing, stomach churning, faintness, and breathlessness (Bighelli et al., 2018). Unless the clinician has a high index of suspicion, panic disorder may remain undetected (Tsuboi and Masuko, 2001). PD is also reported to be associated with comorbid major depression disorder and its prevalence is 24%-88%, which will increase the risk of suicidal behavior (Dannon et al., 2004). Studies were excluded if they included patients with: (1) severe physical disease; (2) another axis I disorder instead of panic disorder; (3) recent major depression disorder (unless panic attacks were deemed to be the predominant disorder and preceded affective symptoms chronologically); (4) Hamilton Depression(HAMD) scale(17-item) total score≥18; (5) drug or alcohol dependence or abuse within the last 6 months; (6) substantial suicide risk; (7) use of anxiolytics and/or other antidepressants within 2 weeks before the randomized treatment; (8) poor response or hypersensitivity to a SSRI in the past; (9) receiving paroxetine or electroconvulsive therapy 3 months before study entry; or cognitive behavioral therapy within 30 days before randomization. Studies from which we could not obtain enough valid information were also removed. METHODS The following work was conducted according to the preferred reporting items for systematic reviews and meta-analyses (PRISMA) statement (Moher et al., 2009). The PROSPERO registration number is: CRD42019145115. Searching Other Resources The references of the relevant systematic reviews, meta-analyses, and all the included literature were also searched in order to avoid missing any possibly necessary literature. Citation: Zhang B, Wang C, Cui L, Gao J, Wang C, Tan X and Fang S (2020) Short-Term Efﬁcacy and Tolerability of Paroxetine Versus Placebo for Panic Disorder: A Meta-Analysis of Randomized Controlled Trials. Front. Pharmacol. 11:275. doi: 10.3389/fphar.2020.00275 Conclusions: Paroxetine is an effective and well-tolerated short-term treatment for adults with panic disorder. March 2020 \| Volume 11 \| Article 275 1 Frontiers in Pharmacology \| www.frontiersin.org Paroxetine and Panic Disorder Zhang et al. Electronic Searches These electronic databases were searched from their inception to July 25, 2019: PubMed, Web of Science, Embase, Cochrane Central Register of Controlled Trials, ScienceDirect, Scopus, PsycINFO, Wanfang, China Biology Medicine disc (CBMdisc), Chongqing VIP, and China National Knowledge Infrastructure (CNKI). We limited the languages to Chinese and English, without any restrictions on publication status. The search terms were: (paroxetine OR seroxat OR cylert OR BRL29060 OR FG-7051 OR brisdelle OR LDMP OR pexeva OR paxil OR aropax) AND (“panic disorder” OR “panic attack” OR agoraphobia OR “acute anxiety”). Medical Subject Headings (MESH) words and free words were used to improve recall ratio. We also searched Clinical Trials.gov to ﬁnd unpublished studies. Details of the search strategy were shown in Supplementary S1 ﬁle and the same search strategy was used to retrieve the above databases again on August 31 to discover if there were any new literature that needed to be included. We have translated the search terms of Chinese databases into corresponding English to show it better. Search Methods for Identiﬁcation of Studies Selective serotonin reuptake inhibitors (SSRIs) are a known ﬁrst-line therapy for PD. Among them, paroxetine was ﬁrst approved by the U.S Food and Drug Administration for the treatment of PD. It can selectively inhibit the re-uptake of serotonin in the presynaptic membrane, and thus increase the concentration of serotonin in the synaptic gap and enhance the central 5-hydroxytryptaminergic neurological function. To date, a series of randomized, double-blind, placebo-controlled clinical trials have been carried out to study its short-term efﬁcacy and tolerability, however there are still relatively few quantitative analyses on it. Therefore, we conducted a meta-analysis and systematically reviewed the relevant literature to evaluate the short-term efﬁcacy and tolerability of paroxetine in order to provide evidence for clinical treatment and research of PD in adults. Frontiers in Pharmacology \| www.frontiersin.org Data Collection and Analysis Selection of Studies The inclusion criteria were as follows: (1) participants had to be patients aged 18 years or older with a principal diagnosis of PD, with or without agoraphobia, and met any of the following criteria: International Classiﬁcation of Disease-10 (ICD-10), or Diagnostic and Statistical Manual of Mental Disorders (DSM-III, DSM-IIIR,DSM-IV); (2) an intervention in which paroxetine was used as monotherapy with a duration of less than 6 months. There was no restriction on dose and frequency; (3) a placebo was used as a comparison; (4) the outcome measures were indicators of efﬁcacy and tolerability. The article might be included in the meta-analysis as long as it had one of the Two authors used End Note X9 and manually removed any duplicate literature independently. Then we browsed the titles and abstracts of remaining documents to exclude literature that did not conform to inclusion criteria. At last, we read the full text of the remaining literature after screening carefully to determine the ﬁnal included literature and clarify the speciﬁc reasons for the removal. If there was any disagreement during this process, it was necessary to reach a consensus after a consultation with the third member. March 2020 \| Volume 11 \| Article 275 Frontiers in Pharmacology \| www.frontiersin.org 2 Zhang et al. Paroxetine and Panic Disorder Assessment of Risk of Bias in Included Studies Assessment of Risk of Bias in Included Studies The Cochrane Collaboration’s tool for assessing risk of bias in randomised trials (Higgins et al., 2011) consists of six domains: selection bias (random sequence generation, allocation concealment), performance bias (blinding of participants and personnel), detection bias (blinding of outcome assessment), attrition bias (incomplete outcome data), reporting bias (selective reporting), and other biases. Two members independently evaluated each domain with “low risk”, “high risk”, or “unclear risk”. If they could not come to an agreement, the decision should be made after discussion with the third member. “High risk of bias” may alter the results seriously, “low risk of bias”, if present, is unlikely to alter the results seriously, and “unclear risk of bias” means it would raise some doubt about the results (Higgins et al., 2011). Data Extraction and Management Phobia Scale(MSPS)overall phobia, total avoidance and fear scores, Montgomery-Asberg Depression Rating Scale (MADRS) score, and Social Adjustment self-report Questionnaire (SAQ) score. These data were summarized in an Excel spreadsheet: the ﬁrst authors, publication year, study design, diagnostic criteria, location, intention-to-treat and completed numbers, gender and age of participants, intervention and control measures, treatment duration, and indicators of efﬁcacy and tolerability. The above work was carried out by two individuals independently and a third person helped them negotiate and resolve any disputes that may have arisen. Dealing With Missing Data For dichotomous variables, if the original literature did not provide the speciﬁc number of events, but provided the events rate, we would calculate the number of events for analysis. For continuous variables, if the literature only provided the standard error (SE) rather than the standard deviation (SD), we would calculate the SD according to the following formula: SE = SD= ﬃﬃﬃn p . (n is the corresponding sample size in each data). If there were still some missing data that affected our analysis in the included literature, we would contact the original authors to obtain the corresponding information. Unit of Analysis Issues The studies we included were all randomized parallel- group trials. If a trial involved multiple treatment groups, such as three or more arms trial that investigated different dose of paroxetine and placebo, or compared two different antidepressants and placebo, we would only compare the paroxetine arm with placebo or each dose of paroxetine with placebo, respectively. Assessment of Heterogeneity 2 We focused on the efﬁcacy and tolerability of paroxetine compared with a placebo in the treatment of PD. The primary efﬁcacy was mainly reﬂected by the mean change compared to the baseline in the total number of full panic attacks and Clinical Global Impression-Severity of Illness (CGI-S) scale score, as well as the proportion of participants with zero full panic attacks and with a 50% or greater reduction in the number of full panic attacks. Secondary efﬁcacy outcomes included mean change compared to the baseline of various scales, the intensity of anticipatory anxiety, as well as response and remission rate. The response rate was deﬁned as a Clinical Global Impression- Improvement (CGI-I) rating of “very much improved” or “much improved” and the remission rate was deﬁned as CGI-S rating of “not at all ill” or “borderline ill” and no Panic and Anticipatory Anxiety Scale (PAAS) full-symptom panic attacks (Pollack et al., 2007b). Tolerability was deﬁned as the withdrawal rate and the incidence of AEs. We used chi-square (Chi2) test and its P value as well as I-square (I2) value to evaluate heterogeneity. I2≥0% is non-heterogeneous, I2≥25% is considered to be mild heterogeneity, I2≥50% is considered to be moderate heterogeneity, and I2≥75% is considered to be severe heterogeneity (Shi et al., 2019). P=0.10 is used as a threshold of statistical signiﬁcance (Bighelli et al., 2018). Frontiers in Pharmacology \| www.frontiersin.org Description of Studies Results of the Search A total of 1421 records were identiﬁed through the electronic database searching and 6 additional documents were found after retrieving the reference list of relevant systematic review, meta- analyses, and included studies. After removing the duplicates by using End Note X9 software and manually, 804 records remained. We next browsed the titles and abstracts of records left and 785 records that did not meet the inclusion criteria were removed.We also read the full-text of 19 articles to evaluate their eligibility, then 8 articles were excluded and 11 studies were included for qualitative synthesis. 13 RCTs were ﬁnally included in the meta-analysis. See Figure 1 for PRISMA ﬂow diagram. Assessment of Reporting Biases b h h d Reporting biases arise when the dissemination of research ﬁndings is inﬂuenced by the nature and direction of results (Sterne et al., 2008). Our meta-analysis mainly involved the following two types of reporting biases: publication bias and outcome reporting bias. The former is the publication or non- publication of research ﬁndings and the latter is selective reporting of some outcomes instead of others, both depending on the nature and direction of the outcomes (Sterne et al., 2008). Dichotomous data: the subjects with zero full panic attacks, the proportion of participants with a 50% or greater reduction in the number of full panic attacks, response and remission rate, total withdrawal rate, withdrawal rate due to a lack of efﬁcacy or relapse, withdrawal rate due to AEs, the incidence of any treatment-emergent adverse events (TEAEs), serious adverse events (SAEs), and common TEAEs. Funnel plots were used to visually reﬂect the presence or absence of publication bias; however, it should not be used when the number of studies was less than 10. Begg’s and Egger’s test were used for quantitative assessment of publication bias. P < 0.05 indicated the publication bias was signiﬁcant (Shi et al., 2019). To detect selective outcome reporting bias, we compared the outcomes of published literature with their trial registries, if they were available. Otherwise, we compared the methods and outcomes in the publications. If all prespeciﬁed outcomes were reported, we could consider it as “low risk”. Continuous data: mean change compared to the baseline for the following data: total number of full panic attacks, intensity of anticipatory anxiety, Hamilton Anxiety (HAMA) Scale total score, Sheehan Disability Scale (SDS) work, social life and family life scores, CGI-S score, CGI-I score, Marks Sheehan March 2020 \| Volume 11 \| Article 275 Frontiers in Pharmacology \| www.frontiersin.org 3 Zhang et al. Paroxetine and Panic Disorder RESULTS The Review Manager (RevMan) 5.3 software was used in our data synthesis. Dichotomous data were pooled by the Mantel- Haenszel (M-H) statistical method and odds ratio (OR) with a 95% conﬁdence interval (CI). Continuous data were processed by the Inverse Variance statistical method and mean difference (MD) with 95% CI. The ﬁxed-effect model was applied by default and the random-effects model was used in case of substantial heterogeneity (I2≥50% and P < 0.10) (Hugues et al., 2017). The overall effect was calculated by Z test, thereof P value ≤0.05 means statistically signiﬁcant. Description of Studies Results of the Search Included Studies All included studies were multicenter, randomized, double-blind, parallel-group placebo-controlled trials. They were conducted from 1994 to 2007. The total number of intention-to-treat (ITT) population in 13 RCTs were 2654, among which 1329 were in the intervention group and 1325 were in the control group. Mean age ranged from 34.7 to 45.0 years. 4 studies were three arms, 4 studies were four arms, and 3 studies were two arms. The ﬁxed dose of paroxetine was in 6 RCTs and ﬂexible dose in 7 RCTs. The maximum dosage reached 75mg. Course of treatment Subgroup Analysis and Investigation of Heterogeneity Subgroup analysis is a common method to deal with heterogeneity. If the pooled data suggested signiﬁcant heterogeneity, we need to look for sources of heterogeneity. In our meta-analysis, if there were enough studies available, we could conduct subgroup analysis based on the dose or duration of paroxetine. FIGURE 1 \| PRISMA ﬂow diagram. Secondary Efﬁcacy Outcomes There was no signiﬁcant difference in the mean change compared to the baseline in intensity of anticipatory anxiety (4RCTs, MD=-0.21, 95%CI -0.69 to -0.28, P=0.40) between the two groups (Figure 4A). No heterogeneity was detected (P=0.66, I2 = 0%). There was also no statistically signiﬁcant difference of its baseline between the two groups (P=0.92). All RCTs described total withdrawal rate, and the results showed that the total withdrawal rate between the two groups was not statistically signiﬁcant (13RCTs, OR=0.91, 95%CI 0.76 to 1.08, P=0.26) and no substantial heterogeneity was detected (P=0.17, I2 = 27%) (Figure 6A).The withdrawal rate due to a lack of efﬁcacy or relapse was reported in 12 RCTs, while no heterogeneity was detected after a pooled analysis(P=0.90, I2 = 0%), and the results showed that fewer patients in the paroxetine group were withdrawn due to a lack of efﬁcacy or relapse than those in the placebo group, which was statistically signiﬁcant (12RCTs, OR=0.44, 95%CI 0.31 to 0.63, P < 0.00001) (Figure 6B). There was no obvious difference in withdrawal rates due to AEs between the two groups (11RCTs, OR=1.29, 95%CI 0.97 to 1.72, P=0.07), and we didn’t ﬁnd any substantial heterogeneity (P=0.16, I2 = 31%) (Figure 6C). Mean change compared to the baseline of HAMA total score (9RCTs, MD=-2.96, 95%CI -3.87 to -2.05, P < 0.00001) (Figure 4B) and CGI-I total score (4RCTs, MD=-0.37, 95%CI -0.64 to -0.10, P=0.008) (Figure 4C) in the paroxetine group was higher than that in placebo group. No substantial heterogeneity was identiﬁed in the former (P=0.28, I2 = 18%). For the latter, on account of its signiﬁcant heterogeneity (P=0.07, I2 = 57%), we applied the random-effects model. There was no statistically signiﬁcant difference of former baseline between the two groups (P=0.21). Risk of Bias in Included Studies Mean change compared to the baseline of SDS work (5RCTs, MD=-1.15, 95%CI -1.59 to -0.71], P < 0.00001) (Figure 5A), family life (5RCTs, MD=-1.21, 95%CI -1.64 to -0.77], P < 0.00001) (Figure 5B), and social life (5RCTs, MD=-1.14, 95% CI -1.57 to -0.70], P < 0.00001) (Figure 5C)scores in the paroxetine group were greater than that in the placebo group. No substantial heterogeneity was found in the three items (work: P=0.56, I2 = 0%; family life: P=0.24, I2 = 27%; social life: P=0.58, I2 = 0%). There was no statistical difference in baseline values among the three domains (work: P=0.13; family life: P=0.45; social life: P=0.58). The outcomes of the risk of bias assessment were presented in two forms: risk of bias graph (Figure 2A) and risk of bias summary (Figure 2B). Sensitivity Analysis To assess the robustness of our outcomes and to explore the contribution of each included trial, sensitivity analysis was conducted by omitting the studies one by one and recalculating the data using Stata version 15.1 (Tabrizi et al., 2019). FIGURE 1 \| PRISMA ﬂow diagram. FIGURE 1 \| PRISMA ﬂow diagram. March 2020 \| Volume 11 \| Article 275 Frontiers in Pharmacology \| www.frontiersin.org 4 Paroxetine and Panic Disorder Zhang et al. ranged from 9 weeks to 12 weeks. Details of the included RCTs were shown in Table 1. -1.02 to -0.13, P=0.01) (Figure 4E) scores in the paroxetine group were higher than that in the placebo group. No heterogeneity was identiﬁed in the overall phobia item (P=0.46, I2 = 0%). However, another item had substantial heterogeneity (P=0.02, I2 = 57%), therefore we used the random-effects model. There was no statistical difference in baseline values between the two items (avoidance: P=0.49; overall phobia: P=0.70). Otherwise, the baseline values provided by the fear item were statistically different (P=0.007), therefore we didn’t make any further comparisons on it. Excluded Studies 8 articles were excluded after reading the full-text for the following reasons: non-placebo control arm (n=3), non- randomized controlled trial (n=3), unable to obtain valid data (n=2). Effects of Interventions Primary Efﬁcacy Outcomes Primary Efﬁcacy Outcomes y y Mean change compared to the baseline in the total number offull panic attacks (6RCTs, MD=-1.96, 95%CI -3.45 to -0.47, P=0.010) (Figure 3A) and CGI-S total score (5RCTs, MD=-0.37, 95%CI -0.74 to -0.01, P=0.05) (Figure 3B)in the paroxetine group was higher in comparison with the placebo group. No heterogeneity was detected in the former (P=0.83, I2 = 0%), while the heterogeneity was evident in the latter (P=0.01, I2 = 69%), therefore we changed to the random-effects model. There was no statistically signiﬁcant difference of their baseline between the two groups (former: P=0.77; latter: P=0.68). Mean change compared to the baseline of MADRS total score (6RCTs, MD=-3.27, 95%CI -4.40 to -2.14], P < 0.00001) (Figure 5D), and SAQ total score (4RCTs, MD=-4.75, 95%CI -7.58 to -1.92, P=0.0010) (Figure 5E) in the paroxetine group was higher in comparison with the placebo group. No heterogeneity was identiﬁed (MADRS: P=0.33, I2 = 13%; SAQ: P=0.86, I2 = 0%). There was no statistically signiﬁcant difference in baseline values between the two groups (MADRS: P=0.17; SAQ: P=0.80). The proportion of patients with a 50% or greater reduction in the number offull panic attacks (4RCTs, OR=1.66, 95%CI 1.08 to 2.55, P=0.02) (Figure 3C) and the subjects with zero full panic attacks (10RCTs, OR=1.67, 95%CI 1.26 to 2.22, P=0.0004) (Figure 3D) in the paroxetine group was higher in comparison with the placebo group. The former had no heterogeneity (P=0.50, I2 = 0%), while the latter had substantial heterogeneity (P=0.02, I2 = 55%), therefore the random-effects model was applied. The response (8RCTs, OR=2.40, 95%CI 1.98 to 2.92], P < 0.00001) (Figure 5F) and remission rate (2RCTs, OR=2.14, 95% CI 1.52 to 3.00, P < 0.0001) (Figure 5G) in the paroxetine group were better than in the placebo group. No substantial heterogeneity was identiﬁed (response: P=0.17, I2 = 33%; remission: P=0.29, I2 = 11%). Paroxetine dose (mg/d) 20-60 40 40 10 20 40 10-60 20-60 12.5-75 12.5-75 12.5-75 10-60 60 orders; Y, yes; CR, of full panic attacks; eehan Phobia Sca 13) remission rate; adverse events. The Incidence of AEs h d f Mean change compared to the baseline of MSPS overall phobia (2RCTs, MD=-1.51, 95%CI -2.19 to -0.83, P < 0.0001) (Figure 4D) and total avoidance (8RCTs, MD=-0.57, 95%CI The incidence of any TEAEs in the paroxetine group was higher than that in the placebo group (10RCTs, OR=1.32, 95%CI 1.05 to March 2020 \| Volume 11 \| Article 275 Frontiers in Pharmacology \| www.frontiersin.org 5 Publication Bias 1.64, P=0.02) (Figure 6D). No substantial heterogeneity (P=0.05, I2 = 48%) was found. The visual examination of the funnel plot (Figure 7A) suggested that there might be publication bias and the cause was mainly the existence of small sample studies. But the Begg’s and Egger’s test (Figure 7B) did not support the result (Begg’s: P=1.000, Egger’s: P=0.579). The incidence of SAEs in the paroxetine group was lower than that in the placebo group (9RCTs, OR=0.42, 95%CI 0.23 to 0.79, P=0.007) (Figure 6E) and no heterogeneity was detected (P=0.65, I2 = 0%). Common TEAEs reported in the included RCTs were analyzed. There was no signiﬁcant difference in sinusitis, constipation, respiratory disorder, decreased appetite, nervousness, headache, and dizziness between the two groups. The incidence of dysmenorrhea, infection, and dyspepsia in the paroxetine group was lower than that in the placebo group. Finally, the incidence of sweating, nausea, dry mouth, somnolence, insomnia, diarrhea, female genital disorders, decreased libido, erectile dysfunction, asthenia, tremor, and abnormal ejaculation in the paroxetine group was higher than that in the placebo group. In the pooled analyses of sinusitis and constipation, we used the random effects model due to the heterogeneity detected. See Table 2 for details. We listed the common AEs associated with the use of paroxetine in Table 3 based on the rank of incidence. Sensitivity Analyses We conducted sensitivity analyses for all the results with substantial heterogeneity, and the results were stable (see Supplementary S2 ﬁle). Paroxetine dose (mg/d) Durance (weeks) Outcome Multicenter Study design 20-60 12 (1)(6)(8)(9)(10)(14)(15)(16)(17)(18) Y RCT 40 12 (4)(12)(13)(14)(15)(16)(17)(18) Y RCT 40 12 (4)(12)(13)(14)(15)(16) Y RCT 10 10 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12) (14)(15)(16)(17)(18) Y RCT 20 10 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12) (14)(15)(16)(17)(18) Y RCT 40 10 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12) (14)(15)(16)(17)(18) Y RCT 10-60 12 (14)(17) Y RCT 20-60 12 (1)(2)(6)(8)(10)(14)(15) Y RCT 12.5-75 10 (4)(6)(8)(12)(14)(15)(16)(17)(18) Y RCT 12.5-75 10 (4)(6)(8)(12)(14)(15)(16)(17)(18) Y RCT 12.5-75 10 (4)(6)(8)(12)(14)(15)(16)(17)(18) Y RCT 10-60 10 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(14) (15)(16)(17)(18) Y RCT 60 9 (4)(14)(15)(16) Y RCT isorders; Y, yes; CR, controlled release; HCL, hydrochloride; (1) total number of full panic attacks; (2) Clinical r of full panic attacks; (4) the proportion of patients with zero full panic attacks; (5) the intensity of anticipatory Sheehan Phobia Scale (MSPS) scores; (9) Sheehan Disability Scale (SDS) score; (10) Montgomery-Asberg (13) remission rate; (14) total withdrawal rate; (15) withdrawal rate due to lack of efﬁcacy or relapse; (16) s adverse events. Zhang et al. Paroxetine and Panic Disorder A B FIGURE 2 \| (A) Risk of bias graph: review authors’ judgements about each risk of bias item presented as percentages across all included studies, (B) Risk of bias summary: review authors’ judgements about each risk of bias item for each included study. FIGURE 2 \| (A) Risk of bias graph: review authors’ judgements about each risk of bias item presented as percentages across all included studies, (B) Risk of bias summary: review authors’ judgements about each risk of bias item for each included study. DISCUSSION Summary of Main Results Summary of Main Results Through the quantitative analyses of 13RCTs, we found patients who received paroxetine experienced more signiﬁcant improvement in the frequency offull panic attacks, HAMA, MADRS, SAQ, MSPS, March 2020 \| Volume 11 \| Article 275 Frontiers in Pharmacology \| www.frontiersin.org Zhang et al. Paroxetine and Panic Disorder A B C D GURE 3 \| Forest plot of primary efﬁcacy outcomes. (A) mean change from baseline in total number of full panic attacks, (B) mean change from baseline in CGI-S total core, (C) the proportion of patients with a 50% or greater reduction in the number of full panic attacks, (D) the proportion of patients with zero full panic attacks. A B C D FIGURE 3 \| Forest plot of primary efﬁcacy outcomes. (A) mean change from baseline in total number of full panic attacks, (B) mean change from baseline in CGI-S total score, (C) the proportion of patients with a 50% or greater reduction in the number of full panic attacks, (D) the proportion of patients with zero full panic attacks. B C FIGURE 3 \| Forest plot of primary efﬁcacy outcomes. (A) mean change from baseline in total number of full panic attacks, (B) mean ch score, (C) the proportion of patients with a 50% or greater reduction in the number of full panic attacks, (D) the proportion of patients wi FIGURE 3 \| Forest plot of primary efﬁcacy outcomes. (A) mean change from baseline in total number of full panic attacks, (B) mean change from baseline in CGI-S total score, (C) the proportion of patients with a 50% or greater reduction in the number of full panic attacks, (D) the proportion of patients with zero full panic attacks. mouth, somnolence, female genital disorders, decreased libido, insomnia, asthenia, impotence, tremor, and abnormal ejaculation. Other TEAEs such as sinusitis, constipation, infection, respiratory disorder, dizziness, dyspepsia, headache, dysmenorrhea, decreased appetite, and nervousness were not related to paroxetine. overall phobia and avoidance, CGI-S, CGI-I, SDS work, social life, and family life scores, as well as response and remission rate than those who received the placebo. There was no evident difference in the intensity of anticipatory anxiety, total withdrawal rate, and withdrawal rate due to AEs between the two groups. In addition, withdrawal rate due to a lack of efﬁcacy or relapse and the incidence of SAEs in the paroxetine group was lower in comparison with the placebo group. Summary of Main Results Meanwhile, the incidence of any TEAEs in the paroxetine group was higher in comparison to the placebo group. The most common TEAEs were sweating, nausea, diarrhea, dry Frontiers in Pharmacology \| www.frontiersin.org Frontiers in Pharmacology \| www.frontiersin.org Overall Completeness and Applicability of Evidence (A) SDS work scores, (B) SDS family life score, (C) SDS social life score, (D) MADRS score, (E) SAQ total score, (F) response rate, (G) remission rate. A D G FIGURE 5 \| Forest plot of secondary efﬁcacy outcomes. (A) SDS work scores, (B) SDS family life score, (C) SDS social life score, (D) MADRS score, (E) SAQ total score, (F) response rate, (G) remission rate. between the two groups. Hence, paroxetine was regarded as effective and well- tolerated and as something that could be applicable to clinical practice. evidence was relatively high in theory, although a lot of research did not provide a detailed scheme of random allocation and blinding, so therefore the overall quality was mixed and unclear. It’s still worth noting that the main high risk of bias was in other bias domain. Furthermore, the authors stated the studies were funded by pharmaceutical companies, and so consequently there might be a certain degree of Overall Completeness and Applicability of Evidence We searched eligible trials as comprehensively as possible, especially unpublished trials. All the included 11 RCTs reported the pre-set March 2020 \| Volume 11 \| Article 275 Frontiers in Pharmacology \| www.frontiersin.org Zhang et al. Paroxetine and Panic Disorder A B C D E orest plot of secondary efﬁcacy outcomes.(A)the intensity of anticipatory anxiety, (B) HAMA total score, (C) CGI-I total score, (D) MSPS overall phobia, avoidance scores. A B C D E FIGURE 4 \| Forest plot of secondary efﬁcacy outcomes.(A)the intensity of anticipatory anxiety, (B) HAMA total score, (C) CGI-I total score, (D) MSPS overall phobia, (E) MSPS t t l id B D FIGURE 4 \| Forest plot of secondary efﬁcacy outcomes.(A)the intensity of anticipatory anxiety, (B) HAMA total score, (C) CGI-I total score, (D) MSPS overall phobia, (E) MSPS total avoidance scores. In our research, 2654 participants entered the ﬁnal analysis, and the sample size was large enough that the conclusions could be basically extended to most other patients with similar conditions. Our conclusions were also consistent with the guidelines, and paroxetine is a commonly used clinical medication for adults with panic disorder. In terms of tolerability, we found that patients treated with paroxetine had a lower incidence of SAEs than placebo, although the incidence of any TEAEs was higher than placebo, and there was no marked difference in the withdrawal rate due to AEs results completely and provided detailed data which we could analyze, besides the remaining 2 RCTs which were ﬁnally considered as “unclear”. In short, none of the RCTs were identiﬁed as high risk of bias in terms of selective outcome reporting. Therefore, we could conduct pooled analyses of multiple items on enough trials to prove that paroxetine is effective and well tolerated in the short-term therapy of adults with PD, mainly reﬂected in the frequency of panic attacks, anxiety, depression, social functions, withdrawal rate, AEs, and so on. March 2020 \| Volume 11 \| Article 275 Frontiers in Pharmacology \| www.frontiersin.org Zhang et al. Paroxetine and Panic Disorder A B C D E F G FIGURE 5 \| Forest plot of secondary efﬁcacy outcomes. (A) SDS work scores, (B) SDS family life score, (C) SDS social life score, (D) MADRS score, (E) SA score, (F) response rate, (G) remission rate. A B C D E F G FIGURE 5 \| Forest plot of secondary efﬁcacy outcomes. Quality of the Evidence All the studies that we included were multicenter, randomized, double-blind, placebo-controlled trials, thus the quality of March 2020 \| Volume 11 \| Article 275 Frontiers in Pharmacology \| www.frontiersin.org 10 Zhang et al. Paroxetine and Panic Disorder A B C D E FIGURE 6 \| Forest plot of tolerability outcomes. (A) total withdrawal rate, (B) withdrawal rate due to lack of efﬁcacy or relapse, (C) withdrawal rate due to AEs, (D) the incidence of any TEAEs, (E) the incidence of SAEs. A B C D E FIGURE 6 \| Forest plot of tolerability outcomes. (A) total withdrawal rate, (B) withdrawal rate due to lack of efﬁcacy or relapse, (C) withdrawal rate due to AEs, the incidence of any TEAEs, (E) the incidence of SAEs. A B B C C D D E bility outcomes. (A) total withdrawal rate, (B) withdrawal rate due to lack of efﬁcacy or relapse, (C) withdrawal rate due to AEs, (D) he incidence of SAEs FIGURE 6 \| Forest plot of tolerability outcomes. (A) total withdrawal rate, (B) withdrawal rate due to lack of efﬁcacy or relapse, ( the incidence of any TEAEs, (E) the incidence of SAEs. FIGURE 6 \| Forest plot of tolerability outcomes. (A) total withdrawal rate, (B) withdrawal rate due to lack of efﬁcacy or relapse, (C) withdrawal rate due to AEs, (D) the incidence of any TEAEs, (E) the incidence of SAEs. overestimation of efﬁcacy. Overall, none of the trials had a signiﬁcant methodological bias, thus the quality of evidence could be considered good. analysis on items with substantial heterogeneity, and the results were stable. Secondly, we only included English and Chinese studies, so we might have missed out high-quality studies in other languages. Finally, as for publication bias, the results of the visual examination of the funnel plot and Begg’s as well as Egger’s test were contradictory, although we have searched all eligible published and unpublished studies as thoroughly as possible, we could not rule out that some studies with small samples, negative results, or sponsored by pharmaceutical companies have not been detected. Potential Biases in the Review Process There were some limitations that need to be noted. Firstly, we did not stratify the factors that might cause heterogeneity, such as the dosage and dosage form of paroxetine, although we did sensitivity March 2020 \| Volume 11 \| Article 275 Frontiers in Pharmacology \| www.frontiersin.org Paroxetine and Panic Disorder Zhang et al. TABLE 2 \| Meta-analyses of common TEAEs reported in the included RCTs. TEAEs RCTs (N) OR [95%CI] Heterogeneity Effect model Overall effect (P value) Female genital disorders* 3 11.07[2.03-60.46] P = 0.99 I2 = 0% Fixed P = 0.006 Dysmenorrhea* 3 0.32[0.11-0.91] P = 0.61 I2 = 0% Fixed P = 0.03 Dyspepsia 3 0.22[0.10-0.50] P = 0.33 I2 = 11% Fixed P = 0.0003 Decreased libido 2 2.37[1.14-4.93] P = 0.22 I2 = 32% Fixed P = 0.02 Infection 4 0.57[0.34-0.96] P = 0.12 I2 = 49% Fixed P = 0.03 Decreased appetite 5 1.42[0.84-2.38] P=0.78 I2 = 0% Fixed P = 0.19 Respiratory Disorder 7 0.83[0.60-1.14] P = 0.92 I2 = 0% Fixed P = 0.25 Nervousness 6 0.9[0.62-1.31] P = 0.36 I2 = 9% Fixed P = 0.58 Sinusitis 3 1.11[0.30-4.05] P = 0.08 I2 = 60% Random P = 0.88 Erectile dysfunction* 2 8.85[1.10-71.49] P = 0.63 I2 = 0% Fixed P = 0.04 Abnormal ejaculation* 8 11.5[5.70-23.20] P = 0.96 I2 = 0% Fixed P < 0.00001 Diarrhea 9 1.74[1.27-2.38] P = 0.14 I2 = 35% Fixed P = 0.0005 Somnolence 9 2.11[1.63-2.74] P = 0.32 I2 = 14% Fixed P < 0.00001 Insomnia 8 1.64[1.26-2.13] P = 0.79 I2 = 0% Fixed P = 0.0002 Asthenia 8 1.93[1.41-2.65] P = 0.61 I2 = 0% Fixed P < 0.0001 Tremor 6 3.86[2.12-7.05] P = 0.89 I2 = 0% Fixed P < 0.0001 Constipation 7 1.47[0.73-2.98] P = 0.02 I2 = 60% Random P = 0.28 Dizziness 9 1.05[0.78-1.41] P = 0.27 I2 = 19% Fixed P = 0.75 Headache 8 0.85[0.68-1.06] P = 0.12 I2 = 39% Fixed P = 0.14 Sweating 3 2.79[1.67-4.67] P = 0.19 I2 = 41% Fixed P < 0.0001 Nausea 9 1.3[1.03-1.64] P = 0.73 I2 = 0% Fixed P = 0.03 Dry mouth 9 1.71[1.29-2.26] P = 0.29 I2 = 17% Fixed P = 0.0002 TEAEs, treatment-emergent adverse events; RCTs, randomized controlled trials; OR, odds ratio; CI, conﬁdence interval; *Corrected for gender. TABLE 2 \| Meta-analyses of common TEAEs reported in the included RCTs. Potential Biases in the Review Process TABLE 3 \| Common AEs associated with paroxetine. Common AEs Number Proportion (%) Nausea 200 16.99 Somnolence 196 16.65 Insomnia 167 14.19 Dry mouth 147 12.49 Asthenia 121 10.28 Diarrhea 114 9.69 Abnormal ejaculation 76 6.46 Sweating 57 4.84 Tremor 52 4.42 Decreased libido 26 2.21 Female genital disorders 13 1.10 Erectile dysfunction 8 0.68 Total 1177 100 AE, adverse events. (Andrisano et al., 2013; Bighelli et al., 2018). In addition, there was a contradiction between our meta-analysis and Andrisano’s research regarding the total withdrawal rate, while their results after the sensitivity analysis was so consistent with ours that there was no evident difference between the two groups, which to some extent supported the credibility of our results. TABLE 3 \| Common AEs associated with paroxetine. Agreements and Disagreements With O h S di R i Other Studies or Reviews To our knowledge, this was the ﬁrst meta-analysis of paroxetine versus placebo directly and individually in the treatment of PD, although paroxetine had been included in previous systematic reviews and meta-analyses. Our results were generally consistent with previous studies (Andrisano et al., 2013; Sugarman et al., 2014; Bighelli et al., 2018), however we made a more comprehensive comparison between paroxetine and placebo in terms of improving panic symptoms, anxiety, depression, and social function and other aspects. It’s worth mentioning that the methodological quality of Andrisano’s research might not be very high, because they included both non-randomized and non-placebo controlled studies Implications for Practice Based on the available data, we could conclude that paroxetine was effective and well-tolerated in the short-term treatment of adults with PD. We believe that the evidence we provided will be of some beneﬁt to clinical practice. However, the maximum duration of treatment of studies included in this meta-analysis lasted only 12 weeks and it did not include data on long-term treatment because that might increase heterogeneity, while paroxetine usually lasts for a long time in clinical application, so our results cannot be directly applied to the evaluation of efﬁcacy and tolerability for paroxetine in long-term treatment. Besides, it is important for us to comprehensively consider the actual condition of patients when we choose the drugs due to individual differences in each patient. Implications for Research This meta-analysis only compared paroxetine as a monotherapy with placebo, however there are still many clinical options on treating PD, so head-to-head comparisons of multiple medications for PD (or combined with psychotherapy) and March 2020 \| Volume 11 \| Article 275 Frontiers in Pharmacology \| www.frontiersin.org 12 Zhang et al. Paroxetine and Panic Disorder work meta-analyses will be necessary to determine the order of ir efﬁcacy and tolerability in the future. Further reﬁnement and atiﬁcation of the dose for paroxetine is also required to make ommendations for an optimal therapeutic dose AUTHOR CONTRIBUTI BZ, CW, and LC contributed to th study JG and CLW organized the d GURE 7 \| (A) The funnel plot of publication bias, (B) Egger’s test of publication bias. be necessary to determine the order of y in the future. Further reﬁnement and AUTHOR CONTRIBUTIONS BZ CW d LC ib d h i d d i f h of publication bias, (B) Egger’s test of publication bias. ecessary to determine the order of he future. Further reﬁnement and roxetine is also required to make therapeutic dose. AUTHOR CONTRIBUT BZ, CW, and LC contributed to study. JG and CLW organized th blication bias, (B) Egger’s test of publication bias. FIGURE 7 \| (A) The funnel plot of publication bias, (B) Egger’s test of publication bias. FIGURE 7 \| (A) The funnel plot of publication bias, (B) Egger’s test of publication bias. REFERENCES American Psychiatric Association (2009). Practice guideline for treatment of patients with panic disorder. second edition. Am. J. Psychiatry 166 (1), 1–90. Pollack, M. H., Lepola, U., Koponen, H., Simon, N. M., Worthington, J. J., Emilien, G., et al. (2007b). A double-blind study of the efﬁcacy of venlafaxine extended- release, paroxetine, and placebo in the treatment of panic disorder. Depress Anxiety 24 (1), 1–14. doi: 10.1002/da.20218 Andrisano, C., Chiesa, A., and Serretti, A. (2013). Newer antidepressants and panic disorder: a meta-analysis. Int. Clin. Psychopharmacol. 28 (1), 33–45. doi: 10.1097/YIC.0b013e32835a5d2e Bakker, A., van Dyck, R., Spinhoven, P., and van Balkom, A. J. (1999). Paroxetine, clomipramine, and cognitive therapy in the treatment of panic disorder. J. Clin. Psychiatry 60 (12), 831–838. doi: 10.4088/jcp.v60n1205 Sheehan, D. V., Burnham, D. B., Iyengar, M. K., Perera, P.Paxil CR Panic Disorder Study Group (2005a). A double-blind, placebo-controlled, ﬂexible-dosing trial to evaluate the efﬁcacy of controlled-release paroxetine in the treatment of panic disorder. (GSK Clinical Study Register (www.gsk-clinicalstudyregister. com). Study No.: 29060/495). Ballenger, J. C., Wheadon, D. E., Steiner, M., Bushnell, W., and Gergel, I. P. (1998). Double-blind, ﬁxed-dose, placebo-controlled study of paroxetine in the treatment of panic disorder. Am. J.Psychiatry 155 (1), 36–42. doi: 10.1176/ ajp.155.1.36. (GSK-ClinicalStudyRegister (www.gsk-clinicalstudyregister.com). StudyNo.: MY1047/BRL–029060/1/CPMS–120). Sheehan, D. V., Burnham, D. B., Iyengar, M. K., Perera, P.Paxil CR Panic Disorder Study Group (2005b). A double-blind, placebo-controlled, ﬂexible-dosing trial to evaluate the efﬁcacy of controlled-release paroxetine in the treatment of panic disorder. (GSKClinical Study Register (www.gsk-clinicalstudyregister. com). Study No.: 29060/497). Bergink, V., and Westenberg, H. G. (2005). Metabotropic glutamate II receptor agonists in panic disorder: a double blind clinical trial with LY354740. Int. Clin. Psychopharmacol. 20 (6), 291–293. doi: 10.1097/00004850-200511000- 00001 Sheehan, D. V., Burnham, D. B., Iyengar, M. K., Perera, P.Paxil CR Panic Disorder Study Group (2005c). A double-blind, placebo-controlled, ﬂexible-dosing trial to evaluate the efﬁcacy of modiﬁed-release paroxetine in the treatment of panic disorder. (GSK Clinical Study Register (www.gsk-clinicalstudyregister.com). Study No.: 29060–494). Bighelli, I., Castellazzi, M., Cipriani, A., Girlanda, F., Guaiana, G., Koesters, M., et al. (2018). Antidepressants versus placebo for panic disorder in adults. Cochrane. Database Syst. Rev. 4, CD010676. doi: 10.1002/14651858.CD010676.pub2 Dannon, P. N., Lowengrub, K., Iancu, I., and Kotler, M. (2004). Paroxetine in panic disorder: clinical management and long-term follow-up. Expert. Rev. Neurother. 4 (2), 191–198. doi: 10.1586/14737175.4.2.191 Shi, J., Wu, X., and Chen, Y. (2019). Study on Dalfampridine in the treatment of Multiple Sclerosis Mobility Disability: A meta-analysis. REFERENCES PLoS One 14 (9), e0222288. doi: 10.1371/journal.pone.0222288 GlaxoSmithKline (1994). A double-blind, multicentered, ﬂexible-dose study of paroxetine, alprazolam and placebo in the treatment of panic disorder. (GSK- Clinical Study Register(www.gsk-clinicalstudyregister.com) StudyNo.: MY1048/BRL029060/1/CPMS–223 (PAR223)). Sterne, J. A., Egger, M., and Moher, D. (2008). “Addressing Reporting Biases,” in The Cochrane Handbook for Systematic Reviews of Interventions. Eds. J. P. Higgins and S. Green, 297–333. Sugarman, M. A., Loree, A. M., Baltes, B. B., Grekin, E. R., and Kirsch, I. (2014). The efﬁcacy of paroxetine and placebo in treating anxiety and depression: a meta-analysis of change on the Hamilton Rating Scales. PLoS One 9 (8), e106337. doi: 10.1371/journal.pone.0106337 Higgins, J. P., Altman, D. G., Gotzsche, P. C., Juni, P., Moher, D., Oxman, A. D., et al. (2011). The Cochrane Collaboration’s tool for assessing risk of bias in randomised trials. BMJ 343, d5928. doi: 10.1136/bmj.d5928 Hugues, A., Di Marco, J., Janiaud, P., Xue, Y., Pires, J., Khademi, H., et al. (2017). Efﬁciency of physical therapy on postural imbalance after stroke: study protocol for a systematic review and meta-analysis. BMJ Open 7 (1), e013348. doi: 10.1136/bmjopen-2016-013348 Tabrizi, R., Ostadmohammadi, V., Lankarani, K. B., Akbari, M., Akbari, H., Vakili, S., et al. (2019). The effects of probiotic and synbiotic supplementation on inﬂammatory markers among patients with diabetes: A systematic review and meta-analysis of randomized controlled trials. Eur. J. Pharmacol. 852, 254–264. doi: 10.1016/j.ejphar.2019.04.003 Judge, R., and Dunbar, G. (1995). Paroxetine, clomipramine and placebo in the treatment of panic disorder. Eur. Neuropsychopharmacol. 5 (3), 361. doi: 10.1016/0924-977X(95)90629-R Tsuboi, K., and Masuko, M. (2001). [Panic disorder and antidepressants]. Nihon. Rinsho. 59 (8), 1592–1598. King, M., Nazareth, I., Levy, G., Walker, C., Morris, R., Weich, S., et al. (2008). Prevalence of common mental disorders in general practice attendees across Europe. Br. J. Psychiatry 192 (5), 362–367. doi: 10.1192/bjp.bp.107.039966 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. Lecrubier, Y., Bakker, A., Dunbar, G., and Judge, R. (1997). A comparison of paroxetine, clomipramine and placebo in the treatment of panic disorder. Collaborative Paroxetine Panic Study Investigators. Acta. Psychiatr. Scand. 95 (2), 145–152. doi: 10.1111/j.1600-0447.1997.tb00388.x. (GSKClinicalStudyRegister(www.gsk- clinicalstudyregister.com).StudyNo.:MY1036/BRL029060/1/CPMS–187 (PAR 187)). Copyright © 2020 Zhang, Wang, Cui, Gao, Wang, Tan and Fang. This is an open- access article distributed under the terms of the Creative Commons Attribution License (CC BY). ACKNOWLEDGMENTS the statistical analysis. BZ wrote the ﬁrst draft of the manuscript. CW wrote sections of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version. the statistical analysis. BZ wrote the ﬁrst draft of the manuscript. CW wrote sections of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version. We would like to thank the authors of the original studies for their contributions. FUNDING The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphar.2020. 00275/full#supplementary-material This work was supported by the National Natural Science Foundation of China (81873794). This work was supported by the National Natural Science Foundation of China (81873794). Pollack, M., Mangano, R., Entsuah, R., Tzanis, E., Simon, N. M., and Zhang, Y. (2007a). A randomized controlled trial of venlafaxine ER and paroxetine in the treatment of outpatients with panic disorder. Psychopharmacology(Berl) 194 (2), 233–242. doi: 10.1007/s00213-007-0821-0 AUTHOR CONTRIBUTIONS network meta-analyses will be necessary to determine the order of their efﬁcacy and tolerability in the future. Further reﬁnement and stratiﬁcation of the dose for paroxetine is also required to make recommendations for an optimal therapeutic dose. BZ, CW, and LC contributed to the conception and design of the study. JG and CLW organized the database. LC and XT performed March 2020 \| Volume 11 \| Article 275 Frontiers in Pharmacology \| www.frontiersin.org Zhang et al. Paroxetine and Panic Disorder REFERENCES 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. Moher, D., Liberati, A., Tetzlaff, J., Altman, D. G.Prisma Group (2009). Preferred reporting items for systematic reviews and meta-analyses: the PRISMA statement. BMJ 339, b2535. doi: 10.1136/bmj.b2535 March 2020 \| Volume 11 \| Article 275 Frontiers in Pharmacology \| www.frontiersin.org
https://openalex.org/W4298175759	http://oceanrep.geomar.de/6743/1/835_Egge_2009_PrimaryProductionDuringNutrientinducedBlooms_Artzeit_pubid12548.pdf	English	null	Primary production during nutrient-induced blooms at elevated CO<sub>2</sub> concentrations	HAL (Le Centre pour la Communication Scientifique Directe)	2,007	cc-by	9,199	Primary production during nutrient-induced blooms at elevated CO2 concentrations J. K. Egge1, T. F. Thingstad1, A. Larsen1, A. Engel2, J. Wohlers4, R. G. J. Bellerby3, and U. Riebesell4 1Department of Biology, University of Bergen, 5020 Bergen, Norway 2Alfred Wegener Institute (AWI) for Marine and Polar Research, Am Handelshafen 12, 27570 Bremerhaven, Germany 3Bjerknes Centre for Climate Research, University of Bergen, All´egaten 55, 5007 Bergen, Norway 4IFM-GEOMAR, Leibniz Institute of Marine Sciences, Kiel University, D¨usternbrooker Weg 20, 24105 Kiel, Germany a substantial part of production in the upper layer was not de- graded locally, but either accumulated or exported vertically. a substantial part of production in the upper layer was not de- graded locally, but either accumulated or exported vertically. Abstract. A CO2 enrichment experiment (PeECE III) was carried out in 9 mesocosms in which the seawater carbon- ate system was manipulated to achieve three different levels of pCO2. At the onset of the experimental period, nutrients were added to all mesocosms in order to initiate phytoplank- ton blooms. Primary production rates were measured by in-vitro incubations based on 14C-incorporation and oxygen production/consumption. Size fractionated particulate pri- mary production was also determined by 14C incubation and is discussed in relation to phytoplankton composition. Pri- mary production rates increased in response to nutrient addi- tion and a net autotrophic phase with 14C-ﬁxation rates up to 4 times higher than initial was observed midway through the 24 days experiment before net community production (NCP) returned to near-zero and 14C-ﬁxation rates dropped below initial values. No clear heterotrophic phase was observed during the experiment. Based on the 14C-measurements we found higher cumulative primary production at higher pCO2 towards the end of the experiment. CO2 related differences were also found in size fractionated primary production. The most noticeable responses to CO2 treatments with respect to primary production rates occurred in the second half of the experiment when phytoplankton growth had become nutrient limited, and the phytoplankton community changed from di- atom to ﬂagellate dominance. This opens for two alternative hypotheses that the effects are either associated with mineral nutrient limited growth, and/or with a change in phytoplank- ton species composition. The lack of a clear net heterotrophic phase in the last part of the experiment supports the idea that Biogeosciences, 6, 877–885, 2009 www.biogeosciences.net/6/877/2009/ © Author(s) 2009. This work is distributed under the Creative Commons Attribution 3.0 License. Biogeosciences Published by Copernicus Publications on behalf of the European Geosciences Union. J. K. Egge et al.: Primary productionat at elevated CO2 concentrations 878 Table 1. Experimental period and CO2 and nutrient manipulation of PeECE I, II and III carried out in 2001, 2003 and 2005, respectively. Temperature range and average global radiation (Geophysical institute, University of Bergen) is given. Table 1. Experimental period and CO2 and nutrient manipulation of PeECE I, II and III carried out in 2001, 2003 and 2005, respectively. Temperature range and average global radiation (Geophysical institute, University of Bergen) is given. and CO2 and nutrient manipulation of PeECE I, II and III carried out in 2001, 2003 and 2005, respectively. global radiation (Geophysical institute, University of Bergen) is given. Table 1. Experimental period and CO2 and nutrient manipulation of PeECE I, II and III carried out in 2001, 2003 and 2005, respectively. Temperature range and average global radiation (Geophysical institute, University of Bergen) is given. 2001 2003 2005 Experimental period 31 May–25 June 4 May–24 June 16 May–10 June CO2 concentration 180, 370, 700 µatm 190, 370, 700 µatm 375, 750, 1150 µatm Initial nutrient supply 17 µM N, 0.5 µM P 9 µM N, 0.5 µM P, 12 µM Si 15 µM N, 0.6 µM P Temperature range 10–13 ◦C 8–10 ◦C 9–11.5 ◦C Average global radiation 17.46 MJ m−2 11.45 MJ m−2 12.81 MJ m−2 elements, the consequence would be a change in the stoi- chiometric relationships in the microbial food web. Based on measurements of the removal of inorganic-C and nitrate, the PeECE-experiments have shown such an effect (Riebesell et al., 2007). A possible enhancement of organic carbon ﬁxa- tion at increased CO2 has been attributed to the production of transparent exopolymer particles (Engel 2002; Riebesell et al. 2007). data). In the 2005 experiment (PeECE III), even higher CO2 concentrations of up to 1050 µatm were used (Schulz et al., 2008). Here we report primary production results mainly from PeECE III, with a comparison to corresponding data from the previous experiments. 14C-based particulate pri- mary production, total and in size-fractions 0.2–1, 1–5, 5–10 and >10 µm, and O2 measurements based on incubation in light and dark bottles were used for estimating (gross and net community) production. The impact of increased CO2 on primary production has been investigated theoretically as well as experimentally. Some studies report small, if any, effects (Clark and Flynn, 2000; Tortell et al., 2002), whereas others show increased rates of phytoplankton growth and/or primary production with increasing CO2 (Riebesell et al. J. K. Egge et al.: Primary productionat at elevated CO2 concentrations 1993; Heine and Sand- Jensen, 1997; Schippers et al., 2004). A change in com- munity primary production may be rooted in a change in phytoplankton community composition. While the afﬁnity for CO2 differs among phytoplankton groups (Tortell, 2000), most species are able to regulate their carbon acquisition by CO2concentrating mechanisms (CCM) (Raven, 1991). The efﬁciency and regulation of CCM, however, differs among phytoplankton species and functional groups (Giordano et al., 2005). Changes in CO2 availability might therefore af- fect competition and succession of phytoplankton species (Burkhardt et al., 2001; Rost et al., 2003; Tortell et al., 2002). 2.1 Set-up and sampling The PeECE III mesocosm experiment was carried out at the Marine Biological Station, University of Bergen, Norway be- tween 16 May and 10 June 2005 (see Table 1). 9 mesocosms (volume 27 m3) made of polyethylene were ﬁlled with unﬁltered, nutrient-poor post-bloom water from the fjord, and manipulated to achieve 3 different levels of CO2 in triplicate mesocosms by aeration of the water column and the overlying atmosphere with CO2-enriched air. The levels of CO2 at the start of the experimental period were 350 µatm (1× CO2), 700 µatm (2× CO2) and 1050 µatm (3× CO2). Nutrients, as nitrate and phosphate, were added to the mesocosms on day t−1, the day before we start sam- pling, in order to achieve an increase in growth and biomass of osmotrophic organisms. For further details concerning the set-up of the experiment see Schulz et al. (2008). Three mesocosm experiments, in 2001, 2003 and 2005, have been carried out in the framework of the Pelagic Ecosystem CO2 Enrichment study (PeECE) with the aim to study the effects of elevated levels of CO2 on the planktonic community (Delille et al., 2005; Engel et al., 2004, 2005; Rochell-Newall et al., 2004; Grossart et al., 2006). Although all PeECE mesocosm studies were carried out during the post-bloom period (May–June), blooms of different phyto- plankton groups, e.g. Emiliania huxleyi and/or diatoms, were initiated by the addition of nutrients in different composi- tions. Differences in temperature and light conditions be- tween the three experiments have likely contributed to the ob- served differences in phytoplankton composition and succes- sion (Table 1). Primary production was measured during all PeECE experiments. No differences in primary production were observed in the 2001 and 2003 experiments where CO2 concentration in the mesocosms was manipulated to 180, 370 and 700 µatm (Delille et al., 2005; Egge unpublished 1 Introduction In the upper photic zone where primary production is usu- ally limited by mineral nutrients (e.g. N, P or Fe), the micro- bial food web can be seen as a set of cycles of the limiting elements, grossly described by the import-export and regen- erated nutrient cycles (Dugdale and Goering, 1967). Onto this set of nutrient cycles, the C-cycle is linked via a more or less ﬂexible stoichiometric relationship in organisms at the different trophic levels and in their interactions. Relatively small alterations in either the element cycles or in the stoi- chiometric C:nutrient coupling may have consequences for the ocean’s C-cycle. Increased atmospheric CO2 leads to both an increased pCO2 and a lowered pH (Wolf-Gladrow et al., 1999). It is an a priori possibility that both of these environmental changes may affect either the cycling of the limiting element, and/or its stoichiometric coupling to C. In either case, this would be expected to lead to changes in the rate of C ﬁxation into organic material and in the processes producing and consuming oxygen. CO2 is often quoted as being a non-limiting factor for primary production in seawater (Raven and Johnston, 1991; Clark and Flynn, 2000). The fact that RUBISCO, the pri- mary carboxylating enzyme in marine phytoplankton has a relatively low afﬁnity for CO2 (Raven and Johnston, 1991), however, has led to a discussion of a possible stimulating ef- fect of increased CO2 levels on primary C-ﬁxation in some groups of phytoplankton (Riebesell, 2004). Should this oc- cur without a proportional change in the cycle of limiting Correspondence to: J. K. Egge (jorun.egge@bio.uib.no) Published by Copernicus Publications on behalf of the European Geosciences Union. opernicus Publications on behalf of the European Geosciences Union. J. K. Egge et al.: Primary productionat at elevated CO2 concentrations J. K. Egge et al.: Primary productionat at elevated CO2 concentrations 2.2 14C Primary production Primary production was measured using the 14C method, ac- cording to Steemann Nielsen (1952) and Gargas (1975). In- tegrated water samples were collected 09:00 a.m., prior to the main sampling (Schulz et al., 2008), using a 5 m long ∼3 cm diameter tube. After mixing, the samples were ﬁlled into plastic bottles (76 ml) (NUNC Easyﬂask), spiked with approximately 4 µCi (Carbon 14 Central) and incubated in- vitro between 10:00 and 14:00 h. The concentration of 14C in the bottle was recorded by removing a 25 µl aliquot from the incubation bottle prior to incubation and added to 600 µl 2 molar NaOH in a scintillation vial. Dark uptake of 14C was measured in bottles wrapped in aluminium foil. Triplicate Biogeosciences, 6, 877–885, 2009 www.biogeosciences.net/6/877/2009/ 879 J. K. Egge et al.: Primary productionat at elevated CO2 concentrations bottles from each mesocosm were incubated in the sea out- side the mesocosms, at the irradiance level corresponding to mid-depth of the upper layer of the mesocosms (see Schulz et al., 2008). The incubation depth was determined based on light proﬁles inside and outside the mesocosms. A Li-Cor Li 1000 datalogger with Li 190SA-Quantum sensor and Li- 192SA Underwater Quantum Sensor was used both for pro- ﬁling and logging. In addition to short term incubation, 24 h incubations were conducted 7 times during the experimental period in order to measure primary production in different fractions. For these incubations 118 mL glass bottles were used, and only one mesocosm per treatment was sampled, M2, M5 and M8. The samples were ﬁltered onto Nuclepore ﬁlters with pore sizes of 0.2, 1, 5 and 10 µm. After ﬁltration all ﬁlters were treated with fuming HCl in order to remove in- organic 14C, a scintillation solution (Ecosint O) was added, and the samples were stored overnight before being counted in a Packard Tri Carb Liquid Scintillation Analyser, model 1900 A. Primary production rates were calculated according to Gargas (1975). For determination of total CO2 concen- tration in the different mesocosms see Bellerby et al. (2008). Daily primary production, based on 4 h incubation, was cal- culated as a function of incoming irradiance during the incu- bation period (4 h) and total irradiance over 24 h according to the formula: Daily 14C production = (14C production during incubation period * 100)/Irradiance during incubation period (%). 2.5 Statistical analysis In order to identify statistically signiﬁcant differences be- tween different treatments we used Student t-tests , Paired Two Sample for Means, according to Sokal and Rohlf (2001). The conﬁdence level for all analysis was set at 95%. 2.3 Oxygen production and consumption Biological Oxygen Demand (BOD) bottles were incubated for 24 h and oxygen was measured using the OxyMini® op- tode system (World Precision Instruments). The instrument was two-point calibrated according to the manual and used with automatic temperature compensation. Oxygen con- centration was determined individually in each BOD bottle both before and after incubation. 3 light and 3 dark bottles from each mesocosm were incubated at the same location as the 14C bottles. NCP and community respiration were based on light and dark bottle incubations, respectively, and gross community production calculated by difference assum- ing respiration to be the same in light and dark bottles. 3 Results Initial particulate primary production rates, based on the 14C method (4 h incubations), ranged from 0.33 to 0.37 µmol C L−1 h−1 (Fig. 1a). After the initial addition of nutrients, a rapid increase in production was observed in all treatments. Maximum rates were observed on day 8, rang- ing from 1.6 to 1.8 µmol C L−1 h−1. Two weaker but distinct peaks were observed on day 12 and day 20 before the pro- duction rates decreased to levels lower than initial. In the second half of the experiment there was a tendency of higher production at elevated CO2 levels. This trend is visible from ca. day 10 in the cumulative production, with a signiﬁcant difference between 3× and 1× CO2 (p<0.05) from day 20 onward (Fig. 1b). 2.2 14C Primary production A µmol C l-1 h-1 0,0 0,5 1,0 1,5 2,0 2,5 3x CO2 2x CO2 1x CO2 Day no. 0 5 10 15 20 25 µmol C l-1 0 50 100 150 200 B Fig. 1. Development of 14C primary production based on 4 h incu- bations during the experiment(µmol C L−1 h−1) (A) and cumula- tive production in µmol C L−1for the 24 days experimental period (B). Values are means ±SD of triplicate CO2 treatments with 1× CO2 (green), 2× CO2 (grey) and 3× CO2 (red). A µmol C l-1 h-1 0,0 0,5 1,0 1,5 2,0 2,5 3x CO2 2x CO2 1x CO2 Day no. 0 5 10 15 20 25 µmol C l-1 0 50 100 150 200 B Fig. 1. Development of 14C primary production based on 4 h incu- bations during the experiment(µmol C L−1 h−1) (A) and cumula- tive production in µmol C L−1for the 24 days experimental period (B). Values are means ±SD of triplicate CO2 treatments with 1× CO2 (green), 2× CO2 (grey) and 3× CO2 (red). J. K. Egge et al.: Primary productionat at elevated CO2 concentrations 14C primary production (µmol C L−1 d−1), based on 24 h incubation during the experiment in the fractions 0.2-1 µm (A), 1– 5 µm (C), 5–10 µm (E) and >10 µm (F), and cumulative produc- tion (µmol C L−1) in the same fractions (B, D, F, G). One meso- cosm of each CO2 treatment (M2, M5 and M8) was investigated. Values are means±SD of triplicate incubations in each mesocosm, and colour code as in Fig. 1. observed a small tendency, although not statistically signif- icant (p=0.2), of increased NCP at elevated CO2 (Fig. 2d). Gross production and community respiration did not reveal any clear CO2 effects, neither in terms of the timing nor the level of production/consumption (Fig. 2a, b, e, f). this fraction amounted to 19, 16 and 14 µmol C L−1 in 3×, 2× and 1× CO2, respectively. The difference between treat- ments was small, and not statistically signiﬁcant between 3× and 1× CO2 (p=0.07). The production in fraction 1–5 µm was generally low and similar for all treatments, except for the very last day of the experiment when production in 3× CO2 treatments was signiﬁcantly lower (p<0.05) (Fig. 3c, d). The largest CO2-related differences between treatments were found in the smallest fraction 0.2–1 µm. Here, pro- duction rates showed a decreasing trend from day 6 onwards in all treatments, but were distinctly higher at elevated CO2 throughout the experiment. In addition, the cumulative pro- duction increased from 30 µmol C L−1 at 1× CO2 to 39 and 43 µmol C L−1 at 2× and 3× CO2, respectively. The differ- ence between 3× and 1× CO2 was statistically signiﬁcant from day 12 onward (p<0.05). We also measured primary production during PeECE II (2003), but no consistent responses to CO2 were observed, neither in 14C (Egge, unpublished data) nor in O2 produc- tion (Engel, unpublished data), although there was a differ- ence in phytoplankton community composition (Grossart et al., 2006). In PeECE III (2005), we therefore decided to carry out fractionated primary production in one mesocosm of each treatment 1× (M8), 2× (M5) and 3× CO2 (M2). On average, the size fractions 0.2–1, 1–5, 5–10 and >10 µm ac- counted for 29, 18, 12 and 41% of total primary production, respectively. J. K. Egge et al.: Primary productionat at elevated CO2 concentrations 14C primary production (µmol C L−1 d−1), based on 24 h incubation during the experiment in the fractions 0.2-1 µm (A), 1– 5 µm (C), 5–10 µm (E) and >10 µm (F), and cumulative produc- 0 200 400 600 Gross production X Data 0 20 40 60 80 µmol O2 l-1 0 200 400 600 Day no. 0 5 10 15 20 25 200 400 600 Net community production µmol O2 l-1 d-1 -20 0 20 40 Community respiration Day no. 0 5 10 15 20 0 20 40 60 3xCO2 2x CO2 1x CO2 A B C D E F Fig. 2. Development of Gross- and Net community-production and Community respiration based on oxygen incubations. Daily pro- duction rates are given as µmol O2 L−1 d−1 (A, C, D) and cumula- tive production and consumption for the 24 days experimental pe- riod as µmol O2 L−1 (B, D, E).Values are means ±SD of triplicate CO2 treatments, colour code as in Fig. 1. 0 10 20 30 40 50 0.2-1 µm µmol C l-1d-1 0 1 2 3 4 5 3x CO2 (M2) 2x CO2 (M5) 1x CO2 (M8) 1-5µm 0 1 2 3 4 5 -10 µm 0 1 2 3 > 10 µm Day no. 0 5 10 15 20 0 2 4 6 8 10 12 14 µmol C l-1 0 10 20 30 40 50 Day no. 0 5 10 15 20 25 0 20 40 60 0 10 20 30 40 50 A B C H G E F D 0 200 400 600 Gross production X Data 0 20 40 60 80 µmol O2 l-1 0 200 400 600 Day no. 0 5 10 15 20 25 200 400 600 Net community production µmol O2 l-1 d-1 -20 0 20 40 Community respiration Day no. 0 5 10 15 20 0 20 40 60 3xCO2 2x CO2 1x CO2 A B C D E F D Fig. 2. Development of Gross- and Net community-production and Community respiration based on oxygen incubations. Daily pro- duction rates are given as µmol O2 L−1 d−1 (A, C, D) and cumula- tive production and consumption for the 24 days experimental pe- riod as µmol O2 L−1 (B, D, E).Values are means ±SD of triplicate CO2 treatments, colour code as in Fig. 1. Fig. 3. J. K. Egge et al.: Primary productionat at elevated CO2 concentrations 880 0 10 20 30 40 50 0.2-1 µm µmol C l-1d-1 0 1 2 3 4 5 3x CO2 (M2) 2x CO2 (M5) 1x CO2 (M8) 1-5µm 0 1 2 3 4 5 -10 µm 0 1 2 3 > 10 µm Day no. 0 5 10 15 20 0 2 4 6 8 10 12 14 µmol C l-1 0 10 20 30 40 50 Day no. 0 5 10 15 20 25 0 20 40 60 0 10 20 30 40 50 A B C H G E F D Fig. 3. 14C primary production (µmol C L−1 d−1), based on 24 h incubation during the experiment in the fractions 0.2-1 µm (A), 1– 5 µm (C), 5–10 µm (E) and >10 µm (F), and cumulative produc- tion (µmol C L−1) in the same fractions (B, D, F, G). One meso- cosm of each CO2 treatment (M2, M5 and M8) was investigated. Values are means±SD of triplicate incubations in each mesocosm, and colour code as in Fig. 1. 0 200 400 600 Gross production X Data 0 20 40 60 80 µmol O2 l-1 0 200 400 600 Day no. 0 5 10 15 20 25 200 400 600 Net community production µmol O2 l-1 d-1 -20 0 20 40 Community respiration Day no. 0 5 10 15 20 0 20 40 60 3xCO2 2x CO2 1x CO2 A B C D E F Fig. 2. Development of Gross- and Net community-production and Community respiration based on oxygen incubations. Daily pro- duction rates are given as µmol O2 L−1 d−1 (A, C, D) and cumula- tive production and consumption for the 24 days experimental pe- riod as µmol O2 L−1 (B, D, E).Values are means ±SD of triplicate CO2 treatments, colour code as in Fig. 1. 0 10 20 30 40 50 0.2-1 µm µmol C l-1d-1 0 1 2 3 4 5 3x CO2 (M2) 2x CO2 (M5) 1x CO2 (M8) 1-5µm 0 1 2 3 4 5 -10 µm 0 1 2 3 > 10 µm Day no. 0 5 10 15 20 0 2 4 6 8 10 12 14 µmol C l-1 0 10 20 30 40 50 Day no. 0 5 10 15 20 25 0 20 40 60 0 10 20 30 40 50 A B C H G E F D Fig. 3. 2.4 Transparent exopolymer particles (TEP) The concentration of TEP was determined using the colori- metric approach by Passow and Alldredge (1995). Between 20 and 75 ml of sample water were ﬁltered onto 0.4 µm polycarbonate ﬁlters (Nuclepore) and stained with Alcian Blue, a cationic copper phtalocyanine dye that speciﬁcally binds to carboxyl- and halfester sulphate reactive groups of acidic polysaccharides. Samples were stored in polypropy- lene tubes at −20◦C until analysis. The concentration of TEP is given in units of µg X. eq. L−1. To convert TEP into car- bon units [µmol C L−1] a conversion factor of f ′=0.63 (En- gel 2004) was used. The highest gross production, measured as oxygen pro- duction plus respiration, was observed on day 6 in 1× and 2× CO2, with 56 and 58 µmol O2 L−1d−1, respectively, whereas a similar maximum of 58 µmol O2 L−1 d−1 was observed in 3× CO2 a few days later (Fig. 2a). For all treatments, maximum NCP was observed on day 6, and after Day 14 no net production was found in the system in any of the treat- ments (Fig. 2c). When plotting cumulative O2 production we Biogeosciences, 6, 877–885, 2009 www.biogeosciences.net/6/877/2009/ 4 Discussion In addition to the CO2 manipulation, all mesocosms in PeECE III were supplied with inorganic nitrate and phosphate. The fjord water initially contained about 3 µmol Si L−1, and hence the addition of nutrients resulted in a rapid increase in primary production and a correspondingly enhanced algal biomass dominated by diatoms. A modest bloom of E. huxleyi and other nano- and pico-sized phyto- plankton succeeded the diatoms (Paulino et al., 2008; Schulz et al., 2008). Over the 24 days experimental period, we recorded a trend of increasing primary production at elevated CO2, although differences were not always statistically sig- niﬁcant. In addition to the CO2 manipulation, all mesocosms in PeECE III were supplied with inorganic nitrate and phosphate. The fjord water initially contained about 3 µmol Si L−1, and hence the addition of nutrients resulted in a rapid increase in primary production and a correspondingly enhanced algal biomass dominated by diatoms. A modest bloom of E. huxleyi and other nano- and pico-sized phyto- plankton succeeded the diatoms (Paulino et al., 2008; Schulz et al., 2008). Over the 24 days experimental period, we recorded a trend of increasing primary production at elevated CO2, although differences were not always statistically sig- niﬁcant. In all treatments our in vitro measurements gave higher values for net O2 production than what was obtained from in situ measurements. Differences between production based on bottle incubation and geochemical approaches, e.g. draw- down of DIC, have also been reported by others. In the Scheldt estuary, estimates of NCP in bottle incubation were 2-fold higher than those obtained from DIC budgets (Gazeau et al., 2005). In a net heterotrophic system, direct mea- surements of primary production and respiration were 4-fold higher than obtained from the geochemical approach (Wang et al., 2005). One reason for the discrepancy between the two methodologies in our experiment may therefore be dis- turbances of auto- and/or heterotrophic processes during the 24 h conﬁnement in the 125 ml bottles used for the O2 in- cubation. Other explanations may be the reduction of gas exchange in the production bottles, or the fact that they were incubated outside the mesocosm. Although the incubations were carried out at a light intensity corresponding to mid- depth of the mixed layer inside the mesocosms, the light con- ditions experienced by a plankton community inside a bottle at a ﬁxed depth obviously differ from those in a mixed water column (e.g. J. K. Egge et al.: Primary productionat at elevated CO2 concentrations All fractions showed an increase in production after the onset of the experiment, but during the ﬁrst week organisms in the >10 µm fraction dominated primary pro- duction (Fig. 3g, h). On day 6, 70% of the total production was observed in this fraction, thereafter the contribution of the >10 µm fraction decreased rapidly. Cumulative produc- tion was highest in 3× CO2, followed by 1× and 2× CO2 in this fraction. The difference between 3× and 2× CO2 was statistically signiﬁcant (p<0.05) during the last week, whereas differences were not obtained between 3× and 1× or 2× and 1× CO2. A distinct, but much smaller peak was observed in the fraction 5–10 µm on day 10 (Fig. 3e, f). Over the experimental period, cumulative production in TEP concentration increased after day 6 in all treatments, reached highest values of 400–450 µg X eq. L−1 between days 11 and 13 (Fig. 4a), and declined thereafter. Net production of TEP, calculated as daily changes of TEP concentration, was observed between days 7 and 11 in all treatments and accounted for at most 5 µmol C L−1 d−1 (Fig. 4b). Net production occurred also occasionally during Biogeosciences, 6, 877–885, 2009 www.biogeosciences.net/6/877/2009/ J. K. Egge et al.: Primary productionat at elevated CO2 concentrations 881 A Day no. 0 5 10 15 20 TEP( µg x eq.l-1) 0 100 200 300 400 500 600 3X CO2 2x CO2 1x CO2 B Day no. 0 5 10 15 20 25 ∆ TEP-Carbon (µmol C l-1 d-1) -8 -6 -4 -2 0 2 4 6 8 Fig. 4. Development of TEP concentration during the experiment. Mean TEP concentration ±SD of triplicate CO2 treatments given as µg Xanthan equivalents X eq L−1 (A), and estimated daily changes of TEP-carbon (µmol C L−1 d−1) (B). probes, Pringault et al. (2007) measured light respiration up to 640 % higher than in dark. We acknowledge that these processes potentially inﬂuenced our measurements, but do not have data to quantify such effects. Consistent with the observed CO2 treatment effect on DIC drawdown, Riebesell et al. (2007) reported changes in in situ O2 concentrations to deviate between CO2 treatments during the course of the ex- periment. Although we observed small differences in NCP with the O2 in vitro technique, these differences were not statistically signiﬁcant. J. K. Egge et al.: Primary productionat at elevated CO2 concentrations The in vitro technique thus would suggest a change in the photosynthetic quotient not found in the in vivo measurements. B Day no. 5 10 15 20 25 ∆ TEP-Carbon (µmol C l-1 d-1) -8 -6 -4 -2 0 2 4 6 8 A Day no. 0 5 10 15 20 TEP( µg x eq.l-1) 0 100 200 300 400 500 600 3X CO2 2x CO2 1x CO2 0 A Day no. 0 5 10 15 20 TEP( µg x eq.l-1) 0 100 200 300 400 500 600 3X CO2 2x CO2 1x CO2 B Day no. 0 5 10 15 20 25 ∆ TEP-Carbon (µmol C l-1 d-1) -8 -6 -4 -2 0 2 4 6 8 Fig. 4. Development of TEP concentration during the experiment. Mean TEP concentration ±SD of triplicate CO2 treatments given as µg Xanthan equivalents X eq L−1 (A), and estimated daily changes of TEP-carbon (µmol C L−1 d−1) (B). Using 24 h incubations for both O2 and 14C we found a NCP:14C-ﬁxation ratio of 1:1.3, corresponding well with previous reports of 1:1 (Marra et al., 2002), while our cor- responding gross O2 production: 14C-ﬁxation was high (ca. 4:1). In comparison, Gazeau et al. (2007) found a gross O2- production:14C-ﬁxation ratio of 1:1 ratio when incubating samples for 15 h , from sunrise to sunset. According to Lizon and Lagadeuc (1998), 24 h-14C incubations should approach net primary production, while 40 min incubations come close to gross primary production. Moreover, Lizon and Lagadeuc (1998) showed that increased incubation time from 4 h to 24 h may reduce production by as much as 40%, ﬁtting well with our observed reduction (34–42%) in 14C-based produc- tion estimates when increasing incubating times from 4 to 24 h. the post-bloom period in individual mesocosms. No signif- icant effect of the CO2 treatment on TEP concentration was observed in the mesocosms (p=0.3). Biogeosciences, 6, 877–885, 2009 4 Discussion inside the mesocosm). In situ measurements of dissolved inorganic carbon (DIC), during the current experiment, showed a signiﬁcantly higher DIC consumption at elevated CO2 (Riebesell et al., 2007; Bellerby et al., 2008). Over the course of the experi- ment excess DIC drawdown accumulated to approximately 40 µmol kg−1 higher carbon consumption at 3× CO2 rela- tive to 1× CO2. Plotting our 14C-data as cumulative pro- duction, we found a somewhat smaller but comparable dif- ference of 22+/−18 µmol C L−1 in particulate primary pro- duction (Fig. 1b). Our 4 h incubated 14C-based in vitro re- sults thus show the same trend of increasing C-ﬁxation with rising pCO2-as reported by Riebesell et al. (2007), but dif- fer in terms of absolute numbers. Production of DO14C was not measured this study, and could probably explain at least part of the discrepancy between DIC drawdown and particu- lar 14C production. High productions of DO14C, up to 50% compared to PO14C, was observed by Karl et al. (1998). In addition, respiration of 14C organic products and excretion to the outside or recycling inside the cell, can take place during the incubation period (Williams and Lef`evre, 2008). As sug- gested in Gargas (1975) correction for respiration of 14C of 6% of production is included in the calculation, but can be much higher (Williams and Lef`evre, 2008). Using oxygen Lack of statistical signiﬁcance may reﬂect either the lack of measurement precision or the absence of an effect. With stronger temperature variation during the temperature- sensitive optode measurement of O2 and generally more han- dling steps, there seems to be a higher potential for measure- ment errors in the in vitro compared to the in situ technique. Biogeosciences, 6, 877–885, 2009 www.biogeosciences.net/6/877/2009/ J. K. Egge et al.: Primary productionat at elevated CO2 concentrations 882 We therefore ﬁnd it difﬁcult to conclude whether the failure of our in vitro based O2-measurement to conﬁrm the in situ effect is rooted in a real disturbance of biological processes in the bottles, or just in a lower precision in the measurements. organic matter production of E. huxleyi (Zondervan et al., 2001; Leonardos and Geider, 2005). Elevated CO2 did not have any effect on primary production when E. huxleyi dom- inated the phytoplankton community in PeECE I, however (Delille et al., 2005; Engel et al., 2005). 4 Discussion Tortell et al. (2002) observed increased Si consumption as well as relatively more diatoms compared to other taxa at elevated CO2, but in our experiment nei- ther silicate drawdown nor pigment analyses indicated that CO2 signiﬁcantly inﬂuenced diatom growth (Schulz et al., 2008). Moreover, differences in particulate production due to CO2 treatment were more evident towards the end of the experiment, after the peak in diatom abundance and after the strongest drawdown in pCO2. Our results therefore do not lend support for a CO2 effect on diatom primary production. This is in accordance with observations that photosynthetic carbon ﬁxation rates of most diatoms tested so far are at or close to saturation at present CO2 levels, with only a few species having responded positively to elevated CO2 (Riebe- sell, 2004; Kim et al., 2006; Sobrino et al., 2008). Primary production in the 1–5 µm fraction was low (18% of total) but increased during the experiment, particularly during the last week. This development mirrors the abun- dance of Synechococcus which increased markedly during the last week of the experiment reaching cell numbers be- tween 3 and 4×105 cells ml−1 (Paulino et al., 2008), sug- gesting that Synechococcus was an important contributor to primary production in this size fraction. p y p A rather high primary production (29% of total produc- tion), increasing with rising CO2, was observed in the 0.2– 1 µm size-fraction. A similarly high share of both pri- mary production and chlorophyll in this size fraction has been reported from the northeast Atlantic Ocean (May–June) (Savidge et al., 1995). Børsheim et al. (2005) showed, how- ever, that approximately half of the picocyanobacteria may pass through 1 µm ﬁlters and may thus contribute distinctly to the production of organic carbon in this size fraction. In our study, maximum production in the 0.2–1µm size frac- tion occurred on day 6, when all picophototrophs (Syne- chococcus, picoeukaryotes) were at a minimum (Paulino et al., 2008). Therefore we do not consider it likely these popu- lations contributed signiﬁcantly to the production in this frac- tion. 14C found in the 0.2–1 µm size-fraction could also have been due to bacterial uptake of labelled carbon released from phytoplankton in the light bottles (Li et al., 1993; Børsheim et al, 2005), which however is not supported by the devi- ating trends observed for bacterial production (Allgaier et al., 2008) and 14C uptake in this fraction. 4 Discussion This discrepancy may be explained by differences in the nutrient environment or phytoplankton composition during blooms in the two ex- periments. In the present experiment, both nitrate and phos- phate were available when E. huxleyi peaked, while nitrate was depleted when the E. huxleyi reached maximum num- bers in PeECE I (Engel et al., 2005; Schulz et al., 2008). Sciandra et al. (2003) observed a decreased production of POC in cultures of E. huxleyi at elevated CO2 when nitrate was depleted, which may explain why no effect of increased CO2 on primary production was observed in PeECE I. While E. huxleyi was dominating during PeECE I, other nano-sized species were as numerous as E. huxleyi in PeECE III. These phytoplankton taxa were affected by increased pCO2 as well (Paulino et al., 2008), and their contribution to primary pro- duction were probably comparable to E. huxleyi. We did not observe any changes in primary production between size fractions to indicate a shift in community composition from diatoms to nano-phytoplankton at high CO2 as was demon- strated by Hare et al. (2007). In our study, inorganic nutrient availability changed along with phytoplankton succession and 5 different phases can be identiﬁed (Tanaka et al., 2008): During the ﬁrst 6 days (Phase I days 0–6) all nutrients were detectable. Silicate was the ﬁrst nutrient to become depleted (day 6), followed by phos- phate depletion on day 10 (Phase II days 6–10), and nitrate depletion on day 13 (Phase III days 10–13). The last two phases were characterized by nutrient depletion and increas- ing (Phase IV) or oscillating (Phase V) phosphate turnover times. The highest particulate primary production rates were observed during the ﬁrst two phases. At the time of sili- cate depletion (day 6), 70% of the total production occurred in the largest size fraction (>10 µm), and the same fraction was responsible for 50–70% of the 33PO4-uptake (Tanaka et al., 2008). Pigment analysis showed that diatoms dominated among larger algae during the ﬁrst two phases (Schulz et al., 2008). Since NCP was close to zero from day 14, diatoms can be considered the main contributors to the net primary production in this study. Although cumulative production in the >10 µm fraction was signiﬁcantly higher at 3× than at 2× CO2, there was no clear trend of primary production with CO2 concentration as production was not different between 3× and 1× CO2. www.biogeosciences.net/6/877/2009/ 4 Discussion The absence of a het- erotrophic phase during the course of the experiment pro- vides further evidence for vertical transport of primary pro- duced organic matter. et al. (2007) and Bellerby et al. (2008). Size-fractionated pri- mary production measurements combined with data on phy- toplankton composition further indicated that in some groups or species primary production may be stimulated at elevated CO2 levels. However, in contrast to the CO2 effect on DIC drawdown, which became evident already during the bloom development, the effect of pCO2 on 14C-based total particu- late primary production was visible only after inorganic nu- trients had been depleted and statistically signiﬁcant only on the very last days of the experiment. The key to explaining this discrepancy may be in the early occurrence of silicate depletion, which may have caused the comparatively early production of TEP by diatoms, leading to the sinking of di- atom biomass and TEP at a time when the phytoplankton bloom was still building up (see Schulz et al. 2008). Whereas a CO2 treatment effect on primary production during this phase would still leave a signal in the time-integrating DIC drawdown, the short-term 14C and O2 primary production measurements in bottle enclosures would have difﬁculties detecting such an effect or may miss it completely. This may also explain why in bottle incubations a CO2 treatment ef- fect is detected in the second half of the experiment, at a time when TEP concentrations were much lower and TEP loss due to sinking was probably low. g p y If the effect of rising pCO2 is an increase in the produc- tion of organic C under conditions of mineral nutrient limited phytoplankton growth, this will only have a feedback effect on atmospheric CO2 if the extra material is not respired by bacteria in the photic zone. Excess organic matter may be unavailable to bacterial consumption for several reasons. It may be physically protected inside phytoplankton cells, or it may be in chemical forms resistant to bacterial enzymatic at- tack. It has also been suggested that degradation of otherwise labile DOC may be prevented by mineral nutrient limitation of bacterial growth (Thingstad et al., 1997). A net effect on C-sequestration may therefore depend not only on the physi- ological responses in phytoplankton, but also vary with eco- logical status and limiting factors for bacterial growth in the photic zone (Tanaka et al. 2008). 4 Discussion Another explana- tion may be that 14C-labelled organic material released from phytoplankton aggregated into transparent exopolymer par- ticles (TEP) which originate from dissolved carbohydrates As in several previous mesocosm experiments (Egge and Heimdal, 1994; Engel et al., 2005), an initial pulse of ni- trate and phosphate induced an E. huxleyi bloom – although with relatively low maximum numbers (observed in Phase II) (Paulino et al., 2008). The 5–10 µm size fraction has been shown to represent E. huxleyi quite well in blooms domi- nated by this species (Egge, 1994; Engel et al., 2008). Con- sistent with this, maximum primary production rates in the 5–10 µm fraction were measured when E. huxleyi cell num- bers were at their maximum in PeECE III (day 10, 4.4 to 4.7×103 cells ml−1, Paulino et al., 2008). A tendency, al- though not signiﬁcant, of increased cumulative primary pro- duction at elevated pCO2 (pP=0.07) in the 5–10 µm fraction is in accordance with previous reports of CO2 sensitivity in Biogeosciences, 6, 877–885, 2009 www.biogeosciences.net/6/877/2009/ www.biogeosciences.net/6/877/2009/ J. K. Egge et al.: Primary productionat at elevated CO2 concentrations 883 and extend from 0.4 µm to several 100 µm during contin- ued aggregation. Newly formed TEP should be included in the14C-labeled material of the 0.2–1µm fraction that was ob- served after day 6. As both diatoms and E. huxleyi have been shown to produce TEP (Passow 2002; Engel et al., 2004), and both populations had their maximum in the ﬁrst half of our experiment (Paulino et al., 2008; Schulz et al., 2008), pro- duction observed in the smallest fraction could have had its origin in the larger size fractions and be linked to the mech- anism of carbon overproduction under nutrient limited con- ditions (Engel 2002). TEP have proven to increase at ele- vated CO2 concentrations (Engel 2002; Engel et al., 2004). However, TEP concentration did not reveal a corresponding effect of CO2 in the present experiment and declined during the 2nd half of the experiment, indicating that TEP dynamics after the diatom peak were mainly driven by loss processes, as also reported for excess organic carbon (Riebesell et al., 2007). Similarly, the apparent difference between primary production, determined in bottle enclosures and changes in suspended TEP concentrations in the mesocosms may be ex- plained by differences in loss processes: high loss through rapid sinking of TEP in the mesocosms versus accumula- tion of TEP in the incubation bottles. References Allgaier, M., Riebesell, U., Vogt, M., Thyrhaug, R., and Grossart, H.-P.: Coupling of heterotrophic bacteria to phy- toplankton bloom development at different pCO2 levels: a mesocosm study, Biogeosciences, 5, 1007–1022, 2008, http://www.biogeosciences.net/5/1007/2008/. Engel, A., Schulz, K., Riebesell, U., Bellerby, R., Delille, B., and Schartau, M.: Effects of CO2 on particle size and phytoplankton abundance during a mesocosm bloom ex- periment (PeECE II), Biogeosciences, 5, 509–521, 2008, http://www.biogeosciences.net/5/509/2008/. Bellerby, R. G. J., Schulz. K. G., Riebesell, U., Neil, C., Nondal, G., Johannessen, T., and Brown, K. R.: Marine ecosystem com- munity carbon and nutrient uptake stoichiometry under varying ocean acidiﬁcation during the PeECE III experiment. Biogeo- sciences, 5, 1517–1527, 2008. Gargas, E.: A manual for phytoplankton primary production studies in the Baltic, The Baltic Marine Biologists, Publication No. 2, The Danish Agency of Environmental Protection, Hørsholm, 1– 88, 1975. Brett, M. T. and Goldman, C. R.: A meta-analysis of the freshwa- ter trophic cascade, Proc. Natl. Acad. Sci, USA, 93, 7723–7726, 1996. Gazeau, F, Gattuso, J. P., Middleburg, J. J., Brion, N., Schiette- catte, L. S., Frankignoulle, M., and Borges, A. V.: Planktonic and whole system metabolism in a nutrient-rich estuary (the Scheldt estuary), Estuaries, 28, 868–833, 2005. Burkhardt S., Amoroso, G., Riebesell, U., and S¨ultemeyer, D.: CO2 and HCO− 3 uptake in marine diatoms acclimated to different CO2 concentrations, Limnol. Oceanogr., 46, 1378–1391, 2001. Gazeau, F., Middelburg, J. J., Loijens, M., Vanderborght J.-H., Pizay, M.-D., and Gattuso, J.-P.: Planktonic primary production in estuaries: comparison of 14C, O2 and 18O methods, Aquat. Microb. Ecol., 46, 95–106, 2007. Børsheim, K.I., Vadstein, O., Myklestad S.M., Reinertsen, H., Kirkvold, K., Olsen, Y.: Photosynthetic algal production, ac- cumulation and realease of phytoplankton storage carbohydrates and bacterial production in a gradient in daily nutrient supply, J. Plank. Res., 27, 743–745, 2005. Giordano, M., Beardall, J., and Raven, J. A.: CO2 concentrating mechanisms in algae: Mechanisms, environmental modulation, and evolution, Annu. Rev. Plant. Biol., 56, 99–131, 2005. Carpenter, S. R.: Microcosm experiments have limited relevance for community and ecosystem ecology, Ecology, 77, 677–680, 1996. Grossart, H. P., Allgaier, M., Passow, U., and Riebesell, U.: Testing the effect of CO2 concentration on the dynamics of marine het- erotrophic bacterioplankton, Limnol. Oceanogr., 51, 1–11, 2006. Clark, D. R. and Flynn, K. J.: The relationship between the dis- solved inorganic carbon concentration and growth rate in marine phytoplankton, Proc. R. Soc. Lond., 267, 953–959, 2000. Hare, C. Edited by: J. Middelburg Edited by: J. Middelburg Edited by: J. Middelburg 4 Discussion The lack of any net het- erotrophic phase in PeECE III shows that organic material produced during net autotrophy was not degraded by bacte- ria in the upper layer, but either accumulated or was exported vertically. This supports the interpretation of Riebesell et al. (2007) of a high export of organic material through the py- cnocline in this experiment. This accumulation/export, com- bined with the observation of a CO2 effect on bacterial pro- duction in PeECE II (Grossart et al. 2006), but not in PeECE III (Allgaier et al., 2008), highlights the need to better under- stand the whole microbial community, including ecological mechanisms regulating bacterial growth rate limitation, in or- der to understand the net effects of any increased C-ﬁxation at high pCO2. An increase in semi-labile DOC during the senescent phase of the E. huxleyi-bloom was evident in PeECE I (Joassin et al., 2007), but statistically signiﬁcant CO2 treat- ment effects on the concentration of DOC were not detected in any of the PeECE experiments (Rochelle-Newall et al., 2004; Grossart et al., 2006; Schulz et al., 2008) or in a meso- cosm experiment with similar CO2 treatments (Kim et al., 2006). Higher bacterial production, possibly indicating more available DOC, was observed at the highest CO2 level in PeECE II, but the present experiment did not reveal similar results (Grossart et al., 2006; Allgaier et al., 2008). As our 14C-based measurements did not include DO14C, allowing for the possibility of a conversion of the over-consumption of DIC into DOC by e.g. excretion or leakage from phyto- plankton cells, we are presently not in the position to draw a ﬁrm conclusion on this matter. Experiments with duration of a few weeks do not include all possible responses of a potentially adaptive plankton com- munity. Extrapolation to longer time scales should there- fore be done with caution. It should also be noted that in large scale experiments, which are generally bound to a small number of replicates, there is a risk of erroneously accepting the hypothesis of “no treatment effect” when perturbations are small and variance is large (Brett and Goldman, 1996; Carpenter, 1996). Still, our results demonstrate a small, but statistically signiﬁcant effect of elevated CO2 on daily pri- mary production. The trend found in cumulative 14C-based particulate primary production was consistent with the over- consumption of DIC at elevated CO2 reported by Riebesell Biogeosciences, 6, 877–885, 2009 www.biogeosciences.net/6/877/2009/ J. 4 Discussion K. Egge et al.: Primary productionat at elevated CO2 concentrations 884 Egge, J. K. and Heimdal, B. R.: Blooms of phytoplankton includ- ing Emiliania huxleyi (Haptophyta). Effects of nutrient supply in different N:P ratios, Sarsia, 79, 333–348, 1994. Acknowledgements. The staff at the Marine Biological Station, University of Bergen, in particular Tomas Sørlie and Agnes Aadnesen, and the Bergen Marine Research infrastructure (RI) are gratefully acknowledged for support in mesocosm logistics. Thanks also go to Craig Neill for providing TCO2 data for primary production calculations, and the Geophysical Institute, University of Bergen for providing data for global radiation. We also like to thank Peter Williams and two anonymous referees for valuable comments and their patience. This study was supported by EU-TMR contract no HPRI-CT-2002-00181, NFR project no.158936/I10 Biodiversity patterns: Blooms versus stable coex- istence in the lower part of the marine pelagic food web and the EU project CARBOOCEAN “Marine carbon sources and sinks assessment”(contract no. 511176). Engel, A.: Direct relationship between CO2 uptake and transpar- ent exopolymer particles production in natural phytoplankton, J. Plank. Res., 24, 49–53, 2002. Engel, A.: Distribution of transparent exopolymer particles (TEP) in the northeast Atlantic Ocean and their potential signiﬁcance for aggregation processes, Deep Sea Res., 51, 83–92, 2004. Engel, A., Delille, B., Jacquet, S., Riebesell, U., Rochelle-Newall, E., Terbr¨uggen, A., and Zondervan, I.: Transparent exopolymer particles and dissolved organic carbon production by Emiliania huxleyi exposed to different CO2 concentrations: a mesocosm experiment, Aquat. Microb. Ecol., 34, 93–104, 2004. Engel, A., Zondervan, I., Aerts, K., Baufort, L., Benthien, A. Chou, L., Delille, B., Gattuso, J. P., Harley, J., Heeman, C., Hoffmann, L., Jacquet, S., Nejstgaard, J., Pizay, M. D., Rochelle-Newall, E., Schneider, U., Terbrueggen, A., and Riebesell, U.: Testing the direct effect of CO2 concentration on a bloom of the coccol- ithophorid Emiliania huxleyi in mesocosm experiment, Limnol. Oceanogr., 50, 493–507, 2005. References P.: Response of coccolithophorid Emil- iania huxleyi to elevated partial pressure of CO2 under nitrogen limitation, Mar. Ecol. Prog. Ser., 261, 111–122, 2003. Schippers, P., L¨urling, M., and Scheffer, M.: Increase of atmo- spheric CO2 promotes phytoplankton productivity, Ecological Lett., 446–451, 2004. Leonardos, N. and Geider, R. J.: Elevated atmospheric carbon diox- ide increases organic carbon ﬁxation by Emiliania huxleyi (Hap- tophyta), under nutrient-limited high-light conditions, J. Phycol., 41, 1196–1203, 2005. Schulz, K. G., Riebesell, U., Bellerby, R. G. J., Biswas, H., Mey- erh¨ofer, M., M¨uller, M. N., Egge, J. K., Nejstgaard, J. C., Neill, C., Wohlers, J. and Z¨ollner, E.: Build-up and decline of organic matter during PeECE, Biogeosciences, 5, 707–718, 2008. Li, W. K. W.,and Irwin, B. D., and Dickie, P. M.: Variation related to biomass and productivity of phytoplankton and bacteria, Limnol. Oceanogr., 38, 483–494, 1993. Sobrino, C. S., Ward, M. L., and Neale, P. J.: Acclimation to ele- vated carbon dioxide and ultraviolet radiation in the diatom Tha- lassiosira pseudonana: Effects on growth, photosynthesis, and spectral sensitivity of photoinhibition, Limnol. Oceanogr., 53, 494–505, 2008. Lizon, F. and Lagadeuc, Y.: Comparisons of primary production values estimated from different incubation times in a coastal sea, J. Plank. Res. 2, 371–381, 1998. Passow, U.: Production of transparent exopolymer particles (TEP) by phyto- and bacterioplankton, Mar. Ecol. Prog. Ser. 236, 1–12, 2002. Sokal, R. R. and Rohlf, F. J.: Biometry, the principles and practice of statistics in biological research, 3rd Ed., 7th Printing, W. H. Freeman and Company, 887 pp., 2001. 14 Passow, U. and Alldredge, A. L.: A dye-binding assay for the spec- trophotometric measurement of transparent exopolymer particles (TEP), Limnol. Oceanogr., 40, 1326–1335, 1995. Steemann Nielsen, E.: The use of radioactive (14C) for measuring organic production in the sea, J. Cons. Perm. Int. Expl. Mer., 18, 117–140, 1952. Paulino, A. I., Egge, J. K., and Larsen, A.: Effects of increased atmospheric CO2 on small and intermediate sized osmotrophs during a nutrient induced phytoplankton bloom, Biogeosciences, 5, 739–748, 2008, http://www.biogeosciences.net/5/739/2008/. Tanaka, T., Thingstad, T. F., Løvdal, T., Grossart, H.-P., Larsen, A., Schulz, K., and Riebesell, U.: Availability of phosphate for phytoplankton and bacteria and of labile organic carbon for bacteria at different pCO2 levels in mesocosm study, Biogeo- sciences, 5, 669–687, 2008. Pringault, O., Tassas, V., and Rochelle-Newall, E.: Consequences of respiration in the light on the determination of produc- tion in pelagic systems, Biogeosciences, 4, 105–114, 2007, http://www.biogeosciences.net/4/105/2007/. References E., Leblanc, K., DiTullio, G. R., Kudela, R. M., Zhang, Y., Lee, P. A., Risman, S., Hutchins, D. A.: Consequences of increased temperature and CO2 for phytoplankton community in Bering Sea, Mar. Ecol. Prog. Ser., 352, 9–16, 2007. Delille, B., Harley, J., Zondervan, I., Jacquet, S., Chou, L., Wollast, R., Bellerby. R. G. J., Frankignoulle, M., Borges, A. V., Riebe- sell, U., and Gattuso, J. P.: Response of primary production and calciﬁcation to changes of pCO2 during experimental blooms of the coccolithophorid Emiliania huxleyi, Global Biogeochem. Cy., 19, GB2023, doi:10.1029/2004GB002318, 2005. Hein, M. and Sand-Jensen, K.: CO2 increases oceanic primary pro- duction, Nature, 338, 526–527, 1997. Joassin, P., Delille, B., Soetaert, K., Borges, A. V., Chou, L. Engel, A., Gattuso, J.-P., Harlay, J., Riebesell, U., Suykens, K., and Gregoire, M.: A mathematical modelling of bloom of the coccolithophore Emiliania huxleyi in a meso- cosm experiment, Biogeosciences Discuss., 5, 787–840, 2008, http://www.biogeosciences-discuss.net/5/787/2008/. Dugdale, R. C. and Goering, J. J.: Uptake of new and regenerated forms of nitrogen in primary productivity, Limnol. Oceanogr., 12, 196–206, 1967. Egge, J. K.: Nutrient control of phytoplankton growth: Effects of macronutrients composition (N, P, Si) on species succession. Dr. Scient thesis, University of Bergen, Norway, ISBN: 82-7744- 007-3, 1994. Karl, D. M., Hebel, D. V., and Bj¨ormann, K.: The role of dissolved organic matter release in the productivity of the oligotrophic North Paciﬁc Ocean, Limol. Oceanogr. 43, 1270–1286, 1998. Biogeosciences, 6, 877–885, 2009 www.biogeosciences.net/6/877/2009/ J. K. Egge et al.: Primary productionat at elevated CO2 concentrations J. K. Egge et al.: Primary productionat at elevated CO2 concentrations 885 Oceanogr., 48, 55–67, 2003. Kim, J.-M., Lee, K., Shin, K., Kang, J.-H., Lee, H.-W., Kim, M., Jang, P.-G., and Jang, M.-C.: The effect of seawater CO2 con- centration on growth of natural phytoplankton assemblage in a controlled mesocosm experiment, Limnol. Oceanogr. 51, 1629– 1636, 2006. Oceanogr., 48, 55–67, 2003. Oceanogr., 48, 55–67, 2003. Savidge, G., Boyd, P., Pomroy, A., Harbour, D., and Joint, I.: Phy- toplankton production and biomass estimates in the northeast Atlantic Ocean, May–June 1990, Deep-Sea Res., 42, 599–617, 1995. Marra, J.: Approches to the Measuremrnt of Plankton Produc- tion: Chapter 4 edited by: Williams, P. J. le B., Thomas, D. N., Reynolds, C. S., in: Primary Productivity. Carbon assimilation in marine and freshwater ecosystems, Blackwell Science, Oxford, 78–108, 2002. Sciandra, A., Harley, J., Lef`evre, D., Lem´ee, R., Rimmelin, P., De- nis, M., and Gattuso, J. References Thingstad, T. F., Hagstrom, A., and Rassoulzadegan, F.: Accumula- tion of degradable DOC in surface waters: Is it caused by a mal- functioning microbial loop?, Limnol. Oceanogr., 42, 398–404, 1997. Raven, J. A.: Physiology of inorganic C acquisition and implica- tions for resource use efﬁciency by marine phytoplankton: rela- tion to increased CO2 and temperature, Plant. Cell. Environ., 14, 779–774, 1991. Tortell, P. D.: Evolutionary and ecological perspectives on carbon acquisition in phytoplankton, Limnol. Oceanogr., 45, 744–750, 2000. Raven, J. A. and Johnston, A. M.: Mechanisms of inorganic-carbon acquisition in marine phytoplankton and their implications for the use of other recourses, Limnol. Oceanogr. 36, 1701–1714, 1991. Tortell, P. D., DiTullino, G. R., Sigman, D. M., and Morel, F. M. M.: CO2 effects on taxonomic composition and nutrient utilization in an Equatorial Paciﬁc phytoplankton assemblage, Mar. Ecol. Prog. Ser., 236, 37–43, 2002. Riebesell, U., Wolf-Gladrow, D., and Smetacek, V.: Carbon dioxide limitation of marine phytoplankton growth rates, Nature, 361, 249–251, 1993. Wang, Z. A., Cai, W. J., Wang, Y. C., and Ji, H. W.: The south- eastern continental shelf of the United States as an atmospheric CO2 source and an exporter of inorganic carbon to the ocean, Continental Shelf Res, 25, 1917–1941, 2005. Riebesell, U.: Effects of CO2 enrichment on marine phytoplankton, J. Oceanogr. 60, 719–729, 2004. Williams, P. J. le B. and Lef`evre, D.: An assessment of the mea- surement of phytoplankton respiration rates from dark 14C incu- bations, Limnol. Oceanogr. Methods, 6, 1–11, 2008. Riebesell, U., Schulz, K. G., Bellerby, R. G. J., Botros, M., Fritsche, P., Meyerh¨ofer, M., Neil, C., Nondal, G., Oschies, A., Wohlers, J., and Z¨ollner, E.: Enhanced biological car- bon consumption in a high CO2 ocean, Nature, 450, 545–548, doi:10.1038/nature06267, 2007. Wolf-Gladrow, D., Riebesell, U., Burkhardt, S., and Bijma, J.: Di- rect effects of CO2 concentration on growth and isotopic compo- sition of marine plankton, Tellus, 51B, 461–476, 1999. Rochelle-Newall, E., Delille, B., Frankignoulle, M., Gattuso, J. P., Jacquet, S., Riebesell, U., Terbruggen, A., and Zondervan, I.: Chromophoric dissolved organic matter in experimental meso- cosms maintained under different pCO2 levels, Mar. Ecol. Prog. Ser., 272, 25–31, 2004. Zondervan, I., Zeebe, R. E., Rost, B., and Riebesell, U.: Decreasing marine biogenic calciﬁcation: a negative feedback on rising at- mospheric pCO2, Global Biogeochem. Cy., 15, 507–516, 2001. Rost, B., Riebesell, U., Burkhardt, S. and S¨ultemeyer, D.: Carbon acquisition of bloom-forming phytoplankton, Limnol. Biogeosciences, 6, 877–885, 2009 www.biogeosciences.net/6/877/2009/
https://openalex.org/W1828425454	https://jmedicalcasereports.biomedcentral.com/track/pdf/10.1186/s13256-015-0726-1	English	null	Unusual localization of a primary pleomorphic malignant fibrous histiocytoma on the mitral valve: a case report and review of the literature	Journal of medical case reports	2,015	cc-by	3,096	CASE REPORT Open Access Prifti et al. Journal of Medical Case Reports (2015) 9:246 DOI 10.1186/s13256-015-0726-1 Prifti et al. Journal of Medical Case Reports (2015) 9:246 DOI 10.1186/s13256-015-0726-1 JOURNAL OF MEDICAL CASE REPORTS Abstract Introduction: It has been reported that cardiac malignant fibrous histiocytomas occur more frequently in the left side of the heart, especially in the left atrium, but rarely invade the mitral valve. We present a case with a giant malignant fibrous histiocytoma with an unusual localization involving almost the entire left atrium, mitral valve, and left superior pulmonary vein. Case presentation: We describe the case of a 54-year-old woman from Kosovo admitted to our emergency department with dyspnea. A transthoracic echocardiography demonstrated a giant mass localized on her left atrium. Our patient underwent emergent total surgical removal of the mass. The mass extended between her left superior pulmonary vein, and extended to her left atrium and the posterior mitral valve leaflet. We formulated a surgical plan for total separation of the mass from the endocardium. Total removal was performed and her left side pulmonary veins were entirely freed from the mass. We then performed a mitral valve replacement. The differential diagnosis included other masses of the left atrium, including thrombi, vegetations, and cardiac tumors. Postsurgical histopathologic results showed a pleomorphic malignant fibrous histiocytoma. Six monthly follow-up cardiac and abdominal sonographic examinations revealed no tumor recidivism. Conclusion: We reviewed 90 cases with malignant fibrous histiocytoma reported in the literature. Our case was especially unusual because of the primary location in the mitral valve, the pleomorphic variant, and the dimensions and extension. Complete surgical resection is mandatory to ameliorate symptoms and to obtain histologic information. Keywords: Left atrium, Mitral valve, Pleomorphic malignant fibrous histiocytoma Keywords: Left atrium, Mitral valve, Pleomorphic malignant fibrous histiocytoma Unusual localization of a primary pleomorphic malignant fibrous histiocytoma on the mitral valve: a case report and review of the literature Edvin Prifti1, Fadil Ademaj2,3, Majlinda Ikonomi1 and Aurel Demiraj2 Introduction rarely invade the mitral valve. The pleomorphic variant of MFH is rarely found and comprises a high cellularity with bizarre tumor cells with marked atypia and high mitotic index. Here, we report the case of a patient with a pleomorphic MFH invading her mitral valve that underwent successful surgical removal. To the best of our knowledge, this is the first reported case of a pleo- morphic variant of such dimensions and tumor exten- sion involving the mitral valve. The first reported case of a cardiac malignant fibrous histiocytoma (MFH) was published by O’Brien et al. [1] in 1964 and the first case undergoing surgical removal in 1978 [2]. In 2001, Okamoto et al. [3] analyzed 46 cases of reported MFH. Since then, 44 additional cases of MFH have been reported, resulting in a total of 90 cases of MFH. Cardiac MFH occur more frequently in the left side of the heart, especially in the left atrium [4–6], but Correspondence: fadilademaj1971@gmail.com 2Division of Cardiology, Regional Hospital of Gjakovo, Gjakovo, Kosovo 3Division of Heart Disease, Gjakovo Hospital, Rr. Prizren, Gjakove, Kosove Full list of author information is available at the end of the article © 2015 Prifti et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. * Correspondence: fadilademaj1971@gmail.com 2Division of Cardiology, Regional Hospital of Gjakovo, Gjakovo, Kosovo 3Division of Heart Disease, Gjakovo Hospital, Rr. Prizren, Gjakove, Kosove Full list of author information is available at the end of the article Case presentation A 54-year-old woman from Kosovo was admitted to our hospital with dyspnea. On clinical examination, our A 54-year-old woman from Kosovo was admitted to our hospital with dyspnea. On clinical examination, our Page 2 of 5 Prifti et al. Journal of Medical Case Reports (2015) 9:246 Page 2 of 5 patient’s blood pressure was 100/60 mmHg, and her pulse rate was 130 beats per minute with a regular rate and rhythm. Cardiac auscultation revealed a diastolic murmur. End-inspiratory crackles suggested pulmonary edema. Two-dimensional transthoracic echocardiog- raphy revealed a giant mass originating from her poster- ior mitral valve leaflet, occupying almost her entire left atrial cavity. Cerebral, thoracic, and abdominal com- puted tomography was also performed, showing no evi- dence of additional tumors. aspect of her left atrium. The tumor invaded the posterior leaflet of her mitral valve. The tumor mass was carefully detached from the endocardium (Fig. 1c) and then entirely removed, including the posterior leaflet of her mitral valve, which was replaced with a 29-mm St Jude mechan- ical prosthesis (Fig. 1d). Macroscopically, the excised lesion was composed of multiple irregular soft tissue fragments (Fig. 2). After the surgical excision, the mass was fixed in formalin, paraffin embedded, sectioned at 3-μm thick, and stained conven- tionally with hematoxylin and eosin. Examination of the histology revealed a high grade sarcoma composed of a fusicellular proliferation in a partial storiform pattern, with irregular fascicles, high cellularity, and pleomorphic and bizarre tumor cells with marked atypia and a high mitotic index (Fig. 3a). There were also large areas of necrosis. Immunohistochemical examination results were 25 % positive for Ki-67 in the tumor (Fig. 3b); negative for the muscle markers and the melanocytic markers CD45 and S100 (Fig. 3c); and positive for CD68, Our patient underwent emergency surgical removal of the cardiac tumor. Intraoperative transesophageal echo- cardiography was performed, which confirmed the pres- ence of the tumor (Fig. 1a). Our patient underwent bicaval cannulation. Her aorta was clamped and anterograde car- dioplegia was administered. Then her left atrium was opened at the interatrial groove. A giant yellowish-white tumoral mass was identified, occupying almost her entire left atrial cavity (Fig. 1b). The mass had invaded her left superior pulmonary vein and extended into the posterior Fig. 1 a Intraoperative transesophageal echocardiography. b Yellowish-white mass in the left atrium. c Removal of the mass from the endocardium. Case presentation d Mitral valve replacement with a mechanical prosthesis Fig. 1 a Intraoperative transesophageal echocardiography. b Yellowish-white mass in the left atrium. c Removal of the mass d Mitral valve replacement with a mechanical prosthesis Fig. 1 a Intraoperative transesophageal echocardiography. b Yellowish-white mass in the left atrium. c Removal of the mass from the endocardium. d Mitral valve replacement with a mechanical prosthesis Page 3 of 5 Prifti et al. Journal of Medical Case Reports (2015) 9:246 vimentin, and alpha-1-antitrypsin (Fig. 3d). A diagnosis of pleomorphic MFH was made. Fig. 2 The excised macroscopic tumoral fragments Our patient’s postoperative course was uneventful. Six weeks after surgery, she started a six-course chemotherapy regimen of 1.5 mg/m2 of ifosfamide on days 1 to 3 and 80 mg/m2 of epidoxorubicin on day 1. The treatment was well tolerated with no unacceptable toxicities. Our patient was still alive with no signs of metastasis six months later. Discussion Journal of Medical Case Reports (2015) 9:246 Table 1 Cases reported in the literature with primary cardiac malignant fibrous histiocytoma Variables Total 90 Female 60 (67 %) Age (years) 14–80 Localization Left atrium 49 Pericardium 5 Pulmonary veins 2 Mitral valve 8 Right ventricle 8 Right atrium 9 Left ventricle 5 Pulmonary artery 3 Inferior vena cava 1 Symptoms Dyspnea 63 (70 %) Palpitations 40 (45 %) Pleomorphic 12 (13 %) sarcoma (MFH), occur most often as endocardium-based pathologies. The differential diagnosis of cardiac sar- coma in these circumstances is cardiac myxoma, especially in the case of a left atrial intimal-type sar- coma. MFH is a pleomorphic sarcoma composed of fibroblasts, myofibroblasts, and histiocyte-like cells. The pleomorphic variant of the MFH is rarely re- ported and only had mitral valve involvement in two of the 90 cases we found in the literature [8, 9]. The treatment plan for MFH depends on the tumor status, size, histology, location, and metastatic spread. Complete surgical resection is mandatory to ameliorate symptoms and to obtain histologic information. In our case, once we had started surgery, we suspected that we had a malignant tumor. However, owing to the fact that the tumor had invaded her posterior mitral valve leaflet, causing severe mitral valve obstruction, we had to re- move her mitral valve as this was the only measure to complete total tumoral mass removal and relieve the mi- tral valve stenosis. Although the prognosis of MFH is poor and the possi- bility of local recurrence and metastasis is high, patients benefit from surgery. Multiple studies have reported a median survival of six months for right-sided tumors, whereas left heart tumors seem to have a better progno- sis [14]. The role of chemotherapy and radiotherapy in the treatment of primary cardiac sarcomas has not proven to be beneficial, and complete surgical excision is the only mode of therapy that has been shown to pro- long survival. manifests as an embolism. Almost 65.5 % of the reported patients presented with a primary MFH in the left atrium. The tumor can also localize in different portions of the left atrium, such as the pulmonary veins [4], septum, and posterior wall. However, the MFH invaded the mitral valve in only eight of the 90 cases [7–13], in- cluding our own. In our case, the tumor occupied almost her entire left atrial cavity. Discussion Primary MFH is the second most common primary car- diac sarcoma, with an estimated incidence of ∼1.7 % of cases. It usually affects people of ages 14 to 77 years [3]. After a careful review of the literature, we found reports of a total of 90 cases of primary MFH (Table 1). Primary MFH is common in the left heart, and it frequently Fig. 2 The excised macroscopic tumoral fragments Fig. 2 The excised macroscopic tumoral fragments Fig. 3 a Histological examination revealed a high grade sarcoma with a fusicellular proliferation organized in a partial storiform pattern, with irregular fascicles, high cellularity, and pleomorphic and bizarre tumor cells with marked atypia and a high mitotic index (hematoxylin and eosin ×20). b Staining was 25 % positive for Ki-67 in the tumor (Ki-67 ×20). c The resected sample was negative for muscle markers (smooth muscle actin ×20) and melanocytic markers CD45 and S10. d The immunohistochemical examinations were positive for CD68, vimentin, and alpha-1- antitrypsin (CD68 ×20) Fig. 3 a Histological examination revealed a high grade sarcoma with a fusicellular proliferation organized in a partial storiform pattern, with irregular fascicles, high cellularity, and pleomorphic and bizarre tumor cells with marked atypia and a high mitotic index (hematoxylin and eosin ×20). b Staining was 25 % positive for Ki-67 in the tumor (Ki-67 ×20). c The resected sample was negative for muscle markers (smooth muscle actin ×20) and melanocytic markers CD45 and S10. d The immunohistochemical examinations were positive for CD68, vimentin, and alpha-1- Fig. 3 a Histological examination revealed a high grade sarcoma with a fusicellular proliferation organized in a partial storiform pattern, with irregular fascicles, high cellularity, and pleomorphic and bizarre tumor cells with marked atypia and a high mitotic index (hematoxylin and eosin ×20). b Staining was 25 % positive for Ki-67 in the tumor (Ki-67 ×20). c The resected sample was negative for muscle markers (smooth muscle actin ×20) and melanocytic markers CD45 and S10. d The immunohistochemical examinations were positive for CD68, vimentin, and alpha-1- antitrypsin (CD68 ×20) Page 4 of 5 Prifti et al. Discussion Our report seems to be the first reported case with such large tumor dimensions and extension involving the pulmonary vein, left atrium, and mitral valve. The non-septal origin of the mass strongly supports the suspicion of sarcoma. Multiple at- tachment sites and infiltration of the mitral valve and the atrial and ventricular walls are also indicative of malignancy. Conclusion Our case was especially unusual because of the primary location of an MFH in the mitral valve, the pleomorphic variant, and the dimensions and extension. Complete surgical resection, including mitral valve replacement, is mandatory to improve symptoms and to obtain histo- logic information. Clinical manifestations of primary MFH are dependent on their size and location. With small tumors, no clinical manifestations are evident. As the tumor grows, the most frequent symptoms are shortness of breath, palpi- tation, or chest discomfort. The MFH in our case is, to the best of our knowledge, the largest reported. Competing interests The authors declare that they have no competing interests. Consent Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal. Histologically, MFH is a variously shaped and multilo- bulated mass, sessile or pedunculated. It is a tumor of the fibroblasts, with giant cells and atypical nuclear and cytoplasmic findings. The term MFH has now become synonymous of undifferentiated high-grade pleomorphic sarcoma, because the fibrohistiocytic differentiation is not characteristic of a specific tumor type. Sarcomas of myoblastic or fibroblastic origin, including leiomyosarcoma, fibrosarcoma, myxoid fibrosarcoma, and pleomorphic Competing interests The authors declare that they have no competing interests. Authors’ contributions EP performed the surgical procedure. FA preoperatively diagnosed the patient. MI performed the histopathological examination. AD performed the literature review. All authors were involved in drafting the manuscript or revising it critically for important intellectual content and have read and approved the final version. Page 5 of 5 Page 5 of 5 Prifti et al. Journal of Medical Case Reports (2015) 9:246 Prifti et al. Journal of Medical Case Reports (2015) 9:246 Author details 1 1Division of Cardiac Surgery, University Hospital Center of Tirana, Tirana, Albania. 2Division of Cardiology, Regional Hospital of Gjakovo, Gjakovo, Kosovo. 3Division of Heart Disease, Gjakovo Hospital, Rr. Prizren, Gjakove, Kosove. Kosovo. 3Division of Heart Disease, Gjakovo Hospital, Rr. Prizren, Gjakove, Kosove. Received: 2 May 2015 Accepted: 7 October 2015 Acknowledgement We would like to thank Mrs Enida Hoxha/Prifti for her support regarding the linguistic editing of the manuscript. References References 1. O’Brien JE, Stout AP. Malignant fibrous xanthomas. Cancer. 1964;17:1155–445. 1. O’Brien JE, Stout AP. Malignant fibrous xanthomas. Cancer. 1964;17:1155–445. 2. Shah AA, Churg A, Sbarbaro JA, Sheppard JM, Lamberti J. Malignant fibrous histiocytoma of the heart presenting as an atrial myxoma. Cancer. 1978;42:2466–71. 2. Shah AA, Churg A, Sbarbaro JA, Sheppard JM, Lamberti J. Malignant fibrous histiocytoma of the heart presenting as an atrial myxoma. Cancer. 1978;42:2466–71. 3. Okamoto K, Kato S, Katsuki S, Wada Y, Toyozumi Y, Morimatsu M, et al. Malignant fibrous histiocytoma of the heart: case report and review of 46 cases in the literature. Intern Med. 2001;40:1222–6. 3. Okamoto K, Kato S, Katsuki S, Wada Y, Toyozumi Y, Morimatsu M, et al. Malignant fibrous histiocytoma of the heart: case report and review of 46 cases in the literature. Intern Med. 2001;40:1222–6. 4. Bandyopadhyay S, Banerjee S, Paul A, Das RK. Primary malignant fibrous histiocytoma involving the left pulmonary vein presenting as a left atrial tumor. Ann Card Anaesth. 2013;16(4):293–5. 5. Milicic D, Juretic A, Bulum J, Saric N, Bisof V, Jelic I, et al. Primary malignant fibrous histiocytoma of the heart with skeletal muscles metastases. J Card Surg. 2007;22(6):513–6. 6. Dorobantu M, Fruntelata A, Constantinescu D, Racoveanu I, Ardeleanu C, Tatu-Chitoiu G, et al. Primary left heart malignant fibrous histiocytoma. Eur J Echocardiogr. 2005;6(3):225–7. g 7. Auer J, Berent R, Gurtner F. “Malignant” mitral stenosis. J Cardiothorac Surg 2012;8:7–19. 8. Soo WM, Pang YH, Poh KK. Lethal mass mimicking myxoma in the heart. Asian Cardiovasc Thorac Ann. 2014;22(8):962–4. 7. Auer J, Berent R, Gurtner F. “Malignant” mitral stenosis. J Cardiothorac Surg. 2012;8:7–19. 2012;8:7 19. 8. Soo WM, Pang YH, Poh KK. Lethal mass mimicking myxoma in the heart. Asian Cardiovasc Thorac Ann. 2014;22(8):962–4. 9. Fontana A, Sciuchetti JF, Boffi L, Colagrande L, Trocino G. Unusual localization of a malignant fibrous histiocytoma on the mitral valve. Eur J Echocardiogr. 2010;11(1):77–9. 9. Fontana A, Sciuchetti JF, Boffi L, Colagrande L, Trocino G. Unusual localization of a malignant fibrous histiocytoma on the mitral valve. Eur J Echocardiogr. 2010;11(1):77–9. 10. Wang CH, Yu HY, Chi NS, Chen YS, Lee KK, Cheng YJ, et al. Complete excision of primary cardiac malignant fibrous histiocytoma involving the left atrial free wall and mitral annulus by modified autotransplantation. J Thorac Cardiovasc Surg. 2006;131(3):731–3. 10. Wang CH, Yu HY, Chi NS, Chen YS, Lee KK, Cheng YJ, et al. References Complete excision of primary cardiac malignant fibrous histiocytoma involving the left atrial free wall and mitral annulus by modified autotransplantation. J Thorac Cardiovasc Surg. 2006;131(3):731–3. 11. Sasaki K, Tanaka S, Ikeshita M, Shoji T, Fujita Y, Katagiri K. Malignant fibrous histiocytoma of the heart–a case report and review of the literature. Nihon Kyobu Geka Gakkai Zasshi. 1991;39(12):2240–5. y 12. Baba T, Tanemoto K, Kuinose M, Kanaoka Y, Murakami T. A case of primary cardiac malignant fibrous histiocytoma. Kyobu Geka. 1998;51(10):849–52. 13. Fukumitsu T, Tsunekawa A, Watanabe M, Iwase M, Takeuchi E, Abe T. Primary malignant fibrous histiocytoma of the left atrium with acute mitral regurgitation. Am Heart J. 1988;115(3):691–3. g g 14. Schena S, Caniglia A, Agnino A, Caruso G, Ferlan G. Survival following treatment of a cardiac malignant fibrous histiocytoma. Chest. 2000;118:271–3. 14. Schena S, Caniglia A, Agnino A, Caruso G, Ferlan G. Survival following treatment of a cardiac malignant fibrous histiocytoma. Chest. 2000;118:271–3. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review
https://openalex.org/W2157730094	https://europepmc.org/articles/pmc3365021?pdf=render	English	null	Prediction of Molecular Targets of Cancer Preventing Flavonoid Compounds Using Computational Methods	PloS one	2,012	cc-by	6,647	Introduction polyphenol found in red wine, is known to possess antioxidant activities as well as anticancer activities explained by its inhibition of the cyclooxygenase proteins [9]. Recently, we reported that resveratrol can suppress leukotriene A4 hydrolase (LTA4H) activity [10], which is over-expressed in lung and colon cancer cells [11]. The consumption of fruits and vegetables has long been believed to decrease the risk of developing various types of human cancers [1]. A major class of compounds within foods that possess these activities are the polyphenols [2]. The phenylpropanoid metabolic pathways of plants generate these polyphenolic com- pounds from thousands of secondary plant metabolites [3]. Flavonoids are the most common family of polyphenolic compounds with up to 8,000 individual compounds identified [4]. Flavonoids found in vegetables, cereals, legumes, fruits, and beverages such as wine, teas, and coffees can be subdivided into 14 different categories based on chemical structure. These categories include the chalcones, dihydrochalcones, aurones, flavones, flavonols, dihydroflavonols, flavanones, flavanols, flavandiols, anthocyanidins, isoflavonoids, biflavonoids, and proanthocyani- dins [5]. Flavonoids are promiscuous in that they can suppress the growth of many different types of cancer cells through a variety of mechanisms. This nonspecificity is compounded by the fact that thousands of flavonoids exist and therefore their study has provided a very rich area of research. Only a few cases of computational work focusing on flavonoids exist [12,13,14]. Using conventional methods to select the best single or group of best chemicals for identifying compounds that are effective in on treating or preventing a disease like cancer is difficult. Compu- tational strategies for determining protein targets of flavonoids have not yet received a great deal of attention. Over the last 3 years, our laboratory has utilized computational strategies that include virtual screening, shape similarity-screening, and molec- ular docking to identify potential protein targets of flavonoids and other phytochemicals [15]. These computational-based method- ologies have provided efficient and inexpensive tools to gain further understanding of the anticancer and therapeutic effects exerted by polyphenols. Herein we present our process for combining those computational strategies with experimental methodologies for validating specific flavonoids and their respec- tive protein targets. The anticarcinogenic potential of a variety of well-characterized flavonoids has been well documented [1]. Isoflavonoids, such as anthraquinones, chalcones, and prenylflavonoids, are all capable of promoting estrogenic activity in mammals [6]. They have also been shown to possess anticancer properties [7]. Abstract Flavonoids are promiscuous in that they can suppress the growth of many different types of cancer cells through a variety of mechanisms. This nonspecificity is compounded by the fact that thousands of flavonoids exist and therefore their study has provided a very rich area of research. Only a few cases of computational work focusing on flavonoids exist [12,13,14]. Using conventional methods to select the best single or group of best chemicals for identifying compounds that are effective in on treating or preventing a disease like cancer is difficult. Compu- tational strategies for determining protein targets of flavonoids have not yet received a great deal of attention. Over the last 3 years, our laboratory has utilized computational strategies that include virtual screening, shape similarity-screening, and molec- ular docking to identify potential protein targets of flavonoids and other phytochemicals [15]. These computational-based method- ologies have provided efficient and inexpensive tools to gain further understanding of the anticancer and therapeutic effects exerted by polyphenols. Herein we present our process for combining those computational strategies with experimental methodologies for validating specific flavonoids and their respec- tive protein targets. Hanyong Chen1., Ke Yao1., Janos Nadas1., Ann M. Bode1, Margarita Malakhova1, Naomi Oi1, Haitao Li1, Ronald A. Lubet2, Zigang Dong1* Hanyong Chen1., Ke Yao1., Janos Nadas1., Ann M. Bode1, Margarita Malakhova1, Naomi Oi1, Haitao Li1, Ronald A. Lubet2, Zigang Dong1* 1 The Hormel Institute, University of Minnesota, Austin, Minnesota, United States of America, 2 The National Cancer Institute, Bethesda, Maryland, United States of America tute, University of Minnesota, Austin, Minnesota, United States of America, 2 The National Cancer Institute, Bethesda, Maryland, Un ty of Minnesota, Austin, Minnesota, United States of America, 2 The National Cancer Institute, Bethesda, Maryland, United States o The Hormel Institute, University of Minnesota, Austin, Minnesota, United States of America, 2 The National Cancer Institute, Bethe merica PLoS ONE \| www.plosone.org Abstract Plant-based polyphenols (i.e., phytochemicals) have been used as treatments for human ailments for centuries. The mechanisms of action of these plant-derived compounds are now a major area of investigation. Thousands of phytochemicals have been isolated, and a large number of them have shown protective activities or effects in different disease models. Using conventional approaches to select the best single or group of best chemicals for studying the effectiveness in treating or preventing disease is extremely challenging. We have developed and used computational-based methodologies that provide efficient and inexpensive tools to gain further understanding of the anticancer and therapeutic effects exerted by phytochemicals. Computational methods involving virtual screening, shape and pharmacophore analysis and molecular docking have been used to select chemicals that target a particular protein or enzyme and to determine potential protein targets for well-characterized as well as for novel phytochemicals. Citation: Chen H, Yao K, Nadas J, Bode AM, Malakhova M, et al. (2012) Prediction of Molecular Targets of Cancer Preventing Flavonoid Compounds Using Computational Methods. PLoS ONE 7(5): e38261. doi:10.1371/journal.pone.0038261 Editor: Niall James Haslam, University College Dublin, Ireland Editor: Niall James Haslam, University College Dublin, Ireland Received January 13, 2012; Accepted May 4, 2012; Published May 31, 2012 Received January 13, 2012; Accepted May 4, 2012; Published May 31, 2012 Received January 13, 2012; Accepted May 4, 2012; Published May 31, 2012 Copyright: 2012 Chen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Hormel Foundation and National Institutes of Health grants R37 CA081064, CA120388, ES016548, CA0227501 and National Cancer Institute Contract No. HHSN-261200533001C-NO1-CN-53301. The funders had no role in study design,data collection and analysis, decision to publish, or preparation of the manuscript. rests: The authors have declared that no competing interests exis Competing Interests: The authors have declared that no competing interests exist. * E-mail: zgdong@hi umn edu * E-mail: zgdong@hi.umn.edu . These authors contributed equally to this work. polyphenol found in red wine, is known to possess antioxidant activities as well as anticancer activities explained by its inhibition of the cyclooxygenase proteins [9]. Recently, we reported that resveratrol can suppress leukotriene A4 hydrolase (LTA4H) activity [10], which is over-expressed in lung and colon cancer cells [11]. Virtual Screening Virtual screening is a computational technique used in drug discovery research in recent years and it has become an important step in the drug discovery process. The screening involves the identification and compilation of relevant chemical structures from large chemical libraries. The chemicals identified are those most likely to bind to a protein target, typically a protein receptor selected by using various computer programs or identified experimentally. Virtual screening by molecular docking is the major computational method employed in drug discovery for ‘‘hit’’ identification [16]. The primary methodol- ogy used is structure-based virtual screening, which involves docking of thousands of candidate ligands into a protein target followed by scoring the protein-ligand binding interaction to estimate the binding energy of the ligand [17]. Structure-based virtual screening requires the 3D structure of the ligands. The ZINC (i.e., an acronym for ‘‘ZINC is not commercial’’) Database contains over 13 million purchasable compounds in 3D docking format that are freely available for virtual screening [18]. From this huge database, smaller and more specific high- quality libraries can be built for targeted virtual screening [19]. Another available database containing 2D forms of molecules is the National Institute of Health’s PubChem online database comprising over 27 million unique structures (http://pubchem. ncbi.nlm.nih.gov/). In our laboratory, we have created a smaller flavonoid compound database of 2,620 compounds, including aurones, chalcones, flavones, flavanones, isoflavones, biflavo- noids, anthocyanidins, dihydrochalcones and proanthocyanidins. These flavonoid compounds were collected and compiled from the NCI PubChem database using structure based searching. This database was used in the screening for potential inhibitors targeting a number of cancer-related proteins, including p90 ribosomal S6 kinase 2 (RSK2), cyclin dependent kinase (Cdk), mitogen-activated protein kinase kinase 1 (MEK1), epidermal growth factor receptor (EGFR) and phosphatidylinositol 3- kinase/protein kinase B (PI3-K/PKB). Our laboratory has solved and reported the crystal structures of the C-terminal and N-terminal kinase domains of RSK2 [29,30]. The N-terminal kinase domain was bound with ANP at the ATP binding site. Thus, this structure (PDB ID:3G51) was downloaded from the PDB Bank for virtual screening studies. Crystal structures or homology models of the target protein to which a small molecule will be docked are downloaded from the Protein Databank (PDB). Waters, metals, and ligands are then stripped from the structure and hydrogens and atom charges are added to the structures using the protein preparation module in Schro¨din- ger’s Maestro v9.2 GUI. Materials and Methods important for chemoprevention or therapeutic drug development. Therefore, herein we used RSK2 as an example to demonstrate how computational strategies and experimental methodologies can be combined effectively to identify selective flavonoid inhibitors. To date, a number of potential RSK2 inhibitors have been reported, including eriodictyol [24] and kaempferol [23,25,26], two flavonoid compounds and SL0101, a synthesized compound not found in plants [27]. These flavonoids are ubiquitously found in fruits and vegetables as well as popular beverages, including wine, tea, and coffee and exhibit antioxidant, antitumor, and anti- inflammatory effects [28]. Computational Strategies Finding Protein Targets Computational Strategies Finding Protein Targets Virtual Screening An ATP binding site-based pocket was generated within a 30-A˚ 3 grid. The 2D structure database of flavonoids was converted to a 3D structure database using the LigPrep module of the Schro¨dinger Suite of software. High throughput virtual screening (HTVS) and docking are usually performed first because they are intended for the rapid screening of large numbers of ligands followed by standard and extra precision (SP and XP) docking. Here, for our flavonoid database, only SP and XP docking were performed because of the smaller number of ligands involved. All compounds were docked flexibly and a top-20 list of compounds was generated and organized based on the docking score (i.e., lower score is best). A list of the top-6 ranked compounds was compiled (Table 1) and kaempferol (Fig. 1A) and quercetin (Fig. 1B) were purchased for experimental validation. Kaempferol and quercetin are natural flavonols found in apples, onions and other plants. Introduction Genistein, for example, a major member of the isoflavonoid family derived from soybeans [7], specifically inhibits the epidermal growth factor receptor (EGFR) tyrosine kinase activity, which plays an important role in cell proliferation and transformation [8]. New findings continue to be reported related to these compounds. Resveratrol, a May 2012 \| Volume 7 \| Issue 5 \| e38261 1 Shape-Similarity Screening Method The Ras/extracellular signal-regulated kinase (ERK) pathway regulates cell proliferation, survival, growth and motility and tumorigenesis [20]. RSK2, a member of the p90RSK family, is a direct substrate kinase of ERKs and is an important direct effector for transcriptional activation of downstream target transcription factors. Furthermore, RSK2 is reportedly involved in prostate cancer cell proliferation [21] and c-fos–dependent osteosarcoma development [22]. RSK2 protein abundance is increased in many human cancer cell lines and in various human skin tumors, including melanomas and squamous cell carcinomas [23]. Therefore, identifying a selective RSK2 inhibitor is extremely Shape Similarity Screening Method The theory of shape-similarity screening is derived from the idea that molecules possessing similar shapes and electrostatic capabil- ities might exhibit analogous biological activity. The method involves consideration of the atomistic and spatial characteristics of the target molecule. The pharmacophore and physical features of the molecule are quantitatively compared with a library of compounds. When searching for potential target proteins, the compound library used is composed of crystallized ligands extracted from the most recent version of the PDB [31]. The ligand conformation in the crystal structure is used because Table 1. Results of virtual screening for RSK2 inhibitors. Table 1. Results of virtual screening for RSK2 inhibitors. Table 1. Results of virtual screening for RSK2 inhibitors. Compound Name Docking Score (kcal/mol) Activity Validation Pedalitin 210.20 * Quercetin 3-sulfate 29.93 * Quercetin 29.40 Yes 5-Hydroxy-49-methoxy-7-methylflavone 29.28 * Kaempferol 28.86 Yes 3,39-di-O-ethylquercetin 28.50 * , Not commercially available. doi:10.1371/journal.pone.0038261.t001 PLoS ONE \| www.plosone.org 2 May 2012 \| Volume 7 \| Issue 5 \| e38261 Activity Validation Figure 1. Chemical structures. (A) Chemical structure of kaempferol; (B) Chemical structure of quercetin. (C) Chemical structure of myricetin. (D) Chemical structure of LY294002. (E) Chemical structure of isorhamnetin. doi:10.1371/journal.pone.0038261.g001 Computational Strategies Finding Protein Targets Computational Strategies Finding Protein Targets Figure 1. Chemical structures. (A) Chemical structure of kaempferol; (B) Chemical structure of quercetin. (C) Chemical structure o Chemical structure of LY294002. (E) Chemical structure of isorhamnetin. doi:10.1371/journal.pone.0038261.g001 Figure 1. Chemical structures. (A) Chemical structure of kaempferol; (B) Chemical structure of quercetin. (C) Chemical structure of myricetin. (D) Chemical structure of LY294002. (E) Chemical structure of isorhamnetin. doi:10.1371/journal.pone.0038261.g001 the atoms are oriented in a manner optimized for binding to the protein. Any available database can be used when searching for similar compounds. protein structures complexed with ligands, were used for shape- similarity screening. PLoS ONE \| www.plosone.org PLoS ONE \| www.plosone.org Computational Strategies Finding Protein Targets Computational Strategies Finding Protein Targets Figure 2. Quercetin binds with RSK2 and inhibits RSK2 activity in vitro. (A) Quercetin binds at the ATP pocket of RSK2 most likely in an ATP- competitive manner. (B) RSK2 binds with quercetin. A lysate prepared from JB6 C41 cells or commercially available active RSK2 was incubated with Sepharose 4B-quercetin beads or with Sepharose 4B beads alone, and the pulled down proteins were analyzed by Western blot. (C) Quercetin binds with either the NTD or the CTD of RSK2. To identify the RSK2 domain that binds with quercetin, RSK2 proteins, as indicated, were incubated with Sepharose 4B-quercetin beads or with Sepharose 4B beads alone. The pulled down proteins were analyzed by Western blot. (D) Active RSK2 (10 ng) was combined with GST-NFAT3-261-365 (2 mg), 10 mM unlabeled ATP, 10 mCi [c-32P]ATP, and different doses of quercetin (0–50 mM). An in vitro kinase assay was performed and the32P-labeled phosphorylated NFAT3 was visualized by autoradiography. Band density was quantified using the Image J software program (NIH) and the band intensity of active RSK2 and GST-NFAT3-261-365 (100%) was compared. doi:10.1371/journal.pone.0038261.g002 We also attempted to identify novel inhibitors targeting PI3-K, which is a well known kinase involved in cellular functions such as growth, proliferation, differentiation, motility, survival and intra- cellular trafficking, all of which are associated with cancer development. Wortmannin and LY294002 are broad inhibitors that target all PI3-K family members. LY294002 (Fig. 1D) is a morpholine derivative of quercetin and although its IC50 is about 500-fold higher than that of wortmannin, it is still widely used in cell biology as a specific PI3-K inhibitor because of its stability in solution [37]. We therefore used the structure of LY294002 to carry out the screening for potential PI3-K inhibitors in our flavonoid database using the PHASE module of the Schro¨dinger Suite 2011. To perform the screening, myricetin and LY294002 were first prepared separately using the LigPrep module of the Schro¨dinger Suite 2011 under the OPLS_2005 force field and a specifying pH value of 7.0. Then shape-similarity screening was carried out using PHASE. Different protocols for docking are attempted before determin- ing the correct set of parameters to be used for docking. The correct re-docking of the ligand that was crystallized with the target protein is typically used as validation of the chosen parameters. Computational Strategies Finding Protein Targets When more than one crystal structure of a target protein is available, cross-docking is performed to determine which crystal structure is most suitable for docking [39]. In our laboratory, we had found that isorhamnetin (Fig. 1E), an O-methylated flavonol in herbal medicinal plants such as red turnip, goldenrod, mustard leaf and gross Hippophaer hamnoides L., could inhibit the kinase activity of MEK1 or PI3-K and the inhibition was due to isorhamnetin’s direct binding with these kinases [40]. Molecular docking was used to further study the binding of isorhamnetin with MEK1. First an X-ray structure of the human MEK1 in a complex with ligand and MgATP (PDB 1S9J) with a 2.4 A˚ resolution was downloaded from the PDB. The protein was prepared for docking using the Protein Preparation Wizard. All crystallographic waters were deleted and a 30-A˚ 3 grid was generated based on the ATP noncompetitive ligand (BBM) binding site of the protein receptor. MacroModel was used to build and energetically minimize isorhamnetin to create the most energetically favorable conformation needed for docking studies. , Protein targets that been validated in our laboratory. Shape-Similarity Screening Method We also created a specific kinase database of about 4,000 structures complexed with ligands that we collected from the PDB for screening kinase targets separately. The PHASE [32] module of Schro¨dinger’s molecular modeling software package is one program that can perform this type of shape-similarity search [33]. The atom type information is included for consideration of not only shape similarity but also to align potential pharmacophore points between the queries and targets. The cutoff to be used for the results takes the top aligned structure for each molecule returned and all conformers possessing a Tanimoto similarity coefficient below 0.7 are deleted [34,35]. The smaller flavonoid compound database that we created and the latest version of the PDB, which contains more than 500,000 Our previous studies showed that myricetin (Fig. 1C), a flavonoid found in many grapes, berries, fruits, vegetables, herbs, as well as other plants, and one of the phenolic compounds present in red wine, exhibits potent anticancer and chemopreventive effects, especially on UVB-induced skin cancer [36], but its molecular mechanisms and targets are unclear. Thus, to identify off-target protein affected by myricetin, shape-similarity screening was performed using the PDB ligand database and our special kinase database. PLoS ONE \| www.plosone.org May 2012 \| Volume 7 \| Issue 5 \| e38261 May 2012 \| Volume 7 \| Issue 5 \| e38261 3 Computational Strategies Finding Protein Targets l 4 M 2012 \| V l 7 \| I PLoS ONE \| www.plosone.org PLoS ONE \| www.plosone.org May 2012 \| Volume 7 \| Issue 5 \| e38261 4 , Only the top 3 hits are shown here. y p doi:10.1371/journal.pone.0038261.t002 , Only the top 3 hits are shown here. doi:10.1371/journal.pone.0038261.t002 Discussion Thousands of individual flavonoid compounds exist in various vegetables, fruits, and other plants. Flavonoids, such as catechins found in strawberries and green and black teas, kaempferol from brussel sprouts and apples, and quercetin from beans, onions and apples, are believed to exert anti-inflammatory and anticancer activities. For example, all of these compounds reportedly might reduce the risk of developing lung cancer [45]. Flavonoids inhibit many different types of cancers through a variety of mechanisms and thousands of flavonoids exist, which makes the screening of their potential anticancer effects and identification of their specific protein targets extremely challenging using conventional ap- proaches. Fortunately, the development of computational simula- tion techniques and other computational strategies has simplified and streamlined the overall process. Virtual screening can easily generate results from all flavonoid compounds that can bind with and affect the activity of a specific protein target. Shape-screening can assist in finding potential ‘‘off-target’’ proteins of a specific flavonoid compound. Molecular docking methods can provide a better indication of how a compound interacts with its protein target and influence the activity of the targeted protein. These three processes can lead to the rapid discovery of potential lead compounds for anticancer treatment and chemoprevention. Shape-Similarity Screening. In our study, the shape- similarity screening was performed using Schro¨dinger’s PHASE module to examine the database comprised of protein complexes with ligands and the specific kinase database. Myricetin, a flavonoid found in grapes, berries, fruits, vegetables, herbs, as well as other plants, and a phenolic compounds present in red wine, was determined to target many potential kinases (Table 2). The data indicate six different kinase/ligand complexes with an average score of shape-similarity with myricetin greater than 0.7. The ligand with the greatest similarity to myricetin is QUE or quercetin (Fig. 1B), which is bound with Pim-1 (PDB ID: 2O3P). The similarity score is 0.90 and quercetin is a reported inhibitor of Pim-1 [41]. A total of 8 Pim-1 structures were ‘‘hit’’ by myricetin with a similarity score of greater than 0.7 (only the top 3 hits are shown in Table 2). Almost all the ‘‘hit’’ ligands are reported In recent years, with the help of the Blue Gene/L [46,47] supercomputer from IBM, our laboratory has studied the effectiveness and mechanisms of several lead flavonoid com- pounds in cancer chemoprevention and treatment by using computational simulation strategies. Computational Strategies Finding Protein Targets Table 3. Sixflavonoid candidate inhibitors for PI3-K. inhibitors of their targeted kinase and have a similarity score of greater than 0.7. Thus, we believe that these related kinases might be possible targets of myricetin and that myricetin might potentially inhibit their activity. Our previous studies indicated that myricetin could inhibit the activity of PI3-K, MEK1 and Raf [36,42,43]. Table 3. Sixflavonoid candidate inhibitors for PI3-K. Compound Name Shape Similarity Score Validation Mitoflaxone 0.84 * Dimeflin 0.83 * Isowogonin 0.76 * Kumatakenin 0.76 * Myricetin 0.75 Yes Isorhamnetin 0.75 Yes , Not commercially available. doi:10.1371/journal.pone.0038261.t003 To find potential flavonoid inhibitors of PI3-K, LY294002, a broad PI3-K inhibitor, was chosen to use as the query structure and shape-similarity screening was performed using our flavonoid database and the PHASE module. Similarity coefficients below 0.75 were deleted and 6 of the top ranked candidate compounds are listed (Table 3). We have previously validated myricetin [42] and isorhamnetin [40] as direct inhibitors of PI3-K. Virtual Screening g Based on our virtual screening results for RSK2, quercetin and kaempferol have binding scores of 29.40 kcal/mol and 28.86 kcal/mol, respectively, with RSK2 (Table 1). These scores are a very good indication of binding compared with 27.73 kcal/ mol for SL0101, a well-known RSK2 inhibitor. Our docking model shows quercetin binding in an ATP-competitive manner within the ATP banding site of RSK2 (Fig. 2A), indicating that quercetin might also be a potential inhibitor of RSK2. To examine our hypothesis, we conjugated quercetin with Sepharose 4B beads and conducted an in vitro pull-down assay using a whole cell lysate or an active RSK2 protein (200 ng; Fig. 2B). The results showed that the RSK2 protein bound with Sepharose 4B-quercetin beads but not with Sepharose 4B beads alone (Fig. 2B). Furthermore, we conducted a pull-down assay with Sepharose 4B-quercetin beads and several bacterially-expressed His-tagged RSK2 protein fragments, including His-RSK2-1-740, His-RSK2-1-373, His- RSK2-328-740, and His-RSK2-399-740. Western blot results indicated that both the NTD and CTD of RSK2 bound with Sepharose 4B-quercetin beads (Fig. 2C). To confirm the results of the virtual screening that identified quercetin as a potential RSK2 inhibitor, we conducted an in vitro kinase assay. Results indicated that quercetin inhibited RSK2 activity in a dose-dependent manner (Fig. 2D). We previously used an in vitro kinase assay, an anchorage-independent cell transformation assay and a pull-down assay with Sepharose 4B-kaempferol beads to show that kaempferol binds with the NTD of RSK2 and inhibits RSK2 activity in vitro and ex vivo [23,26,31]. Molecular Docking docking. All these docking procedures allowed ligand docking flexibility and a total of 20 top ranked structures were analyzed using the IFD method. docking. All these docking procedures allowed ligand docking flexibility and a total of 20 top ranked structures were analyzed using the IFD method. To examine the molecular mechanism of the inhibition of MEK1 by isorhamnetin and to understand how isorhamnetin interacts with MEK1, we docked isorhamnetin in silico to the ATP- noncompetitive binding pocket of MEK1 using several protocols in the Schro¨dinger Suite of software. By studying all the models returned, we found that isorhamnetin formed some favorable connections and docked nicely within the MEK1 ATP-noncom- petitive binding site. Some important hydrogen bonds were formed between isorhamnetin and the backbone of MEK1, including Val127 in the ATP-noncompetitive binding site and Ser212 in the activation loop. Isorhamnetin also formed hydro- phobic interactions with the side chain at Ile99, Phe129, Ile141, Phe209 and Leu118 (Fig. 3A,B). These results would render MEK1 catalytically inactive by stabilizing the inactive conforma- tion of the activation loop. Note that some images were generated with the UCSF Chimera program [44]. Molecular Docking Method Molecular docking has become a standard tool in computa- tional biology for predicting the binding orientation of small molecule drug candidates with their protein targets in order to predict the affinity and activity of the small molecule. Thus, molecular docking plays an important role in the rational design of drugs. GLIDE from the Schro¨dinger Suite 2011 [38] is one of the programs used in our laboratory to perform docking. Several standard procedures included in Schro¨dinger’s GLIDE docking protocols were performed. Procedures included docking with standard precision (SP) or extra precision (XP) in GLIDE, and the more CPU-intensive Induced-Fit Docking (IFD) method with the default parameters were conducted with SP and XP Table 2. Potential kinase targets of myricetin. Table 2. Potential kinase targets of myricetin. Table 2. Potential kinase targets of myricetin. Protein PDB ID Ligand Code Shape Similarity Score Reported Inhibitor Average Similarity Score Hits Pim-1 2O3P QUE 0.90 yes 0.82 8* 2O63 MYC 0.88 yes 2O64 MYU 0.86 yes GSK-3b 3DU8 553 0.83 yes 0.79 5** 1Q41 IXM 0.80 yes 3ZRL ZRL 0.78 yes PI3-K* 3PRZ 3RZ 0.81 yes 0.77 6 3DPD 41A 0.79 yes 3PS6 3PS 0.78 yes Cdk2 1E1V CMG 0.83 yes 0.76 16 1H0W 207 0.82 yes 1E1X NW1 0.80 yes Raf* 3PPJ FOI 0.73 yes 0.72 2 3C4C 324 0.71 yes MEK1* 3EQH ADP 0.71 yes 0.71 1 , Protein targets that been validated in our laboratory. *, Only the top 3 hits are shown here. doi:10.1371/journal.pone.0038261.t002 PLoS ONE \| www.plosone.org 5 May 2012 \| Volume 7 \| Issue 5 \| e38261 May 2012 \| Volume 7 \| Issue 5 \| e38261 Computational Strategies Finding Protein Targets References 24. Liu K, Cho YY, Yao K, Nadas J, Kim DJ, et al. (2011) Eriodictyol inhibits RSK2-ATF1 signaling and suppresses EGF-induced neoplastic cell transforma- tion. J Biol Chem 286: 2057–2066. 1. Birt DF, Hendrich S, Wang WQ (2001) Dietary agents in cancer prevention: flavonoids and isoflavonoids. Pharmacology & Therapeutics 90: 157–177. 2. Harborne JB, Williams CA (2000) Advances in flavonoid research since 1992. Phytochemistry 55: 481–504. 25. Lee KM, Lee KW, Jung SK, Lee EJ, Heo YS, et al. (2010) Kaempferol inhibits UVB-induced COX-2 expression by suppressing Src kinase activity. Biochem Pharmacol 80: 2042–2049. 3. Ververidis F, Trantas E, Douglas C, Vollmer G, Kretzschmar G, et al. (2007) Biotechnology of flavonoids and other phenylpropanoid-derived natural products. Part I: Chemical diversity, impacts on plant biology and human health. Biotechnology Journal 2: 1214–1234. 26. Cho YY, Yao K, Kim HG, Kang BS, Zheng D, et al. (2007) Ribosomal S6 kinase 2 is a key regulator in tumor promoter induced cell transformation. Cancer Res 67: 8104–8112. 4. Pietta PG (2000) Flavonoids as antioxidants. Journal of Natural Products 63: 1035–1042. 27. Smith JA, Poteet-Smith CE, Xu Y, Errington TM, Hecht SM, et al. (2005) Identification of the first specific inhibitor of p90 ribosomal S6 kinase (RSK) reveals an unexpected role for RSK in cancer cell proliferation. Cancer Res 65: 1027–1034. 5. Bravo L (1998) Polyphenols: Chemistry, dietary sources, metabolism, and nutritional significance. Nutrition Reviews 56: 317–333. 6. Ososki AL, Kennelly EJ (2003) Phytoestrogens: A review of the present state of research. Phytotherapy Research 17: 845–869. 28. Hollman PC, Katan MB (1999) Dietary flavonoids: intake, health effects and bioavailability. Food Chem Toxicol 37: 937–942. research. Phytotherapy Research 17: 845–869. 7. Reynaud J, Guilet D, Terreux R, Lussignol M, Walchshofer N (2005) Isoflavonoids in non-leguminous families: an update. Natural Product Reports 22: 504–515. 29. Malakhova M, Kurinov I, Liu K, Zheng D, D’Angelo I, et al. (2009) Structural diversity of the active N-terminal kinase domain of p90 ribosomal S6 kinase 2. PLoS ONE 4: e8044. 8. Akiyama T, Ishida J, Nakagawa S, Ogawara H, Watanabe S, et al. (1987) Genistein, a specific inhibitor of tyrosine-specific protein-kinases. Journal of Biological Chemistry 262: 5592–5595. 30. Malakhova M, Tereshko V, Lee SY, Yao K, Cho YY, et al. (2008) Structural basis for activation of the autoinhibitory C-terminal kinase domain of p90 RSK2. Nat Struct Mol Biol 15: 112–113. 9. References Jang MS, Cai EN, Udeani GO, Slowing KV, Thomas CF, et al. (1997) Cancer chemopreventive activity of resveratrol, a natural product derived from grapes. Science 275: 218–220. 31. Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, et al. (2000) The Protein Data Bank. Nucleic Acids Research 28: 235–242. 32. (2011) Phase v3.3 Schro¨dinger Suite: User Manual. Schro¨dinger, LLC. 32. (2011) Phase v3.3 Schro¨dinger Suite: User Ma 10. Oi N, Jeong CH, Nadas J, Cho YY, Pugliese A, et al. (2010) Resveratrol, a red wine polyphenol, suppresses pancreatic cancer by inhibiting leukotriene Ahydrolase. Cancer Res 70: 9755–9764. 33. (2011) Schro¨dinger Suite Schro¨dinger, LLC. 34. Willett P, Barnard JM, Downs GM (1998) Chemical similarity searching. Journal of Chemical Information and Computer Sciences 38: 983–996. 11. Chen X, Wang S, Wu N, Yang CS (2004) Leukotriene A4 hydrolase as a target for cancer prevention and therapy. Current Cancer Drug Targets 4: 267–283. 35. Downs GM, Willett P, Fisanick W (1994) Similarity Searching and Clustering of Chemical-Structure Databases Using Molecular Property Data. Journal of Chemical Information and Computer Sciences 34: 1094–1102. 12. Kinoshita T, Lepp Z, Kawai Y, Terao J, Chuman H (2006) An integrated database of flavonoids. Biofactors 26: 179–188. 13. Rolo-Naranjo A, Codorniu-Hernandez E, Ferro N (2010) Quantum Chemical Associations Ligand-Residue: Their Role to Predict Flavonoid Binding Sites in Proteins. Journal of Chemical Information and Modeling 50: 924–933. 36. Jung SK, Lee KW, Byun S, Kang NJ, Lim SH, et al. (2008) Myricetin suppresses UVB-induced skin cancer by targeting Fyn. Cancer Res 68: 6021–6029. 37. Walker EH, Pacold ME, Perisic O, Stephens L, Hawkins PT, et al. (2000) Structural determinants of phosphoinositide 3-kinase inhibition by wortmannin, LY294002, quercetin, myricetin, and staurosporine. Mol Cell 6: 909–919. 14. Suetsugi M, Su L, Karlsberg K, Yuan YC, Chen S (2003) Flavone and isoflavone phytoestrogens are agonists of estrogen-related receptors. Molecular Cancer Research 1: 981–991. . (2011) Glide,V5.6, Schro¨dinger Suite: User manual. Schro¨dinger, L 15. Lee KW, Bode AM, Dong Z (2011) Molecular targets of phytochemicals for cancer prevention. Nat Rev Cancer 11: 211–218. 39. Cavasotto CN, Abagyan RA (2004) Protein flexibility in ligand docking and virtual screening to protein kinases. Journal of Molecular Biology 337: 209–225. 16. Rester U (2008) From virtuality to reality - Virtual screening in lead discovery and lead optimization: A medicinal chemistry perspective. Current Opinion in Drug Discovery & Development 11: 559–568. 40. References Kim JE, Lee DE, Lee KW, Son JE, Seo SK, et al. (2011) Isorhamnetin suppresses skin cancer through direct inhibition of MEK1 and PI3-K. Cancer Prev Res (Phila) 4: 582–591. g y p 17. Lyne PD (2002) Structure-based virtual screening: an overview. Drug Discovery Today 7: 1047–1055. 41. Holder S, Zemskova M, Zhang C, Tabrizizad M, Bremer R, et al. (2007) Characterization of a potent and selective small-molecule inhibitor of the PIM1 kinase. Mol Cancer Ther 6: 163–172. 18. Irwin JJ, Shoichet BK (2005) ZINC - A free database of commercially available compounds for virtual screening. Journal of Chemical Information and Modeling 45: 177–182. 42. Jung SK, Lee KW, Byun S, Lee EJ, Kim JE, et al. (2010) Myricetin inhibits UVB-induced angiogenesis by regulating PI-3 kinase in vivo. Carcinogenesis 31: 911–917. 19. Hartshorn MJ, Verdonk ML, Chessari G, Brewerton SC, Mooij WTM, et al. (2007) Diverse, high-quality test set for the validation of protein-ligand docking performance. Journal of Medicinal Chemistry 50: 726–741. 43. Jung SK, Lee KW, Kim HY, Oh MH, Byun S, et al. (2010) Myricetin suppresses UVB-induced wrinkle formation and MMP-9 expression by inhibiting Raf. Biochem Pharmacol 79: 1455–1461. 20. Cho YY, Bode AM, Mizuno H, Choi BY, Choi HS, et al. (2004) A novel role for mixed-lineage kinase-like mitogen-activated protein triple kinase alpha in neoplastic cell transformation and tumor development. Cancer Res 64: 3855–3864. 44. Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, et al. (2004) UCSF Chimera–a visualization system for exploratory research and analysis. J Comput Chem 25: 1605–1612. 21. Clark DE, Errington TM, Smith JA, Frierson HF, Jr., Weber MJ, et al. (2005) The serine/threonine protein kinase, p90 ribosomal S6 kinase, is an important regulator of prostate cancer cell proliferation. Cancer Res 65: 3108–3116. 45. Cui Y, Morgenstern H, Greenland S, Tashkin DP, Mao JT, et al. (2008) Dietary flavonoid intake and lung cancer–a population-based case-control study. Cancer 112: 2241–2248. 46. Fitch BG, Rayshubskiy A, Eleftheriou M, Ward TJC, Giampapa M, et al. (2006) Blue matter: Strong scaling of molecular dynamics on blue Gene/L. Computational Science - Iccs 2006, Pt 2, Proceedings 3992: 846–854. 22. David JP, Mehic D, Bakiri L, Schilling AF, Mandic V, et al. (2005) Essential role of RSK2 in c-Fos-dependent osteosarcoma development. J Clin Invest 115: 664–672. 23. Cho YY, Yao K, Pugliese A, Malakhova ML, Bode AM, et al. (2009) A regulatory mechanism for RSK2 NH(2)-terminal kinase activity. Discussion doi:10 1371/journal pone 0038261 g003 doi:10.1371/journal.pone.0038261.g003 May 2012 \| Volume 7 \| Issue 5 \| e38261 PLoS ONE \| www.plosone.org 7 Computational Strategies Finding Protein Targets Computational Strategies Finding Protein Targets phenolic phytochemicals [53], caffeic acid [54] and delphinidin [55]. All these compounds exert their inhibitory effect on specific proteins, including RSK2, PI-3K, MEK1, Pin-1 and Fyn, which are highly expressed or overactivated in some cancers such as skin and colon cancer. specific kinase targets. These results can be easily queried by compound identification or by protein type. We have also screened for lead compounds targeting several specific kinase targets related to skin cancer, colon cancer and lung cancer using virtual screening and will use these results for further mechanistic studies in cancer chemoprevention and therapy. To identify the potential targets of a variety of flavonoid compounds and to carefully study their mechanism of action, we created a flavonoid database of 2,620 compounds. We are continuing to enlarge the database by adding more and more flavonoid compounds. Based on this database, we have obtained results for every compound and its potential off-target proteins by using shape-similarity screening. We have more than 4.1 million records for all kinds of protein targets and 374,000 records for the Author Contributions Conceived and designed the experiments: HC KY JN AB MM NO HL RL ZD. Performed the experiments: HC KY JN AB MM NO. Analyzed the data: AM RL ZD. Contributed reagents/materials/analysis tools: ZD. Wrote the paper: HC AB. Discussion These compounds include kaempferol [23], isorhamnetin [40], 6,7,49-trihydroxyisoflavone [48], eriodictyol [24], 7,39,49-trihydroxyisoflavone [49], quercetin [50], EGCG [51], [6]-gingerol [52], myricetin [36], coffee PLoS May 2012 \| Volume 7 \| Issue 5 \| e38261 PLoS ONE \| www.plosone.org 6 Computational Strategies Finding Protein Targets Figure 3. Modeling of isorhamnetin binding with MEK1. (A) Isorhamnetin binds to an ATP-noncompetitive pocket of MEK1. The box indicates an enlarged view. Hydrogen bonds are formed between isorhamnetin and the backbone of MEK1 (Val127 in the ATP-noncompetitive binding site and Ser212 in the activation loop). (B) Ligand interaction diagram of the MEK1 and isorhamnetin complex. Residues are represented as colored spheres, labeled with the residue’s name and number. The colors indicate the residue type (green = hydrophobic; blue = polar). The solid pink line shows the hydrogen bond between the ligand and the receptor. Hydrophobic interactions are formed with the side chain at Ile99, Phe129, Ile141, Phe209 and Leu118. doi:10 1371/journal pone 0038261 g003 Figure 3. Modeling of isorhamnetin binding with MEK1. (A) Isorhamnetin binds to an ATP-noncompetitive pocket of MEK1. The box indicates an enlarged view. Hydrogen bonds are formed between isorhamnetin and the backbone of MEK1 (Val127 in the ATP-noncompetitive binding site and Ser212 in the activation loop). (B) Ligand interaction diagram of the MEK1 and isorhamnetin complex. Residues are represented as colored spheres, labeled with the residue’s name and number. The colors indicate the residue type (green = hydrophobic; blue = polar). The solid pink line shows the hydrogen bond between the ligand and the receptor. Hydrophobic interactions are formed with the side chain at Ile99, Phe129, Ile141, Phe209 and Leu118. doi:10.1371/journal.pone.0038261.g003 Figure 3. Modeling of isorhamnetin binding with MEK1. (A) Isorhamnetin binds to an ATP-noncompetitive pocket of MEK1. The box indicates an enlarged view. Hydrogen bonds are formed between isorhamnetin and the backbone of MEK1 (Val127 in the ATP-noncompetitive binding site and Ser212 in the activation loop). (B) Ligand interaction diagram of the MEK1 and isorhamnetin complex. Residues are represented as colored spheres, labeled with the residue’s name and number. The colors indicate the residue type (green = hydrophobic; blue = polar). The solid pink line shows the hydrogen bond between the ligand and the receptor. Hydrophobic interactions are formed with the side chain at Ile99, Phe129, Ile141, Phe209 and Leu118. 48. Lee DE, Lee KW, Jung SK, Lee EJ, Hwang JA, et al. (2011) 6,7,49- trihydroxyisoflavone inhibits HCT-116 human colon cancer cell proliferation by targeting CDK1 and CDK2. Carcinogenesis 32: 629–635. 51. Urusova D, Shim J, Kim DJ, Jung SK, Zykova T, et al. (2011) Epigallocatechin- gallate suppresses tumorigenesis by directly targeting Pin1. Cancer Prev Res (Phila) in press. 52. Jeong CH, Bode AM, Pugliese A, Cho YY, Kim HG, et al. (2009) [6]-Gingerol suppresses colon cancer growth by targeting leukotriene A4 hydrolase. Cancer Res 69: 5584–5591. Hardware/software codesign and system synthesis (CODES+ISSS ‘ 05): 207–212. 53. Kang NJ, Lee KW, Kim BH, Bode AM, Lee HJ, et al. (2011) Coffee phenolic phytochemicals suppress colon cancer metastasis by targeting MEK and TOPK. Carcinogenesis 32: 921–928. References Cancer Res 69: 4398–4406. 47. Germain RS, Fitch BG, Rayshubskiy A, Eleftheriou M, Pitman MC, et al. (2005) Blue Matter on Blue Gene/L: Massively Parallel Computation for Biomolecular Simulation. presented at 3rd IEEE/ACM/IFIP international conference on PLoS ONE \| www.plosone.org May 2012 \| Volume 7 \| Issue 5 \| e38261 May 2012 \| Volume 7 \| Issue 5 \| e38261 8 Computational Strategies Finding Protein Targets 55. Kwon JY, Lee KW, Kim JE, Jung SK, Kang NJ, et al. (2009) Delphinidin suppresses ultraviolet B-induced cyclooxygenases-2 expression through inhibi- tion of MAPKK4 and PI-3 kinase. Carcinogenesis 30: 1932–1940. PLoS ONE \| www.plosone.org g g g 49. Lee DE, Lee KW, Song NR, Seo SK, Heo YS, et al. (2010) 7,39,49- Trihydroxyisoflavone inhibits epidermal growth factor-induced proliferation and transformation of JB6 P+ mouse epidermal cells by suppressing cyclin-dependent kinases and phosphatidylinositol 3-kinase. J Biol Chem 285: 21458–21466. Computational Strategies Finding Protein Targets 53. Kang NJ, Lee KW, Kim BH, Bode AM, Lee HJ, et al. (2011) Coffee phenolic phytochemicals suppress colon cancer metastasis by targeting MEK and TOPK. Carcinogenesis 32: 921–928. g g g 49. Lee DE, Lee KW, Song NR, Seo SK, Heo YS, et al. (2010) 7,39,49- Trihydroxyisoflavone inhibits epidermal growth factor-induced proliferation and transformation of JB6 P+ mouse epidermal cells by suppressing cyclin-dependent kinases and phosphatidylinositol 3-kinase. J Biol Chem 285: 21458–21466. 54. Kang NJ, Lee KW, Shin BJ, Jung SK, Hwang MK, et al. (2009) Caffeic acid, a phenolic phytochemical in coffee, directly inhibits Fyn kinase activity and UVB- induced COX-2 expression. Carcinogenesis 30: 321–330. 50. Lee KW, Kang NJ, Heo YS, Rogozin EA, Pugliese A, et al. (2008) Raf and MEK protein kinases are direct molecular targets for the chemopreventive effect of quercetin, a major flavonol in red wine. Cancer Res 68: 946–955. 55. Kwon JY, Lee KW, Kim JE, Jung SK, Kang NJ, et al. (2009) Delphinidin suppresses ultraviolet B-induced cyclooxygenases-2 expression through inhibi- tion of MAPKK4 and PI-3 kinase. Carcinogenesis 30: 1932–1940. q j 51. Urusova D, Shim J, Kim DJ, Jung SK, Zykova T, et al. (2011) Epigallocatechin- gallate suppresses tumorigenesis by directly targeting Pin1. Cancer Prev Res (Phila) in press. PLoS ONE \| www.plosone.org May 2012 \| Volume 7 \| Issue 5 \| e38261 9
https://openalex.org/W2044668825	https://europepmc.org/articles/pmc1977509?pdf=render	English	null	Screening for psychiatric morbidity in patients with advanced breast cancer: validation of two self-report questionnaires	British journal of cancer	1,991	cc-by	4,502	Screening for psychiatric morbidity in patients with advanced breast cancer: validation of two self-report questionnaires P. Hopwood', A. Howell2 & P. Maguire' 'Cancer Research Campaign Psychological Medicine Group; 2Cancer Research Campaign Department of Medical Oncology, Christie Hospital and Holt Radium Institute, Wilmslow Road, Withington, Manchester M20 9BX, UK. Summary Eighty-one patients with advanced breast cancer completed the Hospital Anxiety and Depression Scale (HADS) and Rotterdam Symptom Checklist (RSCL) to determine how well these questionnaires identified patients suffering from an anxiety state or depressive illness, compared with an independent interview by a psychiatrist who used the Clinical Interview Schedule. A threshold score was defined for each questionnaire which gave the optimal sensitivity and specificity. Seventy-five per cent of patients were correctly identified as suffering from an affective disorder by both the Rotterdam Symptom Checklist and by the Hospital Anxiety and Depression Scale. Twenty-one per cent of 'normal' patients were misclassified by the Rotterdam Checklist and 26% by the Hospital Anxiety and Depression Scale. When the HADs anxiety and depression subscales were analysed separately, the performance of the anxiety items was superior to that of the depression items. Both questionnaires were found to have good predictive value and could be used in patients with advanced cancer to help screen out those with an affective disorder. Clinicians treating women with advanced breast cancer have become increasingly concerned about their quality of life (Baum et al., 1980; Bell et al., 1985; Brinkely, 1985; Coates et al., 1987; Gough et al., 1983; Tannock et al., 1988), yet, the recognition of psychological distress is handicapped by patients' unwillingness to disclose any emotional problems and doctors' and nurses' reluctance to enquire (Maguire et al., 1980). Ways of improving the identification of psycho- logical morbidity need to be found and a self-assessment approach warrants evaluation. Two self-report question- naires, The Hospital Anxiety and Depression Scale (HADS) (Zigmond & Snaith, 1983) and The Rotterdam Symptom Checklist (RSCL) (de Haes et al., 1990), seemed promising since they were both designed for use with physically ill patients. However, there is little published data concerning their performance in homogeneous cancer groups. The RSCL contains three subscales: physical symptomato- logy due to disease and/or treatment (22 items); psycho- logical symptoms (8 items) and activities of daily living (8 items). All items are rated on a 4-point scale (e.g. 'I feel tense'; not at all (0); a little (1); somewhat (2); very much (3)). The psychological subscale yields a maximum score of 24. Br. J. Cancer (1991), 64, 353-356 Br. J. Cancer (1991), 64, 353-356 Br. J. Cancer (1991), 64, 353-356 Br. J. Cancer (1991), 64, 353-356 © Macmillan Press Ltd., 11" Macmillan Press Ltd., 1991 Screening for psychiatric morbidity in patients with advanced breast cancer: validation of two self-report questionnaires The questionnaire assessed symptoms over the preceding 3 days. y The order in which these questionnaires were completed by patients was balanced so that they were equally represented in first and second positions. Their presentation was therefore alternated as each consecutive clinical session in which patients were being recruited. p g Consecutive patients with a high score on either question- naire were asked if they were willing to be interviewed for the research by the psychiatrist. With respect to the HADS, 'high scorers' were those patients with scores at or above the recommended threshold value of 11, on either subscale. The same value for the psychological subscale of the RSCL was selected following discussion with one of its authors (Pruyn, personal communication, 1983). In addition to patients with a high score, some patients with a low questionnaire score were also approached. This normally involved asking the next low scoring patient following a high scorer. Thirty-four patients had high scores on the HADS and 47 patients had low scores. Using the RSCL, 27 patients had scores > 11 on the psychological complaints subscale and 54 had low scored (i.e. < 10). In total 44 patients out of the 81 recruited had a high score on either the HADS (17) or the RSCL (10) or both (17). Thirty-seven patients had low scores on both questionnaires. The interviews were conducted by a clinical psychiatrist (PH), who was blind to the questionnaire scores, using the Clinical Interview Schedule (Goldberg et al., 1970) with additional questions in order to apply standardised psychiatric diagnostic criteria (DSM III) (American Psychiat- ric Association, 1980). p g g p A study was conducted to determine how well these two questionnaires identified depressive illness and anxiety states in patients with advanced cancer of the breast. Received 16 February 1990; and in revised form 2 February 1991. ved 16 February 1990; and in revised form 2 February 199 Performance of the two questionnaires in the study sample The sensitivity and specificity were calculated for the HADS anxiety and depression subscales and the RSCL psycho- logical complaints subscale, using both the recommended threshold score (11) and a range of alternative cut-off scores. Sensitivity indicated the proportion of correctly identified cases (number of true cases/number of true cases plus number of false negatives) and hence the rate of false nega- tives. Specificity indicates the proportion of correctly identi- fied non-cases (number of true non-cases/number of true non-cases plus number of false positives) and hence the false positive rate (1-specificity). The misclassification rate is cal- culated from the ratio false positives + false negatives/total number of sample. Table II Ratio ofcases to non-cases, identified by clinical interview, at the optimum threshold value for each questionnaire p q Psychiatric assessment Non-cases Cases (i) HADS depression Scores 0-10 49 4 11 + 16 12 (ii) HADS anxiety Scores 0-10 62 3 11 + 7 9 (iii) RSCL Psychological complaints Scores 0-10 49 5 11 + 12 15 p Table I shows the performance of the two questionnaires based on the sample interviewed. Optimum sensitivity and specificity was found with a cut off value of 11 for each scale. The raw data used to calculate these values are shown in Table II. The sensitivity and specificity of each questionnaire were comparable and in an acceptable range. The misclass- ification rate for the HADS anxiety subscale (12%), was considerably lower than that for the depression subscale (25%). Table III Ratio ofcases to non-cases as identified by clinical interview, using a cut-off score of 18 for the HADS questionnaire (anxiety and depression scores combined) Table III Ratio ofcases to non-cases as identified by clinical interview, using a cut-off score of 18 for the HADS questionnaire (anxiety and depression scores combined) Psychiatric assessment Non-cases Cases Scores 0-17 45 5 18 + 16 15 Sensitivity = 75%; Specificity = 74%. Table IV 'Weighted' data used to calculate the sensitivity and specificity of the HADS and RSCL questionnaires in true outpatient conditions Psychiatric assessment Non-cases Cases (i) HADS Low scorers 140.30 6.70 High scorers 28.98 28.01 Sensitivity 80.7%; Specificity 88.88%. (ii) RSCL Low scorers 150.06 9.22 High scorers 19.22 24.77 Sensitivity 71.36%; Specificity 88.65%. ( ) An analysis was also carried out on the HADS using combined subscale scores to compare its performance as a unitary 14 item scale. Performance of the two questionnaires in the study sample The best cut-off score was 18, for which the sensitivity was 75%, specificity 74% and misclass- ification rate 26% (see Table III). p ) Psychiatric assessment Non-cases Cases Scores 0-17 45 5 18 + 16 15 Sensitivity = 75%; Specificity = 74%. Treatment Of the 81 patients interviewed 37 were receiving endocrine therapy, 24 were receiving chemotherapy, seven were taking corticosteroids and 13 were not receiving treatment. Table I Sensitivity, specificity and misclassification rate for the RSCL psychological complaints subscale, HADS anxiety and depression subscales, based on the interviewed sample Predictive values The accuracy of a screening instrument is also dependent on its positive predictive value (PPV) (Vecchio, 1966), that is the The accuracy of a screening instrument is also dependent on its positive predictive value (PPV) (Vecchio, 1966), that is the Patients and methods y) Where physical symptoms were clearly attributable to disease or treatment (for example, fatigue) they were ignored. Patients found to have symptoms of depression or anxiety, but insufficient to meet these DSM III criteria were desig- nated 'borderline' cases. p , g y g Patients found to have symptoms of depression or anxiety, but insufficient to meet these DSM III criteria were desig- nated 'borderline' cases. 'weighted' no. cases x 100 total no. patients screened 34.71 x 100 204 'weighted' no. cases x 100 total no. patients screened 34.71 x 100 204 Psychiatric morbidity Twenty (25%) patients were found at interview to have a depressive illness and/or anxiety state while 11 (14%) patients had a borderline mood disorder. p HADS HADS RSCL anxiety depression Sensitivity % 75 75 75 Specificity % 80 90 75 Misclassification rate % 21 12.3 24.7 Results Two hundred and four patients completed the two question- naires and 18 patients refused, giving a compliance rate of 92%. Eight-one patients agreed to be interviewed and there were no refusals. The estimated prevalence for the whole sample screened was 17%. Patients and methods Patients with advanced cancer of the breast attending medical oncology outpatient clinics over a 12 month period were included, providing they were free from dementia or cerebral metastases. A research nurse explained the study and asked each patient to complete the two questionnaires. The HADS is designed to discriminate between anxiety and depression and is made up of a 7-item anxiety subscale and a 7-item depres- sion subscale. Each item (for example, 'I can laugh and see the funny side of things') is rated on a 4-point scale (e.g. as much as-I always do (0); not quite so much (1); definitely not so much (2); and not at all (3), giving maximum subscale scores of 21 for depression and anxiety respectively. The questionnaire assessed symptoms over the preceding week. The authors of the HADS suggested that scores > 11 on either subscale were indicative of a case of depression or a case of clinical anxiety, whilst subscale scores in the 8-10 range represented borderline cases. A diagnosis of depressive illness required the presence of depressed mood for a minimum of 4 weeks, plus at least four of the following symptoms: change in appetite or weight; insomnia or hypersomnia; psychomotor agitation or retarda- tion; loss of interest including loss of libido; worthlessness or guilt; diminished concentration or suidical ideas. The diag- nosis of an anxiety state required the presence of generalised persistent anxiety of at least one month's duration, together with symptoms from three of four categories: motor tension (for example, shakiness, tension, fidgeting); autonomic hyper- activity (for example, dizziness, paraesthesia, diarrhoea); P.H. HOPWOOD et al. 354 Sensitivity and specificity values were then recalculated using these 'weighted' values for cases and non-cases, the results are shown in Table IV. The combined HADS sub- scales were used for this part of the analysis. Whilst the specificity remained relatively unchanged, the sensitivity of the HADS improved from 75% to 81% and that of the RSCL fell from 75% to 71%. The prevalence of probable cases in the whole sample (204) was calculated using the formula apprehensive expectation (for example, worry, fear, anxious foreboding); and vigilance and scanning (for example, dist- ractibility, poor concentration and irritability). apprehensive expectation (for example, worry, fear, anxious foreboding); and vigilance and scanning (for example, dist- ractibility, poor concentration and irritability). y) Where physical symptoms were clearly attributable to disease or treatment (for example, fatigue) they were ignored. Performance of the two questionnaires in true community conditions In other words, both questionnaires used as screening instru- ments would correctly identify one in every two high scorers as a case and have a relatively low risk of misclassifying a low scorer as a case. The ability of the HADS to accurately detect anxiety cases is high but its performance to detect case depression more modest. probability of a high score being a true psychiatric case. In clinical practice this value is important in determining the utility of an instrument, since it indicates the probability of detecting cases in a given population of patients. It is cal- culated from the ratio of the number of correctly identified cases/total number of persons with high scores. The PPV of a screening test is dependent on a prevalence in a given popula- tion and increases as prevalence rises. The PPV for the RSCL was 55.6%, which is high. For the HADS scale overall the value was 49.6% which is also very acceptable; the PPV for the anxiety subscale was 56.3% and that for the depression subscale 42.9%. (Using weighted data, the values for the two questionnaires were 56.3% and 49.2% respectively.) The neg- ative predictive values (NPV) were also calculated, that is, the proportion of low scorers who are not cases. The value for the RSCL was 80.3% and that for the HADS 82.7%. In other words, both questionnaires used as screening instru- ments would correctly identify one in every two high scorers as a case and have a relatively low risk of misclassifying a low scorer as a case. The ability of the HADS to accurately detect anxiety cases is high but its performance to detect case depression more modest. ition may have been too stringent, and hence the cut off score set too high. Secondly, the omission of somatic items that were thought due to disease or treatment may have raised the threshold for the clinical diagnosis. Further work in much larger samples is currently underway to assess the importance of somatic items in the diagnosis of psychiatric morbidity in cancer patients. A current advantage in both questionnaires tested in this study is that they exclude som- atic items from the psychological subscales. p y g Among the false positive scores were two patients with anxious personalities and two with bereavement reactions, and the questionnaires were not expected to discriminate such cases. Discussion The statistical analyses indicated that both questionnaires performed reasonably well and were suitable for use as screening instruments. However, both warrant refinement to improve their accuracy in detecting cases. Using the HADs as a 14-item scale, our results are very similar to those reported by Razavi et al. (1990), who suggested an optimal cut off point of 19 for major depressive disorder, associated with 70% sensitivity and 25% false positive rate. (Our cut off point of 19 gave exactly the same sensitivity and specificity values, but a threshold of 18 was superior from our data.) When using the separate subscales, the performance of the anxiety subscale was superior to that for depression, in terms of its positive predictive power and false positive rate. Our cut off value of 11 with the HADS depression subscale is a little higher than that reported by Razavi, but a lower value gave an unacceptable false positive rate (39%) with our data. When screening for illness, it is desirable to achieve a 100% detection rate. Whilst this can be achieved with these questionnaires, it is only at the expense of including a high proportion of false positives, which would create an unac- ceptable interview load. p The performance of these questionnaires is much superior to the detection rate of doctors and nurses involved in cancer care. Only 22% of patients with psychological problems following mastectomy are recognised by those concerned with their aftercare (Maguire et al., 1980). However, Maguire found that specialist nurses trained to detect such problems, can identify up to 80% of patients with depression or an anxiety state. A two stage process of assessment could be used, in which patients are serially screened for psychiatric disorder using one of these questionnaires. Those with above threshold scores would then be assessed further by a specialist nurse. This would provide a practical way of iden- tifying patients who might need help in the setting of a busy oncology clinic. Such screening questionnaires could also be used in clinical trials to measure the psychological dimension of quality of life. In clinical practice, patients with high scores warrant fur- ther assessment by a brief interview, as it is posisble to discriminate true cases from false positives on this basis. The 'optimal' sensitivity is aimed to identify an acceptable balance in terms of accuracy and clinical feasibility. This study was supported by Organon International BV, OS, Holland. We are most grateful for the help of research nurses; Mrs Anna Leah and Mrs Patricia Phillips and for advice concerning the analysis from Professor D.P. Goldberg. Performance of the two questionnaires in true community conditions Also included were patients with borderline depression or anxiety as judged by a clinical interview. Some of these patients would fulfill a DSM diagnosis of adjustment disorder (not used in this analysis). Such reactions may give rise to substantial psychological distress but this does not necessarily correlate with a formal psychiatric diagnosis of depressive illness or anxiety state. p y The estimated prevalence of psychological illness, from the 'weighted' data, may be considered low at 17% and ideally the prevalence should be derived from an independent sample from the same population. However, this prevalence is double the psychological morbidity reported by Dean (1987) in a sample of patients with early breast cancer. Like Dean, standard methods for defining psychiatric illness were used in this study, and strict criteria for defining a case were applied. Derogatis et al. (1983) reported a prevalence rate for psycho- logical morbidity of 47% in a heterogenous sample of cancer patients, but of these only 13% were ascribed as DSM III diagnosis of depressive disorder or anxiety state. Sixty-eight per cent of their diagnoses were deemed to be adjustment reactions. Borderline affective disorder Other instruments have been produced specifically for use in cancer research or practice (Bell et al., 1985; Coates et al., 1987; Padilla et al., 1981; Priestman & Baum, 1976; Selby et al., 1984) but these have not been validated against a psych- iatric interview. This makes their scores difficult to interpret. Borderline affective disorder The HADS questionnaire has been used to discriminate patients with borderline depression and anxiety using scores in the 8-10 range (Zigmond & Snaith, 1983). Of the patients interviewed, three out of six patients with a diagnosis of borderline anxiety and three out of eight with borderline depression were misclassified as cases by the HADS using the range of scores suggested by Zigmond and Snaith. Further analysis of the HADS in the borderline range of scores is underway but will not be reported here. It proved feasible to use both questionnaires in a busy clinical setting. The HADS has the advantage of being short, and of discriminating to an extent between cases of depres- sion and anxiety. The RSCL has additional useful questions about other aspects of the patients' quality of life, namely physical symptoms, treatment toxicity and functional status. Other instruments have been produced specifically for use in cancer research or practice (Bell et al., 1985; Coates et al., 1987; Padilla et al., 1981; Priestman & Baum, 1976; Selby et al., 1984) but these have not been validated against a psych- iatric interview. This makes their scores difficult to interpret. The performance of these questionnaires is much superior to the detection rate of doctors and nurses involved in cancer care. Only 22% of patients with psychological problems following mastectomy are recognised by those concerned with their aftercare (Maguire et al., 1980). However, Maguire found that specialist nurses trained to detect such problems, can identify up to 80% of patients with depression or an anxiety state. A two stage process of assessment could be used, in which patients are serially screened for psychiatric disorder using one of these questionnaires. Those with above threshold scores would then be assessed further by a specialist nurse. This would provide a practical way of iden- tifying patients who might need help in the setting of a busy oncology clinic. Such screening questionnaires could also be used in clinical trials to measure the psychological dimension of quality of life. It proved feasible to use both questionnaires in a busy clinical setting. The HADS has the advantage of being short, and of discriminating to an extent between cases of depres- sion and anxiety. The RSCL has additional useful questions about other aspects of the patients' quality of life, namely physical symptoms, treatment toxicity and functional status. Performance of the two questionnaires in true community conditions Table IV 'Weighted' data used to calculate the sensitivity and specificity of the HADS and RSCL questionnaires in true outpatient conditions Table IV 'Weighted' data used to calculate the sensitivity and specificity of the HADS and RSCL questionnaires in true outpatient conditions Sensitivity and specificity are dependent on the ratio of high and low scores in the sample. We deliberatedly balanced this ratio in approximately equal proportions for the interview study, in order to include as many potential 'cases' as possi- ble for the validation exercise. The outpatient population, from which the study sample was drawn, included more low scorers (133, 65%) than the interviewed sample (46%). Simi- larly the prevalence of high scorers was lower in the complete sample (71, 35%) than in the interviewed group (54%). It was desirable, therefore, to re-calculate the proportion of 'cases' to 'non-cases' in the interviewed sample to reflect the prevalence of high scorers and low scorers in the 204 out- patient attenders who completed both questionnaires. Psychiatric assessment Non-cases Cases (i) HADS Low scorers 140.30 6.70 High scorers 28.98 28.01 Sensitivity 80.7%; Specificity 88.88%. (ii) RSCL Low scorers 150.06 9.22 High scorers 19.22 24.77 Sensitivity 71.36%; Specificity 88.65%. PSYCHIATRIC MORBIDITY IN BREAST CANCER PATIENTS 355 probability of a high score being a true psychiatric case. In clinical practice this value is important in determining the utility of an instrument, since it indicates the probability of detecting cases in a given population of patients. It is cal- culated from the ratio of the number of correctly identified cases/total number of persons with high scores. The PPV of a screening test is dependent on a prevalence in a given popula- tion and increases as prevalence rises. The PPV for the RSCL was 55.6%, which is high. For the HADS scale overall the value was 49.6% which is also very acceptable; the PPV for the anxiety subscale was 56.3% and that for the depression subscale 42.9%. (Using weighted data, the values for the two questionnaires were 56.3% and 49.2% respectively.) The neg- ative predictive values (NPV) were also calculated, that is, the proportion of low scorers who are not cases. The value for the RSCL was 80.3% and that for the HADS 82.7%. References MAGUIRE, G.P., TAIT, A., BROOKS, M., THOMAS, C. & SELLWOOD, R.S. (1980). Effect of counselling on the psychiatric morbidity associated with mastectomy. Br. Med. J., 281, 1454. AMERICAN PSYCHIATRIC ASSOCIATION, COMMITTEE ON NOMENCLATURE AND STATISTICS (1980). Diagnostic and Stati- stical Manual of Mental Disorders. Washington DC. American Psychiatric Association. PADILLA, G., PRESANT, C.A., GRANT, M., BAER, C. & METTER, G. (1981). Assessment of quality of life in cancer patients. Proc. Am. Assoc. Cancer Res. & Am. Soc. Clin. Oncol., 22, 397. Abstract C-255. y BAUM, M., PRIESTMAN, T., WEST, R. & JONES, E. (1980). A com- parison of subjective responses in a trial comparing endocrine with cytotoxic treatment in advanced carcinoma of the breast. Eur. J. Cancer, Suppl: 223. PRIESTMAN, T.J. & BAUM, M. (1976). Evaluation of quality of life in patients receiving treatment for advanced breast cancer. Lancet, i, 899. , pp BELL, D.R., TANNOCK, I.F. & BOYD, N.F. (1985). Quality of life measurement in breast cancer patients. Br. J. Cancer, 51, 577. BRINKLEY, D. (1985). Quality of life in cancer trials. Br. Med. J., 291, 685. pp BELL, D.R., TANNOCK, I.F. & BOYD, N.F. (1985). Quality of life measurement in breast cancer patients. Br. J. Cancer, 51, 577. BRINKLEY, D. (1985). Quality of life in cancer trials. Br. Med. J., PRUYN, J.F.A. (1983). (Personal Communication). p , BRINKLEY, D. (1985). Quality of life in cancer trials. Br. Med. J., 291, 685. RAZAVI, D., DELVAUX, N., FARVACQUES, C. & RABAYE, E. (1990). Screening for adjustment disorders and major depressive dis- orders in cancer in-patients. Br. J. Psychiatr., 156, 79. COATES, A., GEBSKI, V., STAT, M. & 14 others (1987). Improving the quality of life during chemotherapy for advanced breast cancer. New Engl. J. Med., 317, 1490. p y , , SELBY, P.J., CHAPMAN, J.A.W., ETAXADI-AMOLI-J. DALLEY, D. & BOYD, N.F. (1984). The development of a method for assessing the quality of life of cancer patients. Br. J. Cancer, 50, 13. g DEAN, C. (1987). Psychiatric morbidity following mastectomy: pre- operative predictors and types of illness. J. Psychosom. Res., 31, 385. q y p , TANNOCK, I.F., BOYD, N.F., DE BOER, G. & 6 others (1988). A randomized trial of two dose levels of Cyclophosphamide, Methotrexate and Fluorouracil chemotherapy for patients with metastatic breast cancer. J. Clin. Oncol., 6, 1377. DEROGATIS, L.R., MORROW, G.R., FETTING, J. & 5 others (1983). The prevalence of psychiatric disorders among cancer patients. Discussion An instrument with good predictive power is very valuable, since it will reduce the interview load. The RSCL was superior to the HADS in this respect. In using either questionnaire as a screening instrument careful preparation is advisable: sensitivity, specificity and cut-off values should be checked, and the predictive value should be calculated according to the known prevalence of affective disorder in the population of patients to be screen- ed. Used in this way, these two instruments will provide a valuable clinical tool in the detection of psychological mor- bidity. Patients misclassified by both questionnaires are worth examining in greater detail. Of the five cases who were false negatives according to the RSCL, four were identified by the HADS, completed on the same occasion. Similarly, the RSCL identified two cases missed by the HADS, suggesting that the use of two questionnaires concurrently or sequen- tially may be of benefit. Other factors may have contributed to the misclassification rate: firstly the clinical 'case' defin- P.H. HOPWOOD et al. 356 References JAMA, 249, 751. , , VECCHIO, T.J. (1966). Predictive value of a single diagnostic test in unselected populations. New Engl. J. Med., 274, 1171. , , GOLDBERG, D.P., COOPER, B., EASTWOOD, M.R., KEDWARD, H.B. & SHEPHERD, M. (1970). A standardised psychiatric interview for use in community surveys. Br. J. Prev. Soc. Med., 24, 18. GOUGH, I.R., FURNIVAL, C.M., SCHILDER, L. & GROVE, W. (1983). Assessment of quality of life of patients with advanced cancer. Eur. J. Clin. Oncol., 19, 1161. GOLDBERG, D.P., COOPER, B., EASTWOOD, M.R., KEDWARD, H.B. & SHEPHERD, M. (1970). A standardised psychiatric interview for use in community surveys. Br. J. Prev. Soc. Med., 24, 18. ZIGMOND, A.S. & SNAITH, R.P. (1983). The Hospital Anxiety and Depression Scale. Acta Psychiatr. Scand., 67, 361. y y , , GOUGH, I.R., FURNIVAL, C.M., SCHILDER, L. & GROVE, W. (1983). Assessment of quality of life of patients with advanced cancer. Eur. J. Clin. Oncol., 19, 1161. DE HAES, J.C.J.M., VAN KNIPPENBERG, F.C.E. & NEIJT, J.P. (1990). Measuring psychological and physical distress in cancer patients: structure and application of the Rotterdam Symptom Checklist. Br. J. Cancer, 62, 1034.
https://openalex.org/W3135222706	https://link.springer.com/content/pdf/10.1007/s11846-021-00450-3.pdf	English	null	What factors condition the financial viability of sheltered employment centres? Empirical evidence	Review of managerial science	2,021	cc-by	13,165	Review of Managerial Science (2022) 16:459–482 https://doi.org/10.1007/s11846-021-00450-3 Review of Managerial Science (2022) 16:459–482 https://doi.org/10.1007/s11846-021-00450-3 ORIGINAL PAPER What factors condition the financial viability of sheltered employment centres? Empirical evidence Vera Gelashvili1 · María‑Jesús Segovia‑Vargas2 · María‑del‑Mar Camacho‑Miñano3 Received: 16 June 2020 / Accepted: 22 January 2021 / Published online: 3 March 2021 © The Author(s) 2021 * Vera Gelashvili vera.gelashvili@urjc.es Extended author information available on the last page of the article Keywords Sheltered employment centres · Profitability · Economic viability · Panel data · Decision trees * Vera Gelashvili vera.gelashvili@urjc.es Extended author information available on the last page of the article 1 Introduction Employment plays a central role in the lives of people with disabilities, offering not only monetary rewards but also such benefits as social identity, contacts and support (Shepherd 1989). Some researchers believe that creating opportunities for members of this population to get and keep jobs has a more profound effect on more areas of their lives than any other medical or social intervention (Board- man et al. 2003). Around Europe, there are a variety of programs that support and promote the employment of people with disabilities. One of the ways of combat- ing labour discrimination against people with disabilities is through social firms (Cooney et al. 2016). Social enterprises put social objectives above economic ones, and if the enterprise is profitable, profits are often reinvested to promote the company’s major social objectives (Del Negro 2012). However, these companies are not well known, which means that the great work they do is not always recog- nized. Within the category of social enterprises, are included sheltered employ- ment centres (Díaz-Foncea and Marcuello 2012), companies that promote labour and social integration of people with disabilities in Spain.i g p p p Sheltered employment centres are special firms because their workforce is made up of at least 70% of people with disabilities, according to the Spanish law for disabled people (Royal Decree 2273/1985, approving the regulations on shel- tered employment centres as defined in article 42 of Law 13/1982, of 7 April 1982, on social integration of people with disabilities). They are important com- panies for society and, especially, for disabled people due to the elimination of labour and social inclusion barriers (Calvo 2004; Mendoza et al. 2019). The role played by sheltered employment centres in the social economy of our country is important because almost 7% of social firms are made up of those companies (CEPES 2020). Furthermore, sheltered employment centres have more stable jobs for workers with disabilities than ordinary companies (Rodríguez and Cueto 2013).f The role, evolution, importance, professionalisation, wage differentials and other global aspects of sheltered employment centres have been studied for many years (Visier 1998; Rubio 2003; Laloma 2007; Jordán de Urríes and Verdugo 2010; Rodríguez et al. 2012; López et al. 2014; Manzano Martín et al. 2016; Gelashvili et al. 2015a,b; Monzón-Campos and Herrero-Montagud 2016; Gelash- vili et al. 2018; Mendoza et al. 2019) but their economic and financial aspects have not been investigated in depth. Abstract Nowadays, employment is a challenge for people but more for disabled ones. Prior literature shows that, at a European level, there are different ways for people with disabilities to find a job, such as a quota system, sheltered workshops, supported employment, etc. In Spain, sheltered employment centres are prototypes of sheltered workshops aimed at integrating more people with disabilities into the workplace. This research project aims to give visibility to these firms and to gain an understand- ing of their economic and financial situation. Using the whole sample of sheltered employment centres in Spain, and their financial data from 2004 to 2016, we show which variables explain their viability. Additionally, in light of the imminent world- wide crisis due to the COVID-19 pandemic situation, we want to test the impact of the last economic crisis on the profitability of sheltered employment centres. The main contribution of this study is that the size of these companies, age, financial risk and sales growth, are determining factors for their profitability. And, the economic and financial crisis has conditioned the viability of sheltered employment centres as many firms on the market registered a decrease in their profitability in the years fol- lowing the crisis but survived. This study helps to shed light on the economic and financial situation of this kind of firms as well as their social visibility. JEL Classification J58 · M4 · M41 JEL Classification J58 · M4 · M41 (0123456789) 1 3 456789) 3 V. Gelashvili et al. 460 1 Introduction In the last five years, some empirical stud- ies have analysed the level of economic impact generated by sheltered employ- ment centres in different regions of Spain (López et al. 2014; Manzano Martín et al. 2016; Gelashvili et al. 2015a) but none of them used data for all sheltered employment centres of Spain. Apart from the higher number of workers with disabilities in sheltered employ- ment centres, there is another characteristic differentiating them from normal companies. Sheltered employment centres can receive public subsidies. Accord- ing to Law 13/1982, of 7 April (LISMI), sheltered employment centres receive public subsidies for the labour insertion of people with disabilities. Those 1 3 What factors condition the financial viability of sheltered… 461 subsidies provided to these entities represent important economic flows for them (López et al. 2014). The purpose of the received subsidies is different: discounts for companies’ social security contributions, subsidies to adapt workstations, new investments, creation of new workplaces, etc. (Mallender et al. 2015). Thus, some authors point out that these public subsidies could determine their success in the labour market (Laloma 2007; Jordán de Urríes and Verdugo 2010). However, there are no empirical studies that test this affirmation. fi Other studies on sheltered employment centres have examined the evolution of these companies and have concluded that their numbers continued to grow even dur- ing the economic crisis that hit Spain in 2008 and beyond (Camacho-Miñano and Perez 2012; Gelashvili et al. 2015b). Meanwhile, in Spain, the number of SMEs declined. According to comparative data of SMEs, from 2006 to 2015 the percent- age decrease was − 3.02, almost 101,000 SMEs less than in 2006.1 Bearing all of this in mind, this study aims to analyse which main factors deter- mine the profitability of sheltered employment centres and test the main empirical assumptions about these specific firms such as the role of public subsidies or the impact of the economic crisis on their viability. The main contribution of this study is that the size, age, financial risk and sales growth are determining variables for the profitability of Spanish sheltered employment centres. Moreover, the economic crisis has negatively conditioned their viability. That means that the profitability of sheltered employment centres has decreased in the years following the crisis. 1 Ministry of Industry, Commerce and Tourism. SME reports 2006, 2015. http://www.ipyme.org/es-ES/ AreaEstadisticas/Paginas/InformesPYME.aspx. 2.1 Sheltered workshop and sheltered employment centres in Spain: Literature review Over the years, much has been written about sheltered workshops (Whitehead 1979; Rosen et al. 1993; Visier 1998; Krupa et al. 2003; Migliore et al. 2007; Migliore 2010; Evert et al. 2012; Hoffman 2013; Dlouhy and Mitchell 2015; Mal- lender et al. 2015; Yell et al. 2017; Lukas et al. 2018). In the beginning, they were developed by charities or religious organizations (Migliore 2010), but then the tasks and definition of sheltered workshops changed (Malo 2003; Galer 2014). Sheltered workshops are defined as “entities which specifically employ disabled people and receive subsidies in compensation for the reduced productivity of their workforce” (Mallender et al. 2015). Sheltered workshops aim to help unem- ployed people with disabilities to “prepare” and become competitively employed within the community (Evert et al. 2012). With social and labour integration, the rehabilitation of people with disabilities is also the main issue for sheltered work- shops (Visier 1998; Mallender et al. 2015). Each country has its systems of employability and social/labour inclusion of people with disabilities (Visier 1998). For example, most European countries have quota obligation systems (Greve 2009), sheltered workshops, supported employment for people with disabilities (Egido et al. 2009; Mallender et al. 2015; Hoffmann and Richter 2019) which includes start-up support for entrepreneurship by people with disabilities (Renko et al. 2016), etc. In Spain, sheltered employ- ment centres are prototypes of sheltered workshops aimed at integrating more people with disabilities into the workplace (Royal Decree 2273/1985, approving the regulations on sheltered employment centres as defined in Article 42 of Law 13/1982, of 7 April 1982, on the social integration of people with disabilities). Due to their economic and social importance, sheltered employment centres are referred to as social enterprises at the European level (López et al. 2014). Stud- ies about sheltered employment centres in Spain analyse how the management of these enterprises works (Giménez 2012; López et al. 2014), what the main objectives of these firms are (Martínez 2009; Jordán de Urríes and Verdugo 2010) or how the number of employees with disabilities has grown during last years (Giménez 2012; Díaz-Foncea and Marcuello 2014; Penabad et al. 2019).f Although there are many studies on different aspects of sheltered employment centres (Calderón and Calderón 2012; Rodríguez et al. 2012; López et al. 2014; Penabad et al. 1 Introduction Although their profitability has decreased in times of crisis, these companies have managed to stay in the market, since some studies have shown that the number of these companies has increased during and after the economic and financial crisis. This fact is an important contribution for these special companies, since the cur- rent situation caused by the COVID-19 has begun to destroy employment around the world and will grow even more among the most disadvantaged, who have the least likely to remain in the ordinary labour market, the people with disabilities. There- fore, the importance of these companies is going to increase in the period of the cur- rent crisis for people with disabilities. This paper is organized as follows: the second section includes the literature review about sheltered employment centres in academic studies. The third section shows proposed research questions. The sample of the research, methodology and variables of the study are shown in the fourth section. The results of the financial data analysis and our main conclusions are presented in the fifth and sixth sections. 1 3 V. Gelashvili et al. 462 2.1 Sheltered workshop and sheltered employment centres in Spain: Literature review 2019; Rodríguez 2017), there is a notable lack of literature on the economic and financial viability and profitability of these centres (Manzano Mar- tín et al. 2016; Gelashvili et al. 2015a, b). Thus, it is important to know how the management of these centres works and what factors determine their economic and financial viability.ii i The first study on the profitability of sheltered employment centres was a study carried out by López et al. (2014). This study examines whether the level of eco- nomic impact generated by the sheltered employment centres depends on the 1 3 1 3 What factors condition the financial viability of sheltered… 463 kind of business activities carried out. The sample for the research included 66 sheltered employment centres of Aragón, another specific region of Spain. Their results have shown that the level of the economic impact of sheltered employ- ment centres depends only on their business activities. Their social activities are not linked to their economic impact although the former are essential due to their objective. The study carried out by Gelashvili et al. (2015a) examined 100 sheltered employment centres in the region of Madrid. The main objective of this paper was to know whether public subsidies were one of the main factors to determine their profitability. Their results showed that sheltered employment centres could be pro- ductive enterprises, on average, even without public subsidies. Manzano Martín et al. (2016) studied 103 sheltered employment centres of Castilla-León, a region of Spain. Their principal findings show that in Castilla- León there are a large number of private sheltered employment centres, dominated especially by small and medium-sized centres. Their results showed that sheltered employment centres are able to obtain as many positive results as other ordinary companies. To the best of our knowledge, there is no empirical study that examines the eco- nomic and financial viability of sheltered employment centres using all sheltered employment centres in Spain, which is the whole population in our country. At the same time, public subsidies are an important variable and differentiate them from an SME. For this reason, it is interesting to show their influence on its profitability and viability. 2.1 Sheltered workshop and sheltered employment centres in Spain: Literature review Another issue is that there are additional subsidies in each of the autono- mous communities in which each sheltered employment centre is located (Laloma 2007), so the results of previous research may change by using the financial data of all sheltered employment centres. 2.2 Viability of firms The viability of a company is studied based on its quantitative and qualitative data and the success or failure of a company depends on many factors. The profitability of enterprises is essential for their viability. Indeed, this topic about the factors that have an impact on the profitability of a company has been studied in some papers such as Schmalensee (1985), Rumelt (1991), Fernández et al. (1996); Claver et al. (2002) and González et al. (2002). According to Claver et al. (2002), the profitability of a firm depends on the resources and capabilities of each company, making them different from others, but not only because the management of these resources and capabilities is essential for determining their success or failure. Some other stud- ies have pointed out that the profitability of companies depends on their employees’ capacity for teamwork, training, and skills (Claver et al. 2002; Rubio and Aragón 2002; Isaac et al. 2009), on their size (Suarez 2000) or the sector of their activi- ties (Iglesias et al. 2007). Previous literature presents different economic and finan- cial variables such as solvency, liquidation, borrowed funds, the share of external funding, asset turnover, sales margin, asset rotation, debt, funds generated by sales, etc. to evaluate the profitability of different types of companies (Fernández and 1 3 3 V. Gelashvili et al. 464 García 1991; Fernández et al. 1996; Suarez 2000; González et al. 2002; Isaac et al. 2009). Fariñas and Rodríguez Romero (1986) analyse the future viability of compa- nies from different countries and point out that their profitability depends on their nationality as well. Another variable that could also influence the profitability of the company is its level of corporate social responsibility (CRS) (Waddock and Graves 1997; Tsoutsoura 2004; Mahbuba and Farzana 2013). A recent study by Shahzad and Sharfman (2015) has detected a positive relationship between corporate social responsibility and the financial performance of firms.ii ii There are some studies on the economic and financial viability and profitabil- ity of SMEs or even family enterprises (Claver et al. 2002; González et al. 2002; Luengo et al. 2005; Isaac et al. 2009). One of the main conclusions of those stud- ies is that, for SMEs, profitability has become the most common financial indicator to measure the level of success or failure in business management (González et al. 2002). 2.2 Viability of firms Moreover, the past economic and financial crisis impended the regular opera- tion or even the survival of many companies (Isaac et al. 2009; Cowling et al. 2012; Carmona et al. 2013) and profitability is one of the most important variables for long-term survival in order to evaluate the yields of any company. The studies analyzed above have shown the importance of social enterprises for the most vulnerable people in the market, in particular, the great work done by shel- tered employment centres for people with disabilities even during the past economic and financial crisis. Recent studies indicate that we are at the beginning of a new economic and financial crisis almost all over the world because of the COVID-19 (Baldwin and Mauro 2020; Fernandes 2020). Several studies from different coun- tries have estimated that the COVID -19 will lead to a catastrophic decrease in employment (Beirne et al. 2020; Blustein et al. 2020; Coibion et al. 2020). The latest data from the Public Service of State Employment in Spain has shown that unem- ployment is increasing month by month, the total number of job seekers in Spain currently stands at almost 4 million and the unemployment figures for April rep- resent an increase of 7.97% compared to March.2 Due to the innumerable barriers for people with disabilities to access or keep employment in the ordinary compa- nies (Gannon and Nolan 2004; Parker Harris et al. 2012), they are more likely to be unemployed and, if employed, to be paid less (Lang et al. 2011). For this reason, the study of sheltered employment centres in the times of the previous economic and financial crisis can help to shed light on how they can survive this global crisis that is coming. 2 https://www.lamoncloa.gob.es/serviciosdeprensa/notasprensa/trabajo14/Paginas/2020/050520-paro. aspx. 3 Research questions Previous literature has been published about the influence of size, sector and location on profitability (Fariñas and Rodríguez Romero 1986; González Perez 1997 Suarez 2000; Sanchez and García 2003; Iglesias et al. 2006). Sanchez and García (2003) 3 What factors condition the financial viability of sheltered… 465 point out that competitive advantages help larger companies to obtain greater profit- ability, while the flexibility and adaptability of SMEs allow them to obtain a bet- ter performance. A study on integration companies shows that the success of these companies depends on their location and the sector of their activities (Retolaza et al. 2007). In addition, some studies have highlighted that public subsidies may play an important role in the economic viability of sheltered employment centres (Laloma 2007; Jordán de Urríes and Verdugo 2010). In the case of sheltered employment centres, it would be important to know the effect of these three important variables on their profitability. Thus, our research questions are presented as follows:ii i RQ 1: What are the main financial factors that determine the profitability of shel- tered employment centres?i i RQ 1: What are the main financial factors that determine the profitability of shel- tered employment centres?i The past economic and financial crisis and its expansion over time force compa- nies to survive in the current social and economic circumstances (Carmona et al. 2013). Despite the environmental crisis they are witnessing, sheltered employment centres are managing to create and maintain workplaces for people with disabilities (Manzano Martín et al. 2016). Also, the evolution of sheltered employment centres shows that the number of centres increased during the time of the crisis (Gelash- vili et al. 2015b), although it punished many SMEs in Europe (Kokocinska and Rekowski 2013). From here, the next research question can be formulated:ii RQ 2: Has the economic and financial crisis conditioned the profitability of shel- tered employment centres? It is important to analyze the impact of the previous crisis on these special com- panies since another one is coming and it is necessary to see how they are going to handle the situation so that people with disabilities be less affected. f The next section gives detailed information about the sample, variables and meth- odology of the study. 3 http://www.bvdinfo.com/en-uss/our-products/company-information/national-products/sabi. 4.1 Sample and data collection According to data available for 2016, 1,834 sheltered employment centres repre- sented all those existing in Spain. Through the Monitoring and Management Service for Supporting Employment of People with Disabilities assigned to the Employ- ment Secretary (SEPE), it was possible to access the names of all existing sheltered employment centres at the end of 2016. There is no accessible database of all the sheltered employment centres in Spain on the internet because the employment competencies in our country are distributed in each region. 3i The SABI3 database was used for extracting financial data, which provides quan- titative and qualitative information on Spanish companies. However, it was not pos- sible to access the financial data for all sheltered employment centres. So, finally, the financial statements of 958 sheltered employment centres were collected from 2004 1 3 3 V. Gelashvili et al. 466 Table 1 Independent variables Source: own elaboration ID Variables Definition AGE Age The number of years since its foundation SIZE Size The number of employees SECTOR Sector of activity Manufacturing companies 0; service companies 1 LOCAT Location Autonomous community (autonomous communi- ties with the highest concentration of sheltered employment centres 1; otherwise 0) LIQUID Liquidity (quick ratio) (Current assets-inventory)/current liability INDEBT Indebtedness Total liabilities/total equity F_RISK Financial Risk Financial expenses/sales SALESGR Sales Growth Sales t –sales t-1/sales t-1 SALESEMP Average sales per Employee Sales/number of employees SUBS Subsidies (capital grants) Amount of money received from public institu- tions EF_CRISIS Economic and Financial crisis (years) During 2008–2014 = 1; 0 = otherwise to 2016. This data represents 52% of all sheltered employment centres in Spain. This is the final sample of our research. to 2016. This data represents 52% of all sheltered employment centres in Spain. This is the final sample of our research. 1 3 4.2 Variables The variables used were those that allow the analysis of the current situation of shel- tered employment centres. Our dependent variable is a viability measurement. We use the proxy of return on assets (ROA) because ROA is considered a ratio required for the financial via- bility of firms (Suarez 2000; Retolaza et al. 2014). In theory, like other firms on the market, sheltered employment centres should be profitable in order to survive but this is not their main goal but rather social implications. Furthermore, ROA has been used for other researchers to define the profitability of firms (José et al. 1996; Suarez 2000; García-Teruel and Martínez-Solano 2007; Isaac et al. 2009; Enqvist et al. 2014; Kohlscheen et al. 2018). The sheltered employment centres of our sam- ple have been classified into two groups according to their ROA (operating result/ total assets) values to carry out our analyses. Therefore, the ones that have a positive ROA value will be assigned to class 1. On the contrary, the sheltered employment centres with negative ROA values have been assigned to class 0.il According to most classical studies, profitability, cash flow, liquidity, leverage and efficiency ratios are the most used in failure prediction studies (Dimitras et al. 1996), which is the opposite concerning viability. However, as we want to explain profitability and not failure prediction, we just select the ones not correlated with ROA. First, a liquidity ratio is chosen. Another ratio not correlated with the depend- ent variable is the level of indebtedness. Additionally, the financial risk and growth rate in sales are also included. The financial risk ratio measures the ability of the 1 3 What factors condition the financial viability of sheltered… 467 company to cover its financial expenses through total sales (Serer et al. 2009; Tarrés 2012). This ratio is considered one of the most important to measure the economic and financial situation of the company. Sales growth is considered the main variable to check the company´s performance (Morbey and Reithner 1990). Several investi- gations have studied the relationship between company profitability and sales and have concluded that there is a positive relationship between the company’s ROA and different types of sales (Gill and Mathur 2011; Kouser et al. 2012). f The list of the independent variables for regression is shown in Table 1.ii Other variables that could condition the profitability of firms are sector, size and age. 4.2 Variables According to Claver et al. (2002), the profitability of a company is linked to industry. This variable has been classified according to the NACE (Statistical Clas- sification of Economic Activities in the European Community) classification. After that, they have been divided into two parts: manufacturing activities and service activities. The division of this variable into two groups is mainly since most of the sheltered employment centres operate in the service sector (Jordán de Urríes and Verdugo 2010; Gelashvili et al. 2016). Additionally, many papers evidence the rela- tionship between size and ROA although the results are not yet conclusive. Some papers justify a positive relationship (González Perez 1997; Pervan and Visic 2012), others a negative one (Antón et al. 1990) and, finally, the rest a neutral one (Galán and Vecino 1997). Location is also another important factor to take into account; this variable has been categorised and divided into autonomous communities with the highest concentration of sheltered employment centres and autonomous commu- nities for a lower concentration of these companies. Based on the study elaborated by Gelashvili et al (2015b) higher number of sheltered employment centres were registered in autonomous communities such as Madrid, Catalonia, Andalusia and Castilla-León. In this way, we would like to know if location conditions the profit- ability of these companies because there is a different regulation about grants and subsidies for this type of company for each autonomous region. Finally, to answer research question 2, a dummy variable economic and financial crisis has been cre- ated. The economic and financial crisis in Spain started in 2008, when the overpric- ing of real estate assets played an important role in the process of weakening the banking system (Montalvo 2009; Alonso 2013; Bank of Spain 2017). Therefore, the sample has been divided into two parts, before and after the economic and financial crisis and the period of the crisis. That allowed us to see if the profitability of these companies has been affected by the economic and financial crisis or not. 4.3 Methodology We perform a detailed descriptive analysis of the sample to characterise the varia- bles under study. Additionally, we use a correlation table in order to analyse the rela- tionship between variables. We then generate a residual plot to verify the assumption of normality. To test our research questions, we build a multiple linear regression model that has the ROA of sheltered employment centres as a response variable and the various explanatory variables listed in Table 1. Taking into account that we have different types of variables, we use linear regression with random effects. 1 3 3 3 V. Gelashvili et al. 468 Table 2 Descriptive statistics Source: own elaboration Variable Obs Mean Std. Dev Min Max ROA 9083 1.64 20.21 − 253.46 617.41 AGE 9083 10.24 6.83 1.00 52.00 SIZE 9083 56.58 142.13 1.00 2338.00 LIQUID 9083 2.75 47.68 0.00 4164.31 INDEBT 9083 3.43 72.19 − 393.01 6304.48 F_RISK 9083 − 0.27 3.73 − 53.15 172.57 SALESGR 9083 0.42 8.77 − 1.00 659.91 SALESEMP 9083 51,266.74 316,197.80 0.00 20,800,000.00 SUBS 9083 100,483.70 590,708.10 0.00 10,500,000.00 Regression with random effects is a regression model (panel data) that combines cross-section and time-series data (Baltagi 2008; Hsiao 2014), as is the case of our model. To ensure that this was the right model, the Breusch-Pagan test has been car- ried out to determine whether we should perform the data analysis using the random effect or pooled estimation (Breusch and Pagan 1980). Following the research ques- tion (RQ1), we expect some variables to be significant, that is, lie below the level of significance of 5% (as p-value < 0.05). From the second research question (RQ2), we would expect the coefficient β of the year dummy variable to be significant as well (p-value < 0.05). This will mean that the economic and financial crisis will have an impact on the profitability of sheltered employment centres. i Additionally, to give another view of the problem raised in this work and com- plete the results obtained, a decision tree based on artificial intelligence (AI) meth- odology has been developed. Methods based on AI are widely used to analyse finan- cial problems (Serrano and Martin del Brio 1993; Sanchis et al. 2007; Diaz et al. 2009). Indeed, AI methods are a complement and, in some cases, a substitute for statistical methods. In any case, they can give another point of view on the prob- lems we are investigating. 4 The J48 algorithm is the freely accessible Java implementation of the C4.5 algorithm contained in the WEKA software. In fact, WEKA is the data mining package developed by the University of Waikato (Witten and Frank 2005) with which we have performed our analysis to complete the main result. 5.1 Linear regression result The results of the descriptive analysis show that sheltered employment centres, on average, are companies with a positive rate of profitability (see Table 2). It is highlighted that the average ROA is equal to 1.64%. This means that the assets of sheltered employment centres generate enough profit, although it is not very high. The average age of sheltered employment centres is more than 10 thus, the major- ity of them are companies with experience in the market. The size of sheltered employment centres has been measured by the number of employees. The average number of employees for these companies is 56. This means that these kinds of firms are very labour intensive. i The result of the descriptive analysis has shown that the average of public subsidies is 100,483 euros. That means that the companies that receive public subsidies usually get a fairly high amount of funds. The maximum amount in terms of subsidies received per company is 10 million, while almost half of these companies have not received these subsidies. By analysing the liquidity ratio it can be observed that its average is 2.75, indicating that sheltered employment centres can pay off their short-term obligations. The result of the indebtedness ratio shows that sheltered employment centres are heavily indebted, 3.43, and 100 euros net worth, on average, meaning they have 343 euros of debts. The negative result of the financial risk ratio indicates that the level of financial risk of shel- tered employment centres is high, even though its standard deviation is also high. The positive results of the growth in sales of variables and sales per employee indicate that these companies, on average, have a positive return on sales.ii The descriptive analysis that gives the first image of the economic and finan- cial situation of the companies was followed by the correlation matrix. The link between ROA and the independent variables is shown in Table 3. There is an interesting correlation between ROA and the size of the company. The profitabil- ity of sheltered employment centres is also determined by their business activity. Correlation between the dependent variable and the age of sheltered employment centres, indebtedness, financial risk and economic and financial crisis is also shown.i Another interesting point is that there is no correlation between the profitabil- ity of these companies and public subsidies as capital grants. 4.3 Methodology Consequently, we are going to examine the economic and financial variables that characterise failure, and therefore, the survival of sheltered employment centres using the C4.5 decision tree. Several algorithms develop deci- sion trees, and what differentiates one decision tree from another is the algorithm that generates it. The algorithm developed by Quinlan and implemented in C4.5 (Quinlan 1993) is probably the most popular of all decision tree algorithms. In it, the criterion used to make the partitions is based on a series of Information Theory con- cepts and has experienced a series of notable improvements over time. For a more detailed description, see Quinlan (1993) and Díaz et al. (2009).4 1 3 What factors condition the financial viability of sheltered… 469 5.1 Linear regression result This means that the idea that the viability of sheltered employment centres is due to their public sub- sidies is not true based on the correlation table result. However, the results of the correlation are not conclusive. The next step in our statistical analysis was the elaboration of linear regres- sion for panel data. A total of 953 groups were analysed with 9,083 observations. The result has shown that the explanatory variables explain an overall 87% of the proposed model and 93% within groups. Furthermore, the p-value was 0.00. This allows us to affirm that the independent variables chosen have explanatory power for the model. In Table 4 we can see that the linear regression confirms 1 3 V. Gelashvili et al. 470 Table 3 Correlation matrix S O l b ti ROA AGE SIZE SECTOR LOCAT LIQUID INDEBT F_RISK SALESGR SALESEMP SUBS EF_CRISIS ROA 1 AGE − 0.0269* 1 0.010 SIZE 0.0583* 0.2863* 1 0.000 0.000 SECTOR 0.0357* − 0.0638* 0.0751* 1 0.001 0.000 0.000 LOCAT 0.002 − 0.002 0.0410* 0.1183* 1 0.827 0.836 0.000 0.000 LIQUID − 0.009 − 0.003 − 0.007 0.014 0.017 1 0.398 0.760 0.489 0.198 0.106 INDEBT − 0.0275* − 0.014 − 0.009 0.009 0.010 0.000 1 0.009 0.189 0.374 0.384 0.361 0.972 F_RISK − 0.0658* − 0.0213* − 0.003 − 0.006 − 0.0289* 0.004 − 0.001 1 0.000 0.043 0.810 0.551 0.006 0.697 0.929 SALESGR 0.010 − 0.021 − 0.013 0.015 0.018 − 0.001 0.001 − 0.0018 1 0.342 0.050 0.223 0.161 0.090 0.946 0.927 0.8607 SALESEMP 0.014 0.0479* − 0.0388* 0.013 0.001 − 0.003 0.003 − 0.0147 0.02 1 0.197 0.000 0.000 0.227 0.910 0.803 0.799 0.1604 0.0564 SUBS 0.003 0.2398* 0.3854* 0.006 − 0.0284* 0.001 − 0.006 0.0230* − 0.005 0.0306* 1 0.796 0.000 0.000 0.576 0.007 0.940 0.599 0.0285 0.6311 0.0035 EF_CRISIS − 0.2757* 0.1888* 0.013 0.0363* 0.007 0.0300* 0.0246* − 0.0379* 0.0254* 0.0844* 0.0885* 1 0.000 0.000 0.213 0.001 0.500 0.004 0.019 0.0003 0.0153 0.000 0.000 Table 3 Correlation matrix ROA AGE SIZE SECTOR LOCAT LIQUID INDEBT F_RISK SALESGR SALESEMP SUBS EF_CRISIS 3 Table 3 Correlation matrix S O l b ti ROA AGE SIZE SECTOR LOCAT LIQUID INDEBT F_RISK SALESGR SALESEMP SUBS EF_CRISI ROA 1 AGE − 0.0269* 1 0.010 SIZE 0.0583* 0.2863* 1 0.000 0.000 SECTOR 0.0357* − 0.0638* 0.0751* 1 0.001 0.000 0.000 LOCAT 0.002 − 0.002 0.0410* 0.1183* 1 0.827 0.836 0.000 0.000 LIQUID − 0.009 − 0.003 − 0.007 0.014 0.017 1 0.398 0.760 0.489 0.198 0.106 INDEBT − 0.0275* − 0.014 − 0.009 0.009 0.010 0.000 1 0.009 0.189 0.374 0.384 0.361 0.972 F_RISK − 0.0658* − 0.0213* − 0.003 − 0.006 − 0.0289* 0.004 − 0.001 1 0.000 0.043 0.810 0.551 0.006 0.697 0.929 SALESGR 0.010 − 0.021 − 0.013 0.015 0.018 − 0.001 0.001 − 0.0018 1 0.342 0.050 0.223 0.161 0.090 0.946 0.927 0.8607 SALESEMP 0.014 0.0479* − 0.0388* 0.013 0.001 − 0.003 0.003 − 0.0147 0.02 1 0.197 0.000 0.000 0.227 0.910 0.803 0.799 0.1604 0.0564 SUBS 0.003 0.2398* 0.3854* 0.006 − 0.0284* 0.001 − 0.006 0.0230* − 0.005 0.0306* 1 0.796 0.000 0.000 0.576 0.007 0.940 0.599 0.0285 0.6311 0.0035 EF_CRISIS − 0.2757* 0.1888* 0.013 0.0363* 0.007 0.0300* 0.0246* − 0.0379* 0.0254* 0.0844* 0.0885* 1 0.000 0.000 0.213 0.001 0.500 0.004 0.019 0.0003 0.0153 0.000 0.000 1 What factors condition the financial viability of sheltered… 471 Table 4 Results of the linear regression Source: Own elaboration ROA Coef Robust Std. Err t P > t [95% Conf. Interval] AGE − .0093609 .0008883 − 10.54 0.000 − .0111019 − .0076199 SIZE .0002296 .000048 4.78 0.000 .0001354 .0003237 SECTOR .0159065 .0177012 0.90 0.369 − .0187872 .0506003 LOCAT − .0049561 .015046 − 0.33 0.742 − .0344456 .0245335 LIQUID − .0000233 .0000828 − 0.28 0.778 − .0001857 .000139 INDEBT − .0000744 .0000541 − 1.37 0.169 − .0001805 .0000317 F_RISK − .0071229 .0010577 − 6.73 0.000 − .0091959 − .0050498 SALESGR .0009191 .0004478 2.05 0.040 .0000414 .0017968 SALESEMP 2.13e− 08 1.28e−08 1.67 0.094 − 3.67e−09 4.64e−08 SUBS 8.76e−09 1.00e−08 0.87 0.382 − 1.09e−08 2.84e−08 EF_CRISIS − .1788339 .0079041 − 22.63 0.000 − .1943256 − .1633421 _cons .9434982 .0190962 49.41 0.000 .9060703 .9809261 the result of the correlations, where there is a positive relationship between the dependent variable and the size of the company. Therefore, our results coincide with the statement of González Perez (1997) and Pervan and Visic (2012). Table 3 Correlation matrix ROA AGE SIZE SECTOR LOCAT LIQUID INDEBT F_RISK SALESGR SALESEMP SUBS EF_CRISIS The dummy variable that classifies sheltered employment centres by sector of activ- ity is not significant (p-value = 0.369 > 0.05), although, the correlation table showed that there was a correlation between ROA and the sector of activity of these social companies. That means that the profitability of sheltered employment centres does not depend on the sector in which they operate. The age of sheltered employment centres is one of the variables that explain the proposed model. But, the coefficient of this variable is negative. This means that new companies have more profitability than the companies with experience at the market. Also, the size of these companies and financial risk are the variables that condition their profitability. The results achieved by González Perez (1997), Claver et al. (2002) and Pervan and Visic (2012) are in line with the results of this study. Other variable that explain the ROA of sheltered employment centres is sales growth. This result confirms the findings of Gill and Mathur (2011) and Kouser et al. (2012), although it should be noted that these studies have analysed the sam- ple of companies operating in different countries such as Canada, where company policy and operation are different. Therefore, this result is an important finding for Spanish social firms and especially for sheltered employment centres, since no prior studies were linking the profitability of sheltered employment centres to sales, espe- cially when it is questioned if the profitability of these companies is related to the subsidies received. The correlation between the explanatory variable and these vari- able has also been confirmed in Table 3.i i To answer research question 2, the independent variable “economic and financial crisis” has been introduced into the model. To measure the effect of the economic crisis on the profitability of these companies, the linear regression has been per- formed, considering the period of crisis in the years 2008–2014. Therefore, dummy 1 3 3 V. Gelashvili et al. Table 3 Correlation matrix ROA AGE SIZE SECTOR LOCAT LIQUID INDEBT F_RISK SALESGR SALESEMP SUBS EF_CRISIS 472 F_RISK <= -0.01: 1 (331.0/8.0) F_RISK > -0.01 \| F_RISK <= 0 \| \| INDEBT <= -1.12: 0 (16.0/2.0) \| \| INDEBT > -1.12 \| \| \| SIZE <= 17 \| \| \| \| LIQUID <= 0.03: 1 (10.0) \| \| \| \| LIQUID > 0.03 \| \| \| \| \| SALESEMP <= 18850.97 \| \| \| \| \| \| SALESEMP <= 747.32: 0 (16.0/1.0) \| \| \| \| \| \| SALESEMP > 747.32 \| \| \| \| \| \| \| LIQUID <= 0.94: 0 (12.0/1.0) \| \| \| \| \| \| \| LIQUID > 0.94 \| \| \| \| \| \| \| \| SECTOR = 0: 1 (4.0) \| \| \| \| \| \| \| \| SECTOR = 1 \| \| \| \| \| \| \| \| \| LOCAT = 0 \| \| \| \| \| \| \| \| \| \| LIQUID <= 4.63: 1 (7.0) \| \| \| \| \| \| \| \| \| \| LIQUID > 4.63: 0 (3.0/1.0) \| \| \| \| \| \| \| \| \| LOCAT = 1: 0 (6.0/1.0) \| \| \| \| \| SALESEMP > 18850.97: 1 (36.0/7.0) \| \| \| SIZE > 17: 1 (77.0/9.0) \| F_RISK > 0 \| \| F_RISK <= 0.85: 0 (104.0) \| \| F_RISK > 0.85: 1 (21.0/3.0) Fig. 1 Decision tree obtained by algorithm C4.5, 2008 (I). Source Own elaboration Fig. 1 Decision tree obtained by algorithm C4.5, 2008 (I). Source Own elaboration variable (EF_CRISIS) is created where the non-crisis period were 2004–2007 and 2015–2016 as zero and the economic and financial crisis period from 2008–2014 as one. We have made this division based on the GDP growth in Spain. According to data provided by the INE (2020),5 in the years of financial crisis, Spain’s GDP decreased considerably, although in 2015 it has begun to recover. Bank of Spain (2017) has pointed out that the economic and financial crisis in Spain is understood between the years 2008–2014. The results of the linear regression for panel data indicate that the ROA of shel- tered employment centres is explained by the variable economic and financial cri- sis. However, it should be noted that the coefficient that explains this relationship is negative. The negative coefficient suggests that, as the independent variable “ROA” increases, the dependent variable “economic and financial crisis” tends to decrease. 5 Instituto Nacional de Estadistica (INE)—[Spanish Statistics Institute]. Available at: https://www. ine.es/dyngs/INEbase/es/operacion.htm?c=Estadistica_C&cid=1254736167628&menu=resultados &idp=1254735576581#!tabs-1254736158133. Table 3 Correlation matrix ROA AGE SIZE SECTOR LOCAT LIQUID INDEBT F_RISK SALESGR SALESEMP SUBS EF_CRISIS During the economic and financial crisis, the profitability of these companies was lower than in the other years. Therefore, we can say that sheltered employment cen- tres like the rest of the companies in Spain have been affected by the crisis. f The following section studies the proposed model by using the C4.5 decision tre 1 3 What factors condition the financial viability of sheltered… 473 Fig. 2 Decision tree obtained by algorithm C4.5, 2008 (II). Source Own elaboration. The strongest rules are pointed out with an arrow Fig. 2 Decision tree obtained by algorithm C4.5, 2008 (II). Source Own elaboration. The strongest rules are pointed out with an arrow 5.2 C4.5 decision tree The model to support our results of the main analysis has been compiled through the C4.5 decision tree. As the sample in this article is from panel data, two differ- ent years have been analysed, namely 2008 and 2016. 2016 has been used as the last year of the sample and 2008 as the year when the economic and financial crisis started in Spain. For 2008, 643 sheltered employment centres were used. The result has shown that almost 91% of the sample was correctly classified. Data for the dependent vari- able (ROA) was classified into two groups: group 0 classifies sheltered employment centres with a negative return on assets and group 1 with positive results. The results of the model can be seen in Fig. 1. The strongest branches for each class (0, 1) have been highlighted in grey, i.e. those that verify a greater number of sheltered employment centres. The independent variable such as economic and financial crisis was not examined, since by 2008 all companies were adopting zero and by 2016 one. 1 3 V. Gelashvili et al. 474 F_RISK <= 0 \| F_RISK <= -0.01: 1 (410.0/6.0) \| F_RISK > -0.01 \| \| SALESGR <= -0.26 \| \| \| LOCAT = 0 \| \| \| \| AGE <= 11: 1 (3.0/1.0) \| \| \| \| AGE > 11 \| \| \| \| \| SALESEMP <= 10084.62: 1 (3.0/1.0) \| \| \| \| \| SALESEMP > 10084.62: 0 (6.0) \| \| \| LOCAT = 1 \| \| \| \| INDEBT <= 0.37 \| \| \| \| \| SALESEMP <= 22992.58: 0 (5.0) \| \| \| \| \| SALESEMP > 22992.58: 1 (2.0) \| \| \| \| INDEBT > 0.37: 1 (3.0) \| \| SALESGR > -0.26 \| \| \| SECTOR = 0 \| \| \| \| INDEBT <= 0.9: 1 (25.0/1.0) \| \| \| \| INDEBT > 0.9 \| \| \| \| \| SUBS <= 32229.26: 0 (9.0/1.0) \| \| \| \| \| SUBS > 32229.26: 1 (6.0) \| \| \| SECTOR = 1: 1 (166.0/27.0) F_RISK > 0 \| F_RISK <= 0.8: 0 (101.0/1.0) \| F_RISK > 0.8 \| \| SALESGR <= 0: 0 (4.0/1.0) \| \| SALESGR > 0: 1 (15.0/1.0) Fig. 3 Decision tree obtained by algorithm C4.5, 2016 (I). 5.2 C4.5 decision tree Source Own elaboration F_RISK <= 0 \| F_RISK <= -0.01: 1 (410.0/6.0) \| F_RISK > -0.01 \| \| SALESGR <= -0.26 \| \| \| LOCAT = 0 \| \| \| \| AGE <= 11: 1 (3.0/1.0) \| \| \| \| AGE > 11 \| \| \| \| \| SALESEMP <= 10084.62: 1 (3.0/1.0) \| \| \| \| \| SALESEMP > 10084.62: 0 (6.0) \| \| \| LOCAT = 1 \| \| \| \| INDEBT <= 0.37 \| \| \| \| \| SALESEMP <= 22992.58: 0 (5.0) \| \| \| \| \| SALESEMP > 22992.58: 1 (2.0) \| \| \| \| INDEBT > 0.37: 1 (3.0) \| \| SALESGR > -0.26 \| \| \| SECTOR = 0 \| \| \| \| INDEBT <= 0.9: 1 (25.0/1.0) \| \| \| \| INDEBT > 0.9 \| \| \| \| \| SUBS <= 32229.26: 0 (9.0/1.0) \| \| \| \| \| SUBS > 32229.26: 1 (6.0) \| \| \| SECTOR = 1: 1 (166.0/27.0) F_RISK > 0 \| F_RISK <= 0.8: 0 (101.0/1.0) \| F_RISK > 0.8 \| \| SALESGR <= 0: 0 (4.0/1.0) \| \| SALESGR > 0: 1 (15.0/1.0) Fig. 3 Decision tree obtained by algorithm C4.5, 2016 (I). Source Own elaboration F_RISK <= 0 \| F_RISK <= -0.01: 1 (410.0/6.0) \| F_RISK > -0.01 \| \| SALESGR <= -0.26 \| \| \| LOCAT = 0 \| \| \| \| AGE <= 11: 1 (3.0/1.0) \| \| \| \| AGE > 11 \| \| \| \| \| SALESEMP <= 10084.62: 1 (3.0/1.0) \| \| \| \| \| SALESEMP > 10084.62: 0 (6.0) \| \| \| LOCAT = 1 \| \| \| \| INDEBT <= 0.37 \| \| \| \| \| SALESEMP <= 22992.58: 0 (5.0) \| \| \| \| \| SALESEMP > 22992.58: 1 (2.0) \| \| \| \| INDEBT > 0.37: 1 (3.0) \| \| SALESGR > -0.26 \| \| \| SECTOR = 0 \| \| \| \| INDEBT <= 0.9: 1 (25.0/1.0) \| \| \| \| INDEBT > 0.9 \| \| \| \| \| SUBS <= 32229.26: 0 (9.0/1.0) \| \| \| \| \| SUBS > 32229.26: 1 (6.0) \| \| \| SECTOR = 1: 1 (166.0/27.0) F_RISK > 0 \| F_RISK <= 0.8: 0 (101.0/1.0) \| F_RISK > 0.8 \| \| SALESGR <= 0: 0 (4.0/1.0) \| \| SALESGR > 0: 1 (15.0/1.0) Fig. 3 Decision tree obtained by algorithm C4.5, 2016 (I). Source Own elaboration Fig. 5.2 C4.5 decision tree Finally, another important branch matches the following rule: if the values of financial risk ratio are between − 0.01 and 0.85, the sheltered employment centres are non-prof- itable firms (this rule is supported by 104 companies). Therefore, according to this method, the financial risk ratio is a key variable (Fig. 2).i i The next step is to see which rules qualify these companies to be profitable or not in 2016. For that year it was possible to analyse 758 sheltered employment centres. The good results in terms of cross-validation (91% classified correctly) justify the analysis of the patterns shown in the tree. Therefore, Figs. 3 and 4 are presented.i As the results show, there are two strong rules for class 1. The first rule shows that if the financial risk ratio of these companies is less than or equal to − 0.01, then the sheltered employment centres are profitable firms (this rule is supported by 410 firms, with six errors). The second rule shows that if the financial risk ratio of these companies is greater than − 0.01, the sales growth ratio is greater than − 0.26 and is a service company, then the sheltered employment centres are profitable firms (this rule is supported by 166 firms, with 27 errors). For non-profitable firms, the tree shows a strong rule with the following pattern: if the financial risk ratio is positive but lower than or equal to 0.8, then the sheltered employment centres belong to the non-profitable category (this pattern is satisfied by 101 firms with one error). Simi- larly to the previous decision tree, the key variable is the financial risk ratio. 5.2 C4.5 decision tree 3 Decision tree obtained by algorithm C4.5, 2016 (I). Source Own elaboration Fig. 3 Decision tree obtained by algorithm C4.5, 2016 (I). Source Own elaboration Fig. 4 Decision tree obtained by algorithm C4.5, 2016 (I). Source Own elaboration. The strongest rules are pointed out with an arrow Fig. 4 Decision tree obtained by algorithm C4.5, 2016 (I). Source Own elaboration. The strongest rules are pointed out with an arrow 1 3 What factors condition the financial viability of sheltered… 475 The branches with more sheltered employment centres are those that should be interpreted since they would reflect certain patterns as they are supported by a large number of cases. As it can be observed in the graph, the most important branch that classified profitable sheltered employment centres indicates that if the financial risk ratio of these companies is less than or equal to − 0.01, the sheltered employ- ment centres are profitable companies (this branch is supported by 331 firms, with 8 errors). Taking into account profitable firms, another strong branch shows that if the financial risk ratio is between − 0.01 and 0, the indebtedness ratio is greater than − 1.12 and the number of employees is more than 17, the sheltered employment centres are profitable (this branch is verified by 77 firms, with 9 errors). Finally, another important branch matches the following rule: if the values of financial risk ratio are between − 0.01 and 0.85, the sheltered employment centres are non-prof- itable firms (this rule is supported by 104 companies). Therefore, according to this method, the financial risk ratio is a key variable (Fig. 2).i the financial risk ratio is between 0.01 and 0, the indebtedness ratio is greater than − 1.12 and the number of employees is more than 17, the sheltered employment centres are profitable (this branch is verified by 77 firms, with 9 errors). Finally, another important branch matches the following rule: if the values of financial risk ratio are between − 0.01 and 0.85, the sheltered employment centres are non-prof- itable firms (this rule is supported by 104 companies). Therefore, according to this method, the financial risk ratio is a key variable (Fig. 2).i i − 1.12 and the number of employees is more than 17, the sheltered employment centres are profitable (this branch is verified by 77 firms, with 9 errors). 6 Conclusions This study aimed to investigate the financial viability of all the sheltered employ- ment centres operating in Spain through different accounting ratios. The results of the descriptive analyses show that sheltered employment centres are companies with a low rate of profitability, but their financial risk is low. The explanation could be that they are social firms looking for the labour integration of disabled people, with- out maximising their net income or incurring high risk. However, minimum profit- ability is needed to survive in the market. Based on the regression analysis and decision trees, the key variable for the prof- itability of these companies is financial risk. This variable analyses the relationship between the company’s financial expenses and sales and shows whether or not it is financially stable. Therefore, we can say that sheltered employment centres that can meet their financial expenses with their main income can have optimal profitability, as once the profitability of these companies increases their financial risk decreases. 1 3 3 476 V. Gelashvili et al. The variables like the sales growth for these companies is significant in the regression analysis. This shows that these companies are working hard to carry out their main activity, achieving profitability and viability through it. Although this result has not been confirmed in the decision tree results, we must take into account the time horizon examined in decision trees, which is one year, and in regression, we have worked with panel data for 12 years. The result of correlations and linear regression have shown that the variable economic and financial crisis is a key fac- tor in explaining the profitability of sheltered employment centres. Outcomes have shown that the profitability of these companies was lower during the economic and financial crisis. Even so, we could not verify this result in the decision tree analysis, since this variable was the dummy variable, which we have not managed to analyse through AI. Another variable that is important for the profitability of these compa- nies is the size. Because of the above, we can say that the managers of these companies have to take special care of those factors such as financial risk, size and sales growth (these last two are not confirmed by the results of the decision trees, but they are confirmed by the regression and correlation analysis), if they want to ensure the future viability of these companies. 6 Conclusions In addition to this, the governments of each region must invest more in equal opportunities for people with disabilities. In this sense, the sheltered employment centres are companies whose main challenge is to ensure paid work for people with disabilities and, at the same time, to stay in the market. Finally, we consider that the creation of employment for people with disabilities is highly significant for society in general as well as for the economy of the coun- try. In addition, social and labour integration helps people with disabilities have less social, mental, medical and financial problems. Therefore, the role of the sheltered employment centres is essential, especially in the current or future time of crisis that is destroying and will destroy millions of workplaces. Although the results show that the profitability of these companies has been affected in the time of the past eco- nomic and financial crisis, they have managed to survive and maintained employ- ment for people with disabilities. This shows the importance of these companies, which can help change the world even during the economic and financial crisis. i This study is not free of limitations. One of the limitations is the lack of up-to- date data, especially economic and financial data, and the complete list of sheltered employment centres. Up until now, there is no complete list of how many sheltered employment centres there are in Spain. Several autonomous communities publish their provisional list, but many do not, and this makes it difficult to find the data. The results obtained in this study can be a good basis in the future, when financial data will be available for all sheltered employment centres, to show if there are signifi- cant changes in their economic-financial structure. Consequently, the lack of current economic and financial data for these companies is one of the important limitations. Another limitation of the study is not being able to use more independent varia- bles. Therefore, for future research lines, we would like to investigate all sheltered employment centres, updated by the last year available, taking into account a wide range of already-existing ratios and new ones like a political party that governs an autonomous community. 6 Conclusions Apart from this, future research would entail analysing all social enterprises (sheltered employment centres, social cooperatives and insertion 1 3 What factors condition the financial viability of sheltered… 477 companies) in Spain to see what the main similarities and differences are between them. The analysis of data through other AI algorithms and statistical techniques for panel data is also proposed. Therefore, our main future research line would be to conduct a more in-depth study of these important topics in an attempt to present the updated financial data for social firms and especially for sheltered employment centres. Funding This work was supported in part by Universidad Complutense de Madrid under the Project San- tander-UCM PR87/19–22586. Funding This work was supported in part by Universidad Complutense de Madrid under the Project San- tander-UCM PR87/19–22586. 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 Com- mons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. 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/licen ses/by/4.0/. References Rev Prisma Soc 9:285–310 i Del Negro G (2012) Las empresas sociales de inserción frente a la exclusión social [Social insertion com- i i th f f i l l i ] R P i S 9 285 310 i Del Negro G (2012) Las empresas sociales de inserción frente a la exclusión panies in the face of social exclusion]. Rev Prisma Soc 9:285–310 p Díaz-Foncea M, Marcuello C (2014) Las empresas sociales en España: concepto y características [Social enterprises in Spain: concept and characteristics]. Revista Vasca de Economía Social, GEZKI Díaz-Foncea M, Marcuello C (2014) Las empresas sociales en España: concepto y características [Social enterprises in Spain: concept and characteristics]. Revista Vasca de Economía Social, GEZKI 8:143–164i Díaz Z, Sanchis A, Segovia MJ (2009) Analysis of financial instability by means of decision trees and lists. Bailly RO (ed) Emerging topics in macroeconomics. Nova Publishers, Hauppauge, pp 303–327 Dimitras AI, Zanakis S, Zopounidis C (1996) A survey of business failures with an emphasis on predi tion methods and industrial applications. Eur J Oper Res 90(6):487–513 Dlouhy S, Mitchell P (2015) Upcycling sheltered workshops: a revolutionary approach to transformin workshops into creative spaces. Ohio University Press. Athens Egido, I., Cerrillo, R. & Camina, A. (2009). La inclusión social y laboral de las personas con discapaci- dad intelectual mediante los programas de empleo con apoyo. Un reto para la Orientación [Social and labour inclusion of people with intellectual disability through supported employment pro- grams. A challenge for guidance]. REOP-Revista Española de Orientación y Psicopedagogía 20(2), 135–146. Enqvist J, Graham M, Nikkinen J (2014) The impact of working capital management on firm profitability in different business cycles: evidence from Finland Res Int Bus Finance 32:36 49 Enqvist J, Graham M, Nikkinen J (2014) The impact of working capital management on firm profitabili in different business cycles: evidence from Finland. Res Int Bus Finance 32:36–49 f Evert R, Wehman P, West M, Burgess S (2012) Do sheltered worksh for adults with autism spectrum disorder? Autism 16(1):87–94 f Evert R, Wehman P, West M, Burgess S (2012) Do sheltered workshops enhance employment outcomes for adults with autism spectrum disorder? Autism 16(1):87–94 with autism spectrum disorder? Autism 16(1):87–94 Fariñas J, Rodríguez L (1986) Rentabilidad y crecimiento de las grandes empresas industriales espa- ñolas en comparación con las de la CEE (1973–1982). [Profitability and growth of large Spanish industrial companies compared to those of the EEC (1973–1982)]. References Alonso IF (2013) Crisis financiera y medios públicos en España: impacto de los recortes presupuestarios en las radiotelevisiones públicas autonómicas históricas (2008–2012) [Financial crisis and pub- lic media in Spain: impact of budget cuts on historic regional public broadcasters (2008–2012)]. Derecom 12(2):17–30 Antón C, Cuadrado C, Rodríguez J (1990) Factores explicativos del crecimiento y la rentabilidad [Factors explaining growth and profitability]. Investig Econ 24(2):153–158 i Baldwin RE, di Mauro W (2020) Mitigating the COVID economic crisis. Centre for Economic Policy Research. https://iheid.tind.io/record/298223 p Baltagi B (2008) Econometric analysis of panel data. Wiley, Chichesteri Bank of Spain [Banco de España] (2017) Informe sobre la crisis financiera y bancaria en España, 2008– 2014 [Report on the financial and banking crisis in Spain, 2008–2014]. Other Publications, pp 1–257f Beirne K, Doorley K, Regan M, Roantree B, Tuda D (2020) The potential costs and distributional effect of Covid-19 related unemployment in Ireland. Budg Perspect. https://doi.org/10.26504/bp202101f Blustein DL, Duffy R, Ferreira JA, Cohen-Scali V, Cinamon RG, Allan BA (2020) Unemployment in the time of COVID-19: A research agenda. J Vocat Behav. https://doi.org/10.1016/j.jvb.2020.103436 Boardman J Grove B Perkins R Shepherd G (2003) Work and employment for people with psychiatric Blustein DL, Duffy R, Ferreira JA, Cohen-Scali V, Cinamon RG, Allan BA (2020) Unemployment in th time of COVID-19: A research agenda. J Vocat Behav. https://doi.org/10.1016/j.jvb.2020.103436 f time of COVID-19: A research agenda. J Vocat Behav. https://doi.org/10.1016/j.jvb.2020.103436 Boardman J, Grove B, Perkins R, Shepherd G (2003) Work and employment for people with psychiatric disabilities. Br J Psych 182(6):467–468i Breusch TS, Pagan AR (1980) The Lagrange multiplier test and its applications to model specification in econometrics. Rev Econ Stud 47(1):239–253 Calderón MJ, Calderón B (2012) Los Centros Especiales de Empleo como mecanismo de tránsito hacia el mercado de trabajo ordinario. [The Sheltered Employment Centres as a mechanism of transition to the ordinary labour market]. CIRIEC – España Rev Econ Pública, Soc Coop 75:223–250 Calvo J (2004) Centros Especiales de Empleo: situación jurídica actual y perspectivas de futuro [Shel- tered Employment Centres: current legal situation and future prospects]. Revista Vasca de Economía Social, GEZKI 139–160 Camacho-Miñano M, Pérez R (2012) Centros especiales de empleo: empresas para una sociedad compro- metida responsablemente [Social firms for a responsible committed society]. Revista de Responsa- bilidad Social de la Empresa 12:77–90 1 3 478 V. Gelashvili et al. Carmona P, Martínez J, Pozuelo J (2013) Diagnóstico económico-financiero de la empresa cooperativa. References Un estudio comparado de los años 2004 y 2007. [Economic and financial diagnosis of cooperative enterprises (A comparative study of the years 2004 and 2007)]. REVESCO Revista de Estudios Cooperativos 110:43–95 Carmona P, Martínez J, Pozuelo J (2013) Diagnóstico económico-financiero de la empresa cooperativa. Un estudio comparado de los años 2004 y 2007. [Economic and financial diagnosis of cooperative enterprises (A comparative study of the years 2004 and 2007)]. REVESCO Revista de Estudios Cooperativos 110:43–95 p CEPES. (2020). Análisis del impacto socioeconómico de los valores Y principios de la economía social en españa [Analysis of the socio-economic impact of the values and principles of the social econ- omy in Spain]. Confederación empresarial española de la economia social (CEPES). Madrid. Spain. CEPES. (2020). Análisis del impacto socioeconómico de los valores Y principios de la economía social en españa [Analysis of the socio-economic impact of the values and principles of the social econ- omy in Spain]. Confederación empresarial española de la economia social (CEPES). Madrid. Spain. Claver E, Llopis J, Molina JF (2002) Recursos de la empresa y pertenencia a un sector industrial: un estu- dio empírico de su influencia sobre la rentabilidad empresarial [Company resources and belonging to an industrial sector: an empirical study of its influence on business profitability]. Investigaciones Europeas de Dirección y Economía de la Empresa 8(1):39–52 Claver E, Llopis J, Molina JF (2002) Recursos de la empresa y pertenencia a un sector industrial: un estu- dio empírico de su influencia sobre la rentabilidad empresarial [Company resources and belonging to an industrial sector: an empirical study of its influence on business profitability]. Investigaciones Europeas de Dirección y Economía de la Empresa 8(1):39–52 Coibion, O., Gorodnichenko, Y. & Weber, M. (2020). Labor markets during the COVID-19 Crisis: a pre- liminary view (No. w27017). National Bureau of Economic Research. Cooney K, Nyssens M, O’Shaughnessy M, Defourny J (2016) Public policies and work integration social enterprises: the challenge of institutionalization in a neoliberal era. Nonprofit Policy Forum 7(4):415–433i ( ) Cowling M, Liu W, Ledger A (2012) Small business financing in the UK before and during the current financial crisis. Int Small Bus J 30(7):778–800 i Del Negro G (2012) Las empresas sociales de inserción frente a la exclusión social [Social insertion com- panies in the face of social exclusion]. References Información Comercial Española 636:87–104 Fernandes, N. (2020). Economic effects of coronavirus outbreak (COVID-19) on the world economy. SSRN 3557504. https://doi.org/10.2139/ssrn.3557504i p g Fernández, A. I. A., & García, M. O. (1991). Análisis del comportamiento económico-financiero de los sectores empresariales en España. [Analysis of the economic and financial behaviour of the business sectors in Spain]. ESIC MARKET, 113–128.i p Fernández E, Montes JM, Vázquez CJ (1996) Caracterización económico-financiera de la gran empresa industrial española según su rentabilidad [Economic and financial characterization of the large Spanish industrial company according to its profitability]. Revista Española de Financiación y Con- tabilidad 25(87):343–359i Galán J, Vecino J (1997) Las fuentes de rentabilidad de las empresas [The sources of profitability of com- panies]. Revista Europea de Dirección y Economía de la Empresa 6(1):21–36 Galer D (2014) “A place to work like any other?” Sheltered workshops in Canada, 1970–1985. Can Disabil Stud 3(2):1–30 Gannon B, Nolan B (2004) Disability and labor force participation in Ireland. Econ Soc Re 35(2):135–155 1 3 What factors condition the financial viability of sheltered… 479 García-Teruel J, Martinez-Solano P (2007) Effects of working capital management on SME profitability. Int J Manag Finance 3(2):164–177i Gelashvili V, Camacho-Miñano MM, Segovia-Vargas M (2015a) The profitability of socially reponsi- ble companies: public subsidies for sheltered employment centers. Ramon Llull J Appl Ethics 6:111–123 Gelashvili V, Segovia-Vargas MJ, Camacho-Miñano MM (2015b) Patrones de supervivencia para la gestión de los centros especiales de empleo [Survival patterns for managing Sheltered Employment Centres]. Revista de Estudios Empresariales Segunda Época 1:109–126i Gelashvili V, Camacho-Miñano MM, Segovia-Vargas MJ (2016) Análisis económico-financiero de los centros especiales de empleo de España [Economic and financial analysis of Sheltered Employment Centres in Spain]. Revista Española de Discapacidad (REDIS) 4(2):7–24i Gelashvili V, Aguilar EMP, Segovia-Vargas MJ, Camacho-Miñano MM (2018) The economic and finan- cial viability of sheltered employment centres. Manag Decis 57(9):2261–2283i Gill A, Mathur N (2011) The impact of board size, CEO duality, and corporate liquidity on the profitabil- ity of Canadian service firms. J Appl Finance Bank 1(3):83–95 i Giménez, S. (2012). Sinergias en tiempos de crisis: los centros especiales de empleo. [Synergies in times of crisis: special employment centres]. Quaderns per a la Inclusió Social, 2. Publicaciones URV. Tarragona, Spain. g p González Perez, A. (1997). La rentabilidad empresarial: evaluación empírica de sus factores determi- nantes. [Corporate profitability: an empirical assessment of its determinants]. References Colegio de Registra- dores de la Propiedad y Mercantiles de España; Centro de Estudios Registrales.i González A, Correa A, Acosta M (2002) Factores determinantes de la rentabilidad financiera de las pymes [Determining factors in the financial profitability of SMEs]. Revista Española de Finan- ciación y Contabilidad 31(112):395–429 y Greve, B. (2009). The labour market situation of disabled people in European countries and implemen- tation of employment policies: a summary of evidence from country reports and research stud- ies. Academic Network of European Disability Experts (ANED), 1–46.f Hoffman LC (2013) Employment opportunity or a discrimination dilemma: sheltered workshops and th employment of the disabled. Univ Pa J Law Soc Change 16:151–179f Hoffmann H, Richter D (2019) Supported employment in Switzerland—Are we on track? Psychiatr Rehabil J 43(1):72–75 ( ) siao C (2014) Analysis of panel data, 3rd edn. Cambridge University Press, England Iglesias S, López V, Rodríguez M (2007) Efecto empresa versus efecto sector Un estudio empírico en empresas Gallegas y Portuguesas. [Company effect versus sector effect An empirical study in Gali- cian and Portuguese companies]. Revista Galega de Economía 16(2):1–13 Isaac JF, Flores O, Hernández F, Torres CZL, García CI (2009) La Rentabilidad Económica (ROA) en las Pymes Exportadoras de Monterrey Nuevo León, México. [Economic Profitability (ROA) in Exporting SMEs in Monterrey Nuevo León, Mexico]. Observatorio de la Economía Latinoameri- cana 116:1–8 Jordán de Urríes, F.B. & Verdugo, M.A. (2010). Informe sobre la situación de los Centros Especiales de Empleo en España. [Report on the situation of the Sheltered Employment Centres in Spain]. INICO, Salamanca. José ML, Lancaster C, Stevens JL (1996) Corporate returns and cash conversion cycles. J Econ Financ 20(1):33–46i Kohlscheen E, Murcia Pabón A, Contreras J (2018) Determinants of bank profitability in emerging mar- kets. Bank for International Settlement (BIS) Working Paper 686i Kouser R, Bano T, Azeem M, Ul Hassan M (2012) Inter-relationship between profitability, growth and size: a case of non-financial companies from Pakistan. Pak J Commer Soc Sci 6(2):405–419 i Kokocinska M, Rekowski M (2013) Impact of the World Crisis on the employment and productivity of SMEs: the case of the largest European Economies. Revista de Economía Mundial 35:121–136fi Krupa T, Lagarde M, Carmichael K (2003) Transforming sheltered workshops into affirmative busi- nesses: an outcome evaluation. Psychiatr Rehabil J 26(4):359–367 Laloma, M. (2007). Empleo protegido en España: Análisis de la normativa legal y logros alcanzados. References [Protected employment in Spain: Analysis of legal regulations and achievement]. Ediciones Cinca, Promovida por Telefónica y CERMI. Madrid. Lang R, Kett M, Groce N, Trani JF (2011) Implementing the United Nations Convention on the rights of persons with disabilities: principles, implications, practice and limitations. ALTER-Eur J Disabil Res/Revue Européenne de Recherche sur le Handicap 5(3):206–220 1 1 3 V. Gelashvili et al. 480 López F, Mateos L, Vera J, Suárez I, Pérezgrueso B, José A, Brusca M (2014) Actividades e impactos de la empresa social. Estudio de los Centros Especiales de Empleo aragoneses. [Activities and impacts of the social enterprise. Study of the Sheltered Employment Centres in Aragon]. CIRIEC-Espana, Revista de Economía Pública, Social y Cooperativa 81:217–239 Lukas JF, Lizasoain L, Galarreta J, Etxeberria J (2018) Job satisfaction among disabled people in the shel-f Lukas JF, Lizasoain L, Galarreta J, Etxeberria J (2018) Job satisfaction among disabled peopl tered workshop: Differential analysis. J Appl Res Intellect Disabil 31(6):1174–1185 ain L, Galarreta J, Etxeberria J (2018) Job satisfaction f Luengo P, Renart M, Sánchez J, Vela J (2005) Evaluación de los factores determinantes de la Rentabilidad y Solvencia de la empresa Murciana. [Evaluation of the determining factors of the Profitability and Sol- vency of the Murcian company]. Cuadernos de Investigación 3:1–109i Mahbuba S, Farzana N (2013) Corporate social responsibility and profitability: a case study on Dutch Bangla Bank Ltd. Int J Bus Soc Res; 3(4):139–145i Mallender, J., Liger, Q., Tierney, R., Beresford, D., Eager, J., Speckesser, S. & Nafilyan, V. (2015). Reason- able accommodation and sheltered workshops for people with disabilities: costs and returns of invest- ments. Policy Department A: Economic and Scientific Policy, Directorate General for Internal Policies. European Parliament. Brussels. Malo MÁ (2003) Las personas con discapacidad en el mercado de trabajo español. [People with disabiliti in the Spanish labour market]. Revista del Ministerio de Trabajo e Inmigración 46:99–126 Manzano Martín, M. I., Redondo Martín, M. N. & Robles Paramio, M. P. (2016). Los centros especiales de empleo en Castilla y León, 2007–2013: efectos de la crisis en función de la personalidad jurídica que adoptan. [Special employment centres in Castile and Leon 2007–2013: effects of the crisis in relation to these centres´ registered legal personalities]. REVESCO: Revista de Estudios Cooperativos, (122), 191–218 Martínez J (2009) El empleo protegido: La importancia de los Centros Especiales de Empleo. [Sheltered employment: The importance of Sheltered Employment Centres]. References Boletín Centro de Investigación de Economía y Sociedad-CIES 69:1–15 Mendoza J, Román C, Hernández M (2019) La Economía Social y la contratación pública: la herramienta de los contratos reservados [The Social Economy and public procurement: the reserved procurements tool]. CIRIEC-España, Revista de Economía Pública, Social y Cooperativa 97:213–244 de los contratos reservados [The Social Economy and public procurement: the reserved tool]. CIRIEC-España, Revista de Economía Pública, Social y Cooperativa 97:213–244 IRIEC-España, Revista de Economía Pública, Social y Cooperativa 97:213–244 Migliore A, Mank D, Grossi T, Rogan P (2007) Integrated employment or sheltered workshops: preferenc of adults with intellectual disabilities, their families, and staff. J Vocat Rehabil 26(1):5–19 f Migliore, A. (2010). Sheltered workshops. International Encyclopedia of Rehabilitation. Center for Interna- tional Rehabilitation Research Information and Exchange (CIRRIE). Buffalo. USAi f Montalvo JG (2009) Financiación inmobiliaria, burbuja crediticia y crisis financiera. Lecciones a partir de la recesión de 2008–09. [Real estate financing, credit bubble and financial crisis. Lessons from the 2008– 09 recession]. Papeles de Economía Española 122:66–85i Monzón-Campos JL, Herrero-Montagud M (2016) Identificación y análisis de las características identi- tarias de la empresa social europea: aplicación a la realidad de los Centros Especiales de Empleo de la economía española. [Identification and analysis of the basic identity characteristics of European social enterprise: Application to the reality of Sheltered Employment Centres of the Spanish economy]. CIRIEC-España, Revista de Economía Pública, Social y Cooperativa 87:295–326fi i e: Application to the reality of Sheltered Employment C Morbey GK, Reithner RM (1990) How R&D affects sales growth, productivity and profitability. Res-Techn Manag 33(3):11–14 Parker Harris S, Owen R, Gould R (2012) Parity of participation in liberal welfare states: human rights, ne liberalism, disability and employment. Disabil Soc 27(6):823–836i Penabad CL, Sanfiz JMM, Manent JT (2019). Análisis económico y social de los centros especiales d empleo: un estudio en Galicia [Economic and social analysis of sheltered workshops: a study in Ga Penabad CL, Sanfiz JMM, Manent JT (2019). Análisis económico y social de los centros especiales de empleo: un estudio en Galicia [Economic and social analysis of sheltered workshops: a study in Gali- cia]. REVESCO: Revista de Estudios Cooperativos; (132):169–194. empleo: un estudio en Galicia [Economic and social analysis of sheltered workshops: a study in G cia]. REVESCO: Revista de Estudios Cooperativos; (132):169–194.li p y cia]. REVESCO: Revista de Estudios Cooperativos; (132):169–194.li cia]. References Morgan Kaufmann, San Francisco Yell ML, Katsiyannis A, Prince A (2017) Sheltered workshops: United States v. Rhode Island. Interv School Clinic 52(5):311–314 References REVESCO: Revista de Estudios Cooperativos; (132):169–194.li Pervan M, Visic J (2012) Influence of firm size on its business success. Cro Pervan M, Visic J (2012) Influence of firm size on its business success. Croat Oper Res Rev 3(1):213–223 li Quinlan JR (1993) C4.5 Programs for machine, Learning. California, Morgan Kaufmann Renko M, Parker Harris S, Caldwell K (2016) Entrepreneurial entry by people with disabilities. Int Sma Bus J 34(5):555–578 Retolaza J, Ruiz M, Araujo A (2007) Factores estratégicos de éxito de las empresas de inserción. [Strate- gic success factors for insertion companies]. CIRIEC-España, Revista de Economía Pública, Social y Cooperativa 59:61–89ifi Retolaza J, San-Jose L, Araujo A (2014) La eficiencia como reto de las empresas de inserción [The efficiency as a challenge of work insertion social enterprises]. REVESCO: Revista de Estudios Cooperativos; (115):159–185 1 3 What factors condition the financial viability of sheltered… 481 Rodríguez V, Malo MÁ, Cueto B (2012) Diferencias salariales por discapacidad y Centros Especiales de Empleo [Salary differences due to disability and Sheltered Employment Centres]. Cuadernos de Economía 35(98):100–116 Rodríguez V, Cueto B (2013) El trabajo de las personas con discapacidad ante la crisis. Revista Internacional de Organizaciones. [The work of people with disabilities in view of the crisis.]. Int J Organ 11:61–86 Rodríguez V (2017) El empleo de las personas con discapacidad en España: quo venis, quo vadis. [Employ- ment of people with disabilities in Spain: quo venis, quo vadis]. Panorama Social 26:41–55 Rubio AB, Aragón AS (2002) Factores explicativos del éxito competitive. Un estudio empírico en la pyme. [Explanatory factors for competitive success. An empirical study in the SME]. Cuadernos de Gestión 2(1):49–63 Rubio, F. (2003). La construcción social del empleo protegido: los Centros Especiales de Empleo [The social construction of protected employment: Sheltered Employment Centres]. Nómadas: Revista Crítica de Ciencias Sociales y Jurídicas; (8):1–10. Rumelt R (1991) How much does industry matter? Strateg Manag J 12(3):167–185 Rosen M, Bussone A, Dakunchak P, Cramp J (1993) Sheltered employment and the second generation wor shop. J Rehabil 59(1):30–34i Shahzad A, Sharfman M (2015) Corporate social performance and financial performance sample-selection issues. Bus Soc 1:1–30 Sanchis A, Segovia MJ, Gil JA, Heras A, Vilar JL (2007) Rough Sets and the role of the monetary policy in financial stability (macroeconomic problem) and the prediction of insolvency in insurance sector (microeconomic problem). Eur J Oper Res 181(3):1554–1573 Shepherd G (1989) The value of work in the 1980s. References Psychiatr Bull 13:231–233 Serrano C, Martín del Brio B (1993) Predicción de la crisis bancaria mediante el empleo de redes neuronales artificiales [Predicting the banking crisis by using artificial neural networks]. Span J Finance Account 22(74):153–176 Serer GL, Campillo JP, Ferrer EJV (2009) Modelización temporal de los ratios contables en la detección del fracaso empresarial de la PYME española [Time modelling of the accounting ratios for detec- tion of managerial failure in Spanish small and medium size enterprises]. Span J Finance Account 38(143):423–447f Schmalensee R (1985) Do markets differ much? Am Econ Rev 75(3):341–351i f Suarez J (2000) Caracterización económico-financiera de las empresas asturianas en función de su nivel de rentabilidad [Economic and financial characterization of Asturian companies according to their level of i Suarez J (2000) Caracterización económico-financiera de las empresas asturianas en función de su nivel de rentabilidad [Economic and financial characterization of Asturian companies according to their level of profitability]. RAE: Revista Asturiana de Economía; (18):191–222.i Suarez J (2000) Caracterización económico-financiera de las empresas ast rentabilidad [Economic and financial characterization of Asturian com profitability]. RAE: Revista Asturiana de Economía; (18):191–222.i i rentabilidad [Economic and financial characterization of Asturian companies according to their level of profitability]. RAE: Revista Asturiana de Economía; (18):191–222.i rentabilidad [Economic and financial characterization of Asturian co profitability]. RAE: Revista Asturiana de Economía; (18):191–222.i i p g profitability]. RAE: Revista Asturiana de Economía; (18):191–222.i i profitability]. RAE: Revista Asturiana de Economía; (18):191–222.i i Tarrés JP (2012) Los ratios financieros en la concesión de riesgo crediticio a la PYME familiar. [Financi i ratios in the granting of credit risk to family SMEs]. Cuadernos Prácticos de Empresa Familiar 3:23–37i soura, M. (2004). Corporate social responsibility and financial performance. Center for responsible business Working paper series UC Berkeley Tsoutsoura, M. (2004). Corporate social responsibility and financial performance. Center for responsib business. Working paper series. UC Berkeley. Visier L (1998) Relaciones laborales en los sistemas de trabajo protegido para personas minusválidas [Labour relations in sheltered work systems for the people with disabilities]. Revista Internacional del Trabajo 117(3):371–390i Waddock SA, Graves SB (1997) The corporate social performance–financial performance link. Strate Manag J 18(4):303–319 Whitehead C (1979) Sheltered workshops in the decade ahead: Better work and wages, or welfare. J Rehab 45(2):77–90 Witten IH, Frank E (2005) Data mining: practical machine learning tools and techniques, 2nd edn. Legislation consulted Law 13/1982, of 7 April, on the Social Integration of Disabled Persons. (LISMI) (BOE of 30 April). Royal Decree 2273/1985 of 4 December 1985, approving the Regulations on Sheltered Employment Centres as defined in Article 42 of Law 13/1982 of 7 April 1982 on the social integration of the disabled. (BOE 294, of 9 December). 1 3 V. Gelashvili et al. 482 Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Vera Gelashvili1 · María‑Jesús Segovia‑Vargas2 · María‑del‑Mar Camacho‑Miñano3 María‑Jesús Segovia‑Vargas mjsegovia@ccee.ucm.es María‑del‑Mar Camacho‑Miñano marcamacho@ccee.ucm.es María‑Jesús Segovia‑Vargas mjsegovia@ccee.ucm.es María‑Jesús Segovia‑Vargas mjsegovia@ccee.ucm.es María‑del‑Mar Camacho‑Miñano marcamacho@ccee.ucm.es María‑del‑Mar Camacho‑Miñano marcamacho@ccee.ucm.es 1 Department of Business Economics, Faculty of Legal and Social Sciences, King Juan Carlos University, Paseo de los Artilleros s/n, 28032 Madrid, Spain 1 Department of Business Economics, Faculty of Legal and Social Sciences, King Juan Carlos University, Paseo de los Artilleros s/n, 28032 Madrid, Spain 2 Department of Financial and Actuarial Economics & Statistics, Faculty of Economics and Business, Complutense University of Madrid, Campus de Somosaguas, Pozuelo de Alarcó 28223 Madrid, Spain 2 Department of Financial and Actuarial Economics & Statistics, Faculty of Economics and Business, Complutense University of Madrid, Campus de Somosaguas, Pozuelo de Alarcón 28223 Madrid, Spain 2 Department of Financial and Actuarial Economics & Statistics, Faculty of Economics and Business, Complutense University of Madrid, Campus de Somosaguas, Pozuelo de Alarcón, 28223 Madrid, Spain 3 Accounting & Finance Department, Faculty of Economics and Business, Complutense University of Madrid, Campus de Somosaguas, Pozuelo de Alarcón, 28223 Madrid, Spain 3 Accounting & Finance Department, Faculty of Economics and Business, Complutense University of Madrid, Campus de Somosaguas, Pozuelo de Alarcón, 28223 Madrid, Spain 3 Accounting & Finance Department, Faculty of Economics and Business, Complutense University of Madrid, Campus de Somosaguas, Pozuelo de Alarcón, 28223 Madrid, Spain 1 3 1 3 1 3
https://openalex.org/W2971124475	https://scholarsarchive.library.albany.edu/cgi/viewcontent.cgi?article=1025&context=edpsych_fac_scholar	English	null	Psychometric properties of a Korean version of the Perceived Stress Scale (PSS) in a military sample	BMC psychology	2,019	cc-by	9,451	University at Albany, State University of New York University at Albany, State University of New York Scholars Archive Scholars Archive Educational & Counseling Psychology Faculty Scholarship Educational & Counseling Psychology 2019 Psychometric properties of a Korean version of the Perceived Psychometric properties of a Korean version of the Perceived Stress Scale (PSS) in a military sample Stress Scale (PSS) in a military sample Kimberly F. Colvin University at Albany, State University of New York Sung Yong Park University at Albany, State University of New York The University at Albany community has made this article openly available. Please share Please share how this access benefits you. Follow this and additional works at: https://scholarsarchive.library.albany.edu/edpsych_fac_scholar Recommended Citation Recommended Citation Colvin, Kimberly F. and Park, Sung Yong, "Psychometric properties of a Korean version of the Perceived Stress Scale (PSS) in a military sample" (2019). Educational & Counseling Psychology Faculty Scholarship. 25. https://scholarsarchive.library.albany.edu/edpsych_fac_scholar/25 This work is licensed under a Creative Commons Attribution 4.0 International License. This Article is brought to you for free and open access by the Educational & Counseling Psychology at Scholars Archive. It has been accepted for inclusion in Educational & Counseling Psychology Faculty Scholarship by an authorized administrator of Scholars Archive Educational & Counseling Psychology The University at Albany community has made this article openly available. Please share Please share how this access benefits you. Follow this and additional works at: https://scholarsarchive.library.albany.edu/edpsych_fac © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Background: Perceived stress reflects a person’s feeling of how much stress the individual is under at a given time. The Perceived Stress Scale (PSS) is a popular instrument measuring the extent to which individuals perceive situations in their life as excessive relative to the ability to cope. Based on a literature review, however, several issues related to the scale remain: (a) the dimensionality is not established, (b) little information about the individual items exists, and (c) much research is based on university student samples. To address these, this study evaluated the psychometric properties of the Korean version of the Perceived Stress Scale (KPSS) using a military sample. Methods: This study was conducted in South Korea with 373 military personnel, aged 19–30 years. Both classical test theory (CTT) and the Rasch rating scale model were used to examine the psychometric properties of the KPSS, including factor structure, concurrent validity, reliability, and item analyses. Results: Internal consistency reliability for the overall and negative/positive perception subscales was.85, .85 and .86, respectively. Based on Rasch reliability, person and item reliability were .82 and .98, respectively. Person and item separation were 2.13 and 7.19, respectively. Concurrent validity was established, with significantly positive association with the measures of depression and negative association with the measure of life satisfaction. Findings from the CFA suggested that a bifactor model with two group factors was the best fit to the observed data. The RSM showed that all but one item had acceptable infit and outfit statistics, and item difficulty ranged from −.73 to 1.22. Besides, the RSM showed positive and moderate inter-item correlations ranging from .42 to .75. RSM showed that all but one item had acceptable infit and outfit statistics, and item difficulty ranged from .73 to 1.22. Besides, the RSM showed positive and moderate inter-item correlations ranging from .42 to .75. Conclusions: The results provided evidence that a 10-item Korean version of the Perceived Stress Scale was a reliable and valid scale to measure perceived stress in military samples. Conclusions: The results provided evidence that a 10-item Korean version of the Perceived Stress Scale was a reliable and valid scale to measure perceived stress in military samples. rds: Factor structure, Confirmatory factor analysis, Rasch rating scale model, Stress, Young adult Psychometric properties of a Korean version of the Perceived Stress Scale (PSS) in a military sample Sung Yong Park* and Kimberly F. Colvin Sung Yong Park* and Kimberly F. Colvin Backgrounds coping and perceived ability to cope with stressful events, such that levels of perceived stress are measured relative to a subject’s judgment of own coping ability [1]. Due to its widespread use and discussion in the literature, PSS continues to be utilized and tested for the psychometric properties and validity. The scale allows respondents in secondary school and above to indicate levels of perceived stress as a result of its simple questionnaire format and short, direct questions [2]. The validity and psychometric properties of the Korean version of PSS were examined in the case of military personnel in South Korea. The Perceived Stress Scale (PSS) is a self-report instru- ment for measuring the extent to which persons perceive situations in their life as excessively stressful relative to their ability to cope [1]. The PSS was designed for measur- ing individuals with at least a junior high school education level. It incorporates the theoretical perspective that varying levels of perceived stress can affect the actual experience of stressful events into a widely applicable instrument [1]. Perceived stress has also been linked with The PSS was developed to measure global perceived stress experienced outside the bounds of a specific life event and focused on the cognitive appraisal process that * Correspondence: spark25@albany.edu Department of Educational and Counseling Psychology, University at Albany, State University of New York, ED231, 1400 Washington Avenue, Albany, NY 12222, USA * Correspondence: spark25@albany.edu Department of Educational and Counseling Psychology, University at Albany, State University of New York, ED231, 1400 Washington Avenue, Albany, NY 12222, USA Recommended Citation Recommended Citation Colvin, Kimberly F. and Park, Sung Yong, "Psychometric properties of a Korean version of the Perceived Stress Scale (PSS) in a military sample" (2019). Educational & Counseling Psychology Faculty Scholarship. 25. https://scholarsarchive.library.albany.edu/edpsych_fac_scholar/25 This work is licensed under a Creative Commons Attribution 4.0 International License. This work is licensed under a Creative Commons Attribution 4.0 International License. This Article is brought to you for free and open access by the Educational & Counseling Psychology at Scholars Archive. It has been accepted for inclusion in Educational & Counseling Psychology Faculty Scholarship by an authorized administrator of Scholars Archive. Please see Terms of Use. For more information, please contact scholarsarchive@albany.edu. e is brought to you for free and open access by the Educational & Counseling Psychology at Scholars has been accepted for inclusion in Educational & Counseling Psychology Faculty Scholarship by an administrator of Scholars Archive. Please see Terms of Use. For more information, please contact scholarsarchive@albany.edu. Park and Colvin BMC Psychology (2019) 7:58 https://doi.org/10.1186/s40359-019-0334-8 rticle is distributed under the terms of the Creative Commons Attribution Page 2 of 11 Park and Colvin BMC Psychology (2019) 7:58 Park and Colvin BMC Psychology (2019) 7:58 Park and Colvin BMC Psychology (2019) 7:58 Much attention has been given to the dimensionality of the PSS. For example, although factor analyses in a study [3] proposed the two-factor model as best fitting the factor structure of the original 14-item PSS and PSS with 10 items, they argued that the distinction between the two factors was irrelevant for purposes of measuring stress. Several following studies have revealed that a two-factor structure ([19, 20]; see [21]) was more accept- able than a one-factor structure for PSS 14 and 10. One study, supported by confirmatory factor analysis (CFA), demonstrated that a second-order factor model was acceptable as an alternative way to use the total score of the two-factor PSS, where “stress” and “counter-stress” are lower-order factors and “perceived stress” is the higher-order factor [12]. The two-factor and second- order factor models do not contain an underlying single construct for stress that explains responses to each of the observed indicators. Recently, a few studies have proposed a bifactor model that addresses these limita- tions of traditional models used to evaluate the structure of multidimensional constructs [22–25]. As shown in Fig. 1, the bifactor model is different from a second- order model in that subgroup factors are not only in- cluded by a general factor underlying all item variables but are also uncorrelated and unique [26]. includes the appraisal of the stressor and individual’s perceived coping ability [1]. The original PSS included a set of 14 items, consisting of (a) seven items with nega- tive perception of uncontrollability, unpredictability, and inability to cope, and (b) seven items with positive per- ception of capability to handle stress successfully [1]. This was reduced to 10 items after four were found to exhibit low factor loadings [3]. The PSS has achieved wide acceptance and has been administered to a wide range of study participants. Participants At a South Korean military institution, 375 air force sol- diers in South Korea, ranging in age from 19 to 30, com- pleted a survey. All participants were male, and the mean length of military service was 17.24 months (SD = 4.17). Regarding the highest level of educational, of the respondents, 5.9% were high school graduates, 84.5% college students, 7.2% college graduates, and 1.9% had attended or completed graduate school. Consent forms and a research description were sent to the air force. After they consented to participate, they completed a paper version of the survey; the survey took approxi- mately 10 min to complete. All but two of the 375 participants who provided complete responses on the KPSS were included in our analyses. Two participants with more than fifteen missing values in responses to all instruments in this survey were excluded from these analyses, yielding a sample size of 373. The PSS measures general stress and is relatively inde- pendent of content that is specific to any particular population [1]. Indeed, the PSS has been empirically val- idated with various populations as described above, but most studies used college students or workers (e.g., professionals and teachers; [21]). Therefore, it is still ne- cessary to validate the PSS with more diverse popula- tions and in various cultures [21]. For example, although several empirical studies revealed that many soldiers are exposed to stress that impacts on mental health condi- tions [30, 31], no instruments assessing soldiers’ stress levels have been validated in this population. As far as we know, the current study is the first validation study on the PSS for military personnel, in any language. Specifically, South Korean soldiers were and are facing mental and physical health problems, considering the situation in South Korea, where South and North Korea are confronting each other as a divided country, and where the situation changes frequently depending on the interests of the neighboring powers. In addition, given the rigid military culture, soldiers experience difficulties, such as conflicts between ranks, work-related conflicts, and an oppressed group life [30]. Therefore, the Korean military population should be considered distinct from the population of Korean college students who experience stress related to future career plans, intense academic workload and achievement, interpersonal relationships, finance, and personal appearance [32]. Participants In this data set, there were 4 missing values across 10 items and 373 survey respondents, yielding a very low percentage (0.1%) for missing values. Although the Little’s missing completely at random test was signifi- cant, it was considered a missing at random pattern based on a visual inspection that showed there are no clusters of missing values. The 4 missing data were imputed using the Expectation-Maximization (EM) algorithm in SPSS Version 24 [33]. The first author conducted the mental health project for Korean military soldiers with a research team; he then obtained the data from a military counselor of the Republic of Korean Air Force (ROKAF) 10th Wing. The current analysis and publication of the data were approved by the ROKAF 10th Wing’s security review. More than 30 language versions of the PSS have been translated and adapted, including Spanish, Portuguese, Mexican Spanish, Chile Spanish, Danish, Norwegian, Swedish, Hebrew, Greek, Italian, German, Moroccan, Bulgarian, Hungarian, Ser- bian, Korean, Japanese, Mandarin, Taiwanese Mandarin, Thai, Bengali, Malayalam, Tamil, Sinhala, Polish, Lithuanian, Turkish, Russian, Urdu, Arabic, and Finnish [4], and validated on diverse samples, including, for ex- ample, university students [1, 5, 6], the general population [3, 7], survivors of suicide [8], adults that participated in a community smoking-cessation program [1], adults with asthma [9], cardiac patients [10, 11], women with breast cancer [12], pregnant and postpartum women [13], teachers [14, 15], workers [14, 16], policewomen [17], and depressed outpatients [18]. Fig. 1 The bifactor model with a general stress factor and two group factors Fig. 1 The bifactor model with a general stress factor and two group factors Fig. 1 The bifactor model with a general stress factor and two group factors Page 3 of 11 Page 3 of 11 Park and Colvin BMC Psychology (2019) 7:58 Park and Colvin BMC Psychology (2019) 7:58 Park and Colvin BMC Psychology Even though the PSS has been widely used, there is relatively little in the extant literature about the PSS’s psychometric properties [20], nor about the use of the PSS for a Korean population. To our knowledge, only a few studies translated the original PSS into Korean and evaluated its psychometric properties [27–29]. For ex- ample, Park and Seo [29] translated the original 14-item PSS into Korean and evaluated the psychometric proper- ties of the Korean version of PSS (KPSS) with Korean college student samples through both exploratory factor analysis (EFA) and CFA. Their findings revealed that the two-factor structure best fit the data belonging to both positive and negative perception of stress subscales. In addition, as evidence of concurrent validity, negative var- iables, including depression, anxiety, and negative affect, were positively related to the negative perception factor in the subscales, while the positive perception factor was associated with positive affect. model, to the KPSS10. The Rasch analysis allowed an examination of the performance of individual items on the KPSS10, for which there is little documentation. Then, internal consistency for the items was investigated by both CTT and Rasch reliability statistics. Finally, the concurrent validity of the KPSS10 was examined by comparing scores with those from measures of emo- tional distress (i.e., depression) and subjective well-being (i.e., life satisfaction). Perceived stress scale The goal of the present study was to examine the psy- chometric properties of a Korean version of the PSS with 10 items (KPSS10) when administered in a military set- ting, with a specific interest in the dimensionality of the scale. Using classical test theory (CTT) and factor analysis, we evaluated the factor structure of the scale. To further examine dimensionality, we fit the rating scale model (RSM), a polytomous extension of the Rasch The Perceived Stress Scale (PSS; [1]) is a self-report measure consisting of 14 items purported to measure “how unpredictable, uncontrollable, and overloaded respondents find their lives” during the past month [3]. The original version consists of seven negatively stated items and seven positively stated items [1]. Two shortened forms of the PSS 14 were also subsequently developed and Park and Colvin BMC Psychology (2019) 7:58 Page 4 of 11 Park and Colvin BMC Psychology the Korean version of the SWLS, which has been trans- lated and evaluated for psychometric properties in a Korean population [38]. In Kim’s study [38], the Cronbach’s alpha was .84, and the current sample yielded the alpha coefficients of .86. validated [3] —the PSS 10 (six negative items and four positive items) and the PSS 4 (two negative items and two positive items). Lee’s review [21] found that the psycho- metric properties of the PSS 10 were more effective in measuring the perceived stress than those of the PSS 14 and 4 items. Center for epidemiologic studies depression scale Center for epidemiologic studies depression scale There is a growing body of evidence identifying the stress-depression connection (see [21]). To establish concurrent validity, a comparison was made with the CES-D, a self-report scale designed to measure the current level of depressive symptoms for general popula- tion [34]. The scale consists of 20 items using a 4-point scale ranging from 0 (Rarely or none of the time, less than 1 day) to 3 (Most or all of the time, 5–7 days). For example, item 1 is “I was bothered by things that usually don’t bother me.” The CES-D has four subfactors: depressive affect, positive affect, somatic symptoms, and interpersonal difficulties [34]. We used the Korean version of the CES-D translated and validated by Chon, Choi, and Yang [35], which demonstrated the same fac- tor structure with the original CES-D and high internal consistency (α = .91). The internal consistency reliability estimate in the present study was .90. g y We used CFA to examine the dimensionality of the KPSS10. Based on the factor structures reported in the PSS literature, four different factor configurations of the KPSS10 were extracted: (a) a single-factor unidimen- sional model that all 10 items are assumed to measure a single stress factor [8], (b) a two-factor model with two covariate factors [19–21, 27, 29], (c) a bifactor model with a general stress factor and a nuisance factor con- sisting of the five reversed items [23], and (d) a bifactor model with a general stress factor accounting for the commonality shared by the items and two subfactors reflecting the unique variance not accounted for by the general stress factor, as seen in Fig. 1 [22, 24, 25]. The bifactor model allowed us to test whether the KPSS10 was a general measure of perceived stress with another specific underlying dimension. Data analysis h CTT The Korean version translated and evaluated by Park and Seo [29] is made up of five negatively stated items (i.e., 1, 2, 3, 11, and 14 in the original PSS 14) and five positively stated items (i.e., 4, 5, 6, 7, and 10 in the original version) depending on factor loadings over 0.5 among the full 14 items. Participants indicate their re- sponse to the KPSS using a 5-point Likert-type scale ranging from 0 (never) to 4 (very often). To produce the total score, the five positively stated items in question- naires were reversed, thus, higher scores indicate higher perceived stress. For the current items used in the study see the Additional file 1. Park and Seo [29] found that a two-factor solution, with positive and negative percep- tion as the subfactors, was supported (α = .74 for positive perception and .77 for negative perception). Concurrent validity was established by moderate correlations with depression, anxiety, negative affect, and positive affect. y Both CTT and Rasch RSM were used to evaluate the psychometric properties of the KPSS10, including factor structure, concurrent validity, reliability, and item analyses. Reliability of the KPSS10 was reported in two ways using Cronbach’s alpha and item-total correlation. In general, a Cronbach’s alpha value of 0.70 is recom- mended as a minimum acceptable criterion for internal consistency [39]. Furthermore, Rasch-based person and item reliability and separation were reported. The person reliability index refers to the expected replicability of person placement if this sample was given other items measuring the same construct, while the item reliability index indicates the replicability of item placements resulting from other samples who behaved in the same way [40]. Both reliability indices range from 0 to 1, with values greater than .90 for items and .80 for persons being regarded as acceptable [40]. The separation index indicates an estimate of the spread or separation of items or persons along the measured variable, with ad- equate separation in persons or items values of at least 2.0 regarded as acceptable [40]. Concurrent validity was investigated by evaluating the correlational relationship with measures of negative emotion (e.g., depression), using the CES-D and subjective well-being (e.g., satisfac- tion with life), using the SWLS. We expected the KPSS10 to correlate positively with the CES-D and to correlate negatively with the SWLS. Satisfaction with life scale As previous literature suggested that perceived stress was predictive of low levels of life satisfaction [36], the Satisfaction with Life Scale (SWLS; [37]) was also administered to assess concurrent validity. The SWLS was designed to assess cognitive judgments of life satis- faction using a short instrument with only five items. The responses to each item (e.g., “So far I have gotten the important things I want in life”) range from 1 (strongly disagree) to 7 (strongly agree), where higher scores indicate higher levels of life satisfaction. We used To examine the adequacy of model-fit, we reported the comparative fit index (CFI) representing incremental fit, standardized root-mean-square residual (SRMR) for absolute fit, and root-mean-square error of approximation Page 5 of 11 Park and Colvin BMC Psychology (2019) 7:58 Park and Colvin BMC Psychology (2019) 7:58 Park and Colvin BMC Psychology two models. The person reliability is .85 for the PCM and .82 for the RSM, and the item reliability is .98 for both PCM and RSM. Given the similarity of threshold spacing, fit indices, and the theoretical argument that the set of response categories is the same across items, we decided to fit the more parsimonious RSM, rather than the PCM. (RMSEA) identifying parsimonious fit. In our data, Mardia’s multivariate kurtosis coefficient of 17.40 indi- cated the absence of multivariate normality [41]. Given this result and the ordinal nature (a five-point Likert-type scale) of the KPSS, robust maximum likelihood estimation was used in the CFA analyses in EQS 6.1 [42], instead of using maximum likelihood estimator. (RMSEA) identifying parsimonious fit. In our data, Mardia’s multivariate kurtosis coefficient of 17.40 indi- cated the absence of multivariate normality [41]. Given this result and the ordinal nature (a five-point Likert-type scale) of the KPSS, robust maximum likelihood estimation was used in the CFA analyses in EQS 6.1 [42], instead of using maximum likelihood estimator. Finally, after fitting the RSM we used WINSTEPS to conduct a principal components analysis of the stan- dardized residuals [49]. If the underlying factor fit by the RSM accounts for most of the variance in the original data, then it is expected that the resulting components of residuals will represent noise. The results of the analysis can be used to separate items into groups to de- termine if some of the unaccounted variance (variance not accounted for in the RSM) can be explained by an additional factor or factors. Satisfaction with life scale Next, as an indicator of unidimensionality used in a bifactor model, we computed the explained common variance (ECV) that is a ratio of common variance attributable to the general factor (ECV; [43]). High ECV values indicate data that have a strong general factor compared to other specific group factors; when values are greater than .70, the common variance can be con- sidered as unidimensional [43]. To further explore dimensionality and assess the relative location of items and respondents, we used WINSTEPS version 4.01 [44] to fit the rating scale model (RSM; [40, 45]) to our data, while accounting for the dimensionality as found in the factor analyses. Con- trary to CTT, Rasch analyses enable researchers to analyze the properties of items, such as item difficulty and item discrimination. The RSM is an extension of the Rasch model for polytomous data [45, 46]. The RSM es- timates the location of the respondents and the KPSS10 items on the same scale, in this case, the scale of per- ceived stress. The RSM manipulates only one set of threshold parameters of across all items on the scale, in- dicating a common rating scale structure for all items [40]. For each item, the overall location of the item is es- timated, along with the location of the thresholds, that is the location on the scale where the likelihood of a re- sponse in a particular category changes. In other words, the scale is divided into sections based on the most likely response. Therefore, the RSM is suitable when one expects that psychological distances between categories are the same across all items [47]. Results Reliability y As shown in Table 1, Cronbach’s alpha coefficients indi- cated good internal consistency for the overall KPSS10 (α = .85), for the negative perception subscale (α = .85), and for the positive perception subscale (α = .86) [40]. Cronbach’s alpha if item deleted for all ten items ranged from .83 to .87. Item 5 was the only item that would yield a slightly higher alpha if removed. Item-total corre- lations for individual items and each factor were also in- vestigated, and ranged from .45 to .75, showing over the generally adopted cutoff criteria (>.40; [50]). Therefore, all items appeared worthy of retention. These two types of statistics on internal consistency reliability indicate that the KPSS10 contains items that are particularly intercorrelated. Regarding the results from Rasch-based reliability, both person and item reliability indices were acceptable: .82 and .98, respectively. In addition, results pertaining to person and item separation were 2.13 and 7.16, respectively. In general, these reliability results in- dicate good separation in the KPSS10 for both persons and items [40]. However, to conduct the Rasch analysis, we had two choices: the RSM and the partial credit model (PCM). While the PCM allows for the item response categories to differ across items, in the case of Likert-type items a strong case needs to be made to use the PCM over the RSM [48]. Theoretically, we would argue that because respondents were presented with the same response options across all items, the set of responses should be treated the same across all items. However, because it is possible that there was an interaction between the re- spondents and the items leading to a discrepant use of response categories across items, we initially fit both the RSM and PCM. The ordering and spacing of the thresh- olds remained roughly the same across all items in both the PCM and the RSM, indicating that the data would support the selection of the RSM. We next compared the person and item reliability index obtained from the Table 1 Descriptive Statistics and Correlations of Measures Measure 1 2 3 4 5 M SD α 1. KPSS Total 1 2.27 .57 .85 2. KPSS Negative perception .84 1 2.16 .71 .85 3. KPSS Positive perception .81 .36 1 2.38 .66 .86 4. CES-D .62 .56 .45 1 .52 .41 .90 5. Life Satisfaction −.49 −.42 −.38 −.47 1 4.27 1.19 .86 Note. N = 373. Results Reliability All correlation coefficients are significant at p < .01; KPSS = Korean version of the Perceived Stress Scale with 10 items; KPSS negative perception indicates the negatively worded items, and KPSS positive perception means the positively worded items; The KPSS positive items were reverse-coded Table 1 Descriptive Statistics and Correlations of Measures Measure 1 2 3 4 5 M SD α 1. KPSS Total 1 2.27 .57 .85 2. KPSS Negative perception .84 1 2.16 .71 .85 3. KPSS Positive perception .81 .36 1 2.38 .66 .86 4. CES-D .62 .56 .45 1 .52 .41 .90 5. Life Satisfaction −.49 −.42 −.38 −.47 1 4.27 1.19 .86 Note. N = 373. All correlation coefficients are significant at p < .01; KPSS = Korean version of the Perceived Stress Scale with 10 items; KPSS negative perception indicates the negatively worded items, and KPSS positive perception means the positively worded items; The KPSS positive items were reverse-coded Note. N = 373. All correlation coefficients are significant at p < .01; KPSS = Korean version of the Perceived Stress Scale with 10 items; KPSS negative perception indicates the negatively worded items, and KPSS positive perception means the positively worded items; The KPSS positive items were reverse-coded Note. N = 373. All correlation coefficients are significant at p < .01; KPSS = Korean version of the Perceived Stress Scale with 10 items; KPSS negative perception indicates the negatively worded items, and KPSS positive perception means the positively worded items; The KPSS positive items were reverse-coded Page 6 of 11 Page 6 of 11 Park and Colvin BMC Psychology (20 Concurrent validity the positive or negative wording of the item, we con- ducted Rasch analyses focusing on the KPSS10 as a whole in a confirmatory manner, rather than on the two subscales. The two group factors could be considered as superficial and not meaningful [3] because they repre- sented the direction of the wording of the items rather than the content of the item; in addition, most research and clinical contexts generally use a single summed PSS score. Reckase [53] argued that item estimates are de- fensible when the first component of principal compo- nents analysis accounts for at least 20% of the variance; in our data the first component accounted for 44% of the variance. To further confirm that a Rasch analysis on all ten items at once was appropriate, we compared the relative item positions and person estimates from an RSM analysis of all ten items with those from analyses of the positive and negative items separately. The person estimates from an RSM analysis with only the positive items correlated .92 with the person estimates based on all ten items, while the estimates based on the negative items correlated .73 with the estimates based on all ten items. The relative positioning of the items when cali- brated separately as positive and negative items were the same as when all ten items were calibrated simultan- eously. These results, coupled with the fact that the first eigenvalue accounts for 44% of the variance, well over the minimum recommended of 20%, indicated that a single RSM analysis of all ten items was appropriate to generate item and person estimates. As expected, we found statistically significant positive associations between the KPSS total scores and two sub- scale scores and depression: CES-D (r = .61, .56, and 44, respectively, p < .01), as well as a negative association with life satisfaction: SWLS (r = −.48, −.42, and −.37, respectively, p < .01). All correlation coefficients ranged between .37 and .61, which are considered to be medium or strong correlations [51]. In sum, these correlations provide evidence of concurrent validity for the KPSS10 (see Table 1). Confirmatory factor analysis (CFA) Results from the CFA supported a bifactor model for the KPSS10. Fit indices mentioned above for the factor structure including one-factor, two-factor, and bifactor models are provided in Table 2. p The initial one-factor CFA model had poor model fit using Hu and Bentler’s joint criteria [52]. Although the two-factor model yielded an acceptable fit to the data, the bifactor model (A) with the general stress factor and one nuisance factor demonstrated better fit as compared to the two-factor model, ΔS-B χ2 (4) = 35.416, p < .001. All factor loadings were significant for the general and the nuisance factor except for item 5. Considering this, we tried to conduct the second bifactor model (B) in which all 10 items load onto the general stress factor as well as on the two group factors. The bifactor model (B) yielded better fit, S-B χ2 (25) = 52.051, p < .001, CFI = .979, SRMR = .039, RMSEA = .054 [.033, .074], and shown a significant improvement in fit indices, as com- pared to the first bifactor model (A), ΔS-B χ2 (5) = 30.418, p < .001. In contrast to the bifactor model (A), all factor loadings were significant for the general and the two group factors (all ps < .001), as shown in Fig. 1. Our findings supported the bifactor model with the general stress factor and the two group factors labeled as “negative perception and positive perception” as the best fitting model. Rasch rating scale model The RSM was fit to the data to evaluate item perform- ance of the KPSS10 with the military sample of respon- dents based on item difficulty, separation index, item misfit detection, item discrimination, and Pearson point measure correlation (PTMEA). The results are provided in Table 3. Ten items are arranged in item difficulty values, from most difficult item to respond to at the top (item 3), to the least difficult item to respond to at the bottom (item 5). For instance, the item 3 “Cannot overcome pilling up difficulties” was more difficult to endorse, referring to higher stress severity, whereas item 5 “Dealing successfully with day-to-day problems and annoyances” was the most likely to obtain a response of “never,” meaning lower stress severity. In addition, the item separation index of 7.19 is also a good separation in the KPSS items and indicates that these items define adequately a distinct hierarchy of item difficulty [54]. The ECV in our supported model was .45, indicating that the general stress factor accounted for almost half the common variance. Because the bifactor model (B) yielded the best fit and the two group factors related to Table 2 Confirmatory Factor Analyses of the KPSS Model S-B χ2 df CFI SRMR RMSEA [90% CI] One-factor model 480.914 35 .649 .157 .185 [.170, .200] Two-factor model 117.885 34 .934 .063 .081 [.065, .097] Bifactor model (A) 82.469 30 .959 .033 .069 [.051, .086] Bifactor model (B) 52.051 25 .979 .039 .054 [.033, .074] Note. CFI Comparative fit index, SRMR standardized root-mean-square residual, RMSEA room-mean-square error of approximation, CI confidence interval; the bifactor model (A) includes a general stress factor and a nuisance factor, while the bifactor model (B) consists of a general stress factor and two group factors Next, item misfit was evaluated using the following Rasch fit indicators. Mean-square fit statistics (MNSQ) were examined; specifically, infit (weighted mean square) and outfit (unweighted mean square) determine how well each item contributes to defining one common con- struct. Rasch rating scale model In the case of a Likert scale, the expected MNSQ Park and Colvin BMC Psychology (2019) 7:58 Page 7 of 11 Table 3 Rasch Rating Scale Model (RSM) Analyses KPSS Item Difficulty Estimated Discrimination Infit MNSQ Outfit MNSQ PTMEA Item 3 (14) Cannot overcome mounting difficulties 1.22 1.23 0.82 0.80 0.66 Item 1 (2) Unable to control the important things 1.03 .93 1.10 1.14 0.60 Item 6 (5) Effectively cope with important changes in your life 0.02 1.21 0.82 0.81 0.66 Item 7 (6) Confident about your ability to handle your problems 0.02 1.18 0.84 0.83 0.68 Item 9 (10) Feel that you are on top of things −0.17 1.33 0.70 0.69 0.70 Item 4 (1) Upset because of something that happened unexpectedly −0.23 .79 1.21 1.22 0.64 Item 2 (3) Feel nervous or stressed −0.24 .84 1.16 1.14 0.68 Item 8 (7) Feel that things are going your way −0.45 1.38 0.63 0.64 0.75 Item 10 (11) Feel angry because of things that happened that are outside of your control −0.48 .82 1.15 1.17 0.59 Item 5 (4) Deal successfully with day-to-day problems and annoyances −0.73 .40 1.58 1.66 0.42 Note. KPSS10 is Korean version of the Perceived Stress Scale 10 items; numbers in parentheses refer to the original number of the PSS-14 [1]; difficulty means perceived stress severity level; infit/outfit statistics in bold are larger than 1.4 and indicate misfit; PTMEA = the point-measure correlation Note. KPSS10 is Korean version of the Perceived Stress Scale 10 items; numbers in parentheses refer to the original number of the PSS-14 [1]; difficulty means perceived stress severity level; infit/outfit statistics in bold are larger than 1.4 and indicate misfit; PTMEA = the point-measure correlation value is 1.0, infit and outfit values from 0.6 to 1.4 are within acceptable bounds for Likert scale measurements, item discrimination estimates post-hoc [54]. The estimates of the item discrimination distributed all around from .40 (item 5) to 1.38 (item 8), including five under-discriminat- ing items and five over-discriminating items shown in Table 3. Finally, the Probability Curves revealed that the 5-point Likert-type scale in the KPSS10 were ordered as expected, indicating that the differentiation of each cat- egory along the attribute measurement was verified (see Fig. 2). Rasch rating scale model value is 1.0, infit and outfit values from 0.6 to 1.4 are within acceptable bounds for Likert scale measurements, indicating construct homogeneity with other items in a scale [47, 55]. MNSQ values greater than 1.4 may indi- cate a lack of construct homogeneity with other items in a scale, while values less than 0.6 may indicate item redundancy [47, 55]. As shown in the Table 3, all items of the KPSS10 had acceptable infit and outfit statistics between 0.60 and 1.40, except for only one item (item 5) revealing both infit and outfit statistics larger than 1.4. Moreover, most items on the KPSS10 had positive, moderate, inter-item correlations ranging from .42 to .75, indicating that all items on the KPSS10 function as intended (see the PTMEA in Table 3; [54]). Although Rasch models are assumed that all item discrimina- tions are regarded as equal, empirical item discrimi- nations are never equal so that WINSTEPS produces value is 1.0, infit and outfit values from 0.6 to 1.4 are within acceptable bounds for Likert scale measurements, indicating construct homogeneity with other items in a scale [47, 55]. MNSQ values greater than 1.4 may indi- cate a lack of construct homogeneity with other items in a scale, while values less than 0.6 may indicate item redundancy [47, 55]. As shown in the Table 3, all items of the KPSS10 had acceptable infit and outfit statistics between 0.60 and 1.40, except for only one item (item 5) revealing both infit and outfit statistics larger than 1.4. Moreover, most items on the KPSS10 had positive, moderate, inter-item correlations ranging from .42 to .75, indicating that all items on the KPSS10 function as intended (see the PTMEA in Table 3; [54]). Although Rasch models are assumed that all item discrimina- tions are regarded as equal, empirical item discrimi- nations are never equal so that WINSTEPS produces Finally, the principal components analysis of the standardized residuals revealed that of the unexplained variance 35% was attributable to the first component, in- dicating that the component is accounting for more than just noise. In fact, the first component separated the 10 items into two distinct groups: the five items with posi- tive wording and the five items with negative wording. The remaining components accounted for roughly equal Fig. 2 The relative category probability curves for items of the KPSS10 Fig. Appropriateness of item difficulty for military samples Appropriateness of item difficulty for military samples Because the Rasch model estimates person and item lo- cations on the same scale, we can investigate whether the item difficulty level of the KPSS10 is appropriate for the current sample. If the KPSS-10 was appropriately targeted for the level of the sample being tested, there should be considerable overlap between the range of the person trait measures and the total test information curve and some of the item category probability curves. As shown in Fig. 3, the test information curve and the items, depicted by each item’s individual category prob- ability curves, were aligned with most of the current sample’s locations along the stress scale (M = −1.45, SD = 1.46, minimum = −6.60, maximum = 2.99). The one exception is for the few people with the lowest estimate of stress, −6.60, where the items were not targeted to the low end of the stress scale. This means the KPSS10 items could measure a more severe level of perceived stress than was needed for this nonclinical sample of South Korean soldiers, but still more than adequately targeted almost the entire sample. Rasch rating scale model 2 The relative category probability curves for items of the KPSS10 Page 8 of 11 Page 8 of 11 Park and Colvin BMC Psychology (2019) 7:5 Park and Colvin BMC Psychology variance, indicating no additional conceptual dimensions to the data. valid instrument measuring perceived stress within military samples in South Korea. The CFA analyses to compare four competing models’ goodness-of-fit demonstrated that a bifactor model with a general stress factor and two group factors was the best fit to our data. Regarding two group factors, our model was more consistent with the bifactor model supported by previous studies [22, 25], rather than Perera et al.’s [23] model with only one nuisance factor consisting of four negatively worded items. In addition to the general stress factor reflecting the overlap across all items, two group factors in our findings indicate that the five negatively worded items of the KPSS10 were loaded onto the negative perception factor and the posi- tively worded remaining five items were loaded onto the positive perception factor. It is worthy of note that when all the items’ loadings on the general factor will be stronger than those on the group factors, a bifactor structure could be viewed as mostly unidimensional. This underlying hypothesis was not supported by factor loadings in our bifactor model; items loaded more strongly on the group factors than on the general stress factor. The principal components analysis on the resid- uals from the RSM analysis demonstrated the same underlying factor structure as the CFA: one general stress factor with the unexplained variance dividing the items into the positive and negatively worded items. Conclusions original study [3]. Concurrent validity of the full and the subscales of the KPSS was established, with significantly positive associations with the measures of depression and negative association with the measure of life satisfaction. In other words, high KPSS10 scores were correlated with reports of increased depression and dis- satisfaction. These findings were consistent with the prior findings showing significant correlations with measures of distress and subjective well-being constructs [3, 22, 56]. Contrary to the earlier findings, however, the two subscales correlated positively with each other. This finding was consistent with the validation study based on Korean college students [29]. original study [3]. Concurrent validity of the full and the subscales of the KPSS was established, with significantly positive associations with the measures of depression and negative association with the measure of life satisfaction. In other words, high KPSS10 scores were correlated with reports of increased depression and dis- satisfaction. These findings were consistent with the prior findings showing significant correlations with measures of distress and subjective well-being constructs [3, 22, 56]. Contrary to the earlier findings, however, the two subscales correlated positively with each other. This finding was consistent with the validation study based on Korean college students [29]. In a South Korean military sample, the Korean version of the PSS proved to be a reliable instrument with con- current validity. We found evidence that while a bifactor model best fit the data, the data are unidimensional enough to conduct a Rasch analysis. To our knowledge, this is the first study to use the Rasch rating scale model to investigate the PSS. The results indicated a good sep- aration in the KPSS for both persons and items, demon- strated that the KPSS is sensitive enough to discriminate between high and low stressed respondents. Given that the PSS was designed to measure the degree to which individuals perceive their lives as stressful in both clin- ical and non-clinical populations, it is not surprising that we found the Korean version of the PSS to be an ad- equate measure of perceived stress in our non-clinical sample of soldiers. To our knowledge, this is the first study to use the Rasch RSM to investigate the PSS. Our findings were in- dicated by the adequate MNSQ fit of almost items, evenly separated item difficulty, acceptable discrimin- ation, and fairly strong positive PTMEA correlations. Conclusions According to the results showing good separation in the KPSS10 for both persons and items, the KPSS10 may be sensitive enough to discriminate between high and low stressed respondents [54]. The majority of the respon- dents’ scale locations overlapped with the item category probability curves in the middle and at the lower end of the scale. Given that the PSS was designed to measure the degree to which individuals perceive their lives as stressful in both clinical and non-clinical population [1], this finding can be regarded as reasonable, concluding that the KPSS10 items are designed to measure more severe levels of perceived stress than was observed in our non-clinical sample of soldiers. Abbreviations CES- D: Center for Epidemiological Studies; CFI: Comparative fit index; CTT: Classical test theory; ECV: Explained common variance; KPSS: Korean version of Perceived Stress Scale; MNSQ: Mean-square fit statistics; PSS: Perceived Stress Scale; PTMEA: Pearson point measure correlation; RMSEA: Root-mean-square error of approximation; RSM: Rating scale model; SRMR: Standardized root-mean-square residual; SWLS: Satisfaction with Life Scale Acknowledgements h h k l d The authors acknowledge and thank the military personnel for their participation. We are also thankful to Seon-Young Bak, who is a military counselor, and Dr. Kyungmi Kim for collecting the data. There are some limitations to be considered in inter- preting the findings. First, the KPSS10 [29] that we used in this study, is a translated and validated version that is adapted for the Korean population. In this process, the KPSS10 included two items not present in the original English PSS10 [3] so that it will be somewhat difficult to compare directly with other previous findings. Second, considering all the items and all subfactors, positive cor- relations were found, justifying computing a total score of the KPSS10. Another limitation of our study is that is we could not compare KPSS10 scores to another meas- ure of stress to assess convergent validity, instead, we established concurrent validity with expected significant correlations among the mental health measures in this study. Finally, it may be difficult to generalize from our findings, because of our particular sample. The military sample in the study was not representative of the mili- tary population in other countries because of the nature of military service in South Korea, in which participation is mandatory. The KPSS10 was also only administered at one-time point, and the sample only included males, therefore, future studies will have to assess test-retest reliability and include women in the study sample. Availability of data and materials The dataset analyzed during the current study is not publicly available because the data are controlled by the Republic of Korea Air Force 10th Fighter Wing but are available from the corresponding author on reasonable request. Authors’ contributions bl f h SP was responsible for the data analyses and interpretation and wrote the manuscript. KC revised the manuscript and supervised all processes. Both authors read and approved the final manuscript. Not applicable. Not applicable. Discussion In this study, we investigated the psychometric proper- ties of the Korean version of the Perceived Stress Scales in a sample of military personnel in South Korea, using the KPSS 10 items translated and validated by Park and Seo [29]. Overall, both CTT analyses and Rasch model- ing provided evidence that the KPSS10 is a reliable and Regarding the reliability, the overall and two subscales’ Cronbach’s alpha coefficients (.85, .85, and .86, respect- ively) indicate that the KPSS10 had a good internal consistency reliability for the Korean military sample. Our findings were higher than those observed in the Fig. 3 Items’ category probability curves and the total test information curve Page 9 of 11 Park and Colvin BMC Psychology (2019) 7:58 Park and Colvin BMC Psychology (2019) 7:58 Page 9 of 11 Additional file Additional file 1: Korean Version of the Perceived Stress Scale (KPSS). (PDF 168 kb) Ethics approval and consent to participate The survey data collection and publication were approved by the Security Review Board of the Republic of Korea Air Force (ROKAF) 10th Fighter Wing, South Korea (Protocol number: Intelligence and Security Command – 8960 & 5890), referenced by ROKAF regulation 3–21, Article 201–2 “Security review approval procedure”, and “Department of personnel management-9651.” All soldiers who enrolled in the study gave oral and written consent to partici- pate in the study. The study and current analysis were approved by the IRB at the University at Albany, SUNY. (18-X-233-01). References 1. Cohen S, Kamarck T, Mermelstein R. A global measure of perceived stress. J Health Soc Behav. 1983;24:385–96. 25. Wu SM, Amtmann D. Psychometric evaluation of the perceived stress scale in multiple sclerosis. ISRN Rehabil. 2013;2013:1–9. 2. Karam F, Bérard A, Sheehy O, Huneau MC, Briggs G, Chambers C, Einarson A, Johnson D, Kao K, Koren G, Martin B. Reliability and validity of the 4-item perceived stress scale among pregnant women: results from the OTIS antidepressants study. Res Nurs Health. 2012;35(4):363–75. 26. Gustafsson JE, Balke G. General and specific abilities as predictors of school achievement. Multivariate Behav Res. 1993;28(4):407–34. 27. Lee EH, Chung BY, Suh CH, Jung JY. Korean version of the perceived stress scale (PSS-14, 10 and 4): psychometric evaluation in patients with chronic disease. Scand J Caring Sci. 2015;29(1):183–92. 3. Cohen S, Williamson GM. Perceived stress in a probability sample of the United States. In: Spacapan S, Oskamp S, editors. The social psychology of health: Claremont symposium on applied social psychology. Newbury Park, CA: Sage; 1988. p. 31–67. 28. Hong GR, Kang HK, Oh E, Park Y, Kim H. Reliability and validity of the Korean version of the perceived stress Scale-10 (K-PSS-10) in older adults. Res Gerontol Nurs. 2015:45–51. 4. Cohen’s laboratory for the Study of Stress, Immunity, and Disease. Dr. Cohen’s Scales. 2018. http://www.psy.cmu.edu/~scohen/index.html. Accessed 15 Jun 2018. 4. Cohen’s laboratory for the Study of Stress, Immunity, and Disease. Dr. Cohen’s Scales. 2018. http://www.psy.cmu.edu/~scohen/index.html. Accessed 15 Jun 2018. 29. Park JO, Seo YS. Validation of the perceived stress scale (PSS) on samples of Korean university students. Korean J Psychol. 2010;29(3):611–29. 30. Koo SS. A study on mental health of new generation soldiers. Mental Health Soc Work. 2006;24:64–93. 5. Örücü MÇ, Demir A. Psychometric evaluation of perceived stress scale for Turkish university students. Stress Health. 2009;25(1):103–9. 5. Örücü MÇ, Demir A. Psychometric evaluation of perceived stress scale for Turkish university students. Stress Health. 2009;25(1):103–9. 31. Martin PD, Williamson DA, Alfonso AJ, Ryan DH. Psychological adjustment during army basic training. Mil Med. 2006;171(2):157–60. 6. Roberti JW, Harrington LN, Storch EA. Further psychometric support for the 10-item version of the perceived stress scale. J Coll Couns. 2006; 9(2):135–47. 32. Lee DH, Kang S, Yum S. A qualitative assessment of personal and academic stressors among Korean college students: an exploratory study. Coll Stud J. 2005;39(3):442–9. 7. Andreou E, Alexopoulos EC, Lionis C, Varvogli L, Gnardellis C, Chrousos GP, Darviri C. References Perceived stress scale: reliability and validity study in Greece. Int J Environ Res Public Health. 2011;8(8):3287–98. 33. IBM Corp. Released. IBM SPSS statistics for windows, version 24.0. Armonk: IBM Corp; 2016. 8. Mitchell AM, Crane PA, Kim Y. Perceived stress in survivors of suicide: psychometric properties of the perceived stress scale. Res Nurs Health. 2008; 31(6):576–85. 34. Radloff LS. The CES-D scale: a self-report depression scale for research in the general population. Appl Psychol Meas. 1977;1(3):385–401. 35. Chon KK, Choi SC, Yang BC. Integrated adaptation of CES-D in Korea. Korean J Health Psychol. 2001;6(1):59–76. 9. Sharp LK, Kimmel LG, Kee R, Saltoun C, Chang CH. Assessing the perceived stress scale for African American adults with asthma and low literacy. J Asthma. 2007;44(4):311–6. 36. Abolghasemi A, Varaniyab ST. Resilience and perceived stress: predictors of life satisfaction in the students of success and failure. Procedia Soc Behav Sci. 2010;5:748–52. 10. Leung DY, Lam TH, Chan SS. Three versions of perceived stress scale: validation in a sample of Chinese cardiac patients who smoke. BMC Public Health. 2010;10(1):513–20. 37. Diener E, Emmons RA, Larsen RJ, Griffin S. The satisfaction with life scale. J Pers Assess. 1985;49(1):71–5. 11. Pbert L, Doerfler LA, DeCosimo D. An evaluation of the perceived stress scale in two clinical populations. J Psychopathol Behav Assess. 1992;14(4): 363–75. 38. Kim JH. The relationship between life satisfaction/life satisfaction expectancy and stress/well-being: an application of motivational states theory. Korean J Health Psychol. 2007;12:325–45. 12. Golden-Kreutz DM, Browne MW, Frierson GM, Andersen BL. Assessing stress in cancer patients: a second-order factor analysis model for the perceived stress scale. Assessment. 2004;11(3):216–23. y 9. Kline P. A psychometrics primer. London: Free Association Books; 40. Wright BD, Masters GN. Rating scale analysis: Rasch measurement. Chicago: Mesa Press; 1982. 13. Chaaya M, Osman H, Naassan G, Mahfoud Z. Validation of the Arabic version of the Cohen perceived stress scale (PSS-10) among pregnant and postpartum women. BMC Psychiatry. 2010;10(1):111–8. 41. Mardia KV. Measures of multivariate skewness and kurtosis with applications. Biometrika. 1970;57(3):519–30. 42. Bentler PM, Wu EJ. EQS 6.1 for Windows: Users' guide. Encino: Mulivariate Software; 2003. 14. Almadi T, Cathers I, Mansour AM, Chow CM. An Arabic version of the perceived stress scale: translation and validation study. Int J Nurs Stud. 2012; 49(1):84–9. 43. Rodriguez A, Reise SP, Haviland MG. Applying bifactor statistical indices in the evaluation of psychological measures. J Pers Assess. 2016;98(3): 223–37. 15. Competing interests The authors declare that they have no competing interests. Competing interests The authors declare that they have no competing interests. Received: 14 December 2018 Accepted: 21 August 2019 Received: 14 December 2018 Accepted: 21 August 2019 24. Reis D, Lehr D, Heber E, Ebert DD. The German version of the Perceived Stress Scale (PSS-10): evaluation of dimensionality, validity, and measurement invariance with exploratory and confirmatory bifactor modeling. Assessment. 2017; doi:1073191117715731. References Reis RS, Hino AA, Rodriguez Añez CR. Perceived stress scale: reliability and validity study in Brazil. J Health Psychol. 2010;15(1):107–14. 44. Linacre JM. Winsteps® Rasch measurement computer program, Version 4.1 Beaverton: Winsteps.com; 2017. 16. Lesage FX, Berjot S, Deschamps F. Psychometric properties of the French versions of the perceived stress scale. Int J Occup Med Environ Health. 2012; 25(2):178–84. 45. Andrich D. Rasch models for measurement. Newbury Park, CA: Sage Publications; 1988. 17. Wang Z, Chen J, Boyd JE, Zhang H, Jia X, Qiu J, Xiao Z. Psychometric properties of the Chinese version of the perceived stress scale in policewomen. PLoS One. 2011;6(12):e28610. 46. Embretson SE, Reise SP. Item response theory for psychologists. Mahwah, NJ: L. Erlbaum; 2000. 18. Wongpakaran N, Wongpakaran T. The Thai version of the PSS-10: an investigation of its psychometric properties. Biopsychosoc Med. 2010; 4(1):6. 47. Bond TG, Fox CM. Applying the Rasch model: fundamental measurement in the human sciences. 3rd ed. Mahwah, NJ: L. Erlbaum; 2015. 48. Wright BD. Model selection: rating scale model (RSM) or partial credit model (PCM)? Rasch Meas Trans. 1998;12(3):641–2. 48. Wright BD. Model selection: rating scale model (RSM) or par (PCM)? Rasch Meas Trans. 1998;12(3):641–2. 19. Hewitt PL, Flett GL, Mosher SW. The perceived stress scale: factor structure and relation to depression symptoms in a psychiatric sample. J Psychopathol Behav Assess. 1992;14(3):247–57. 49. Linacre JM. Detecting multidimensionality: which residual data-type works best? J Outcome Meas. 1998;2:266–83. 50. Ware JE Jr, Gandek B. Methods for testing data quality, scaling assumptions, and reliability: the IQOLA project approach. J Clin Epidemiol. 1998;51(11):945–52. 20. Taylor JM. Psychometric analysis of the ten-item perceived stress scale. Psychol Assess. 2015;27(1):90–101. 21. Lee EH. Review of the psychometric evidence of the perceived stress scale. Asian Nurs Res. 2012;6(4):121–7. 51. Cohen J. Statistical power analysis for the behavioral sciences. 2nd ed. New York, NY: Routledge; 1988. 22. Jovanović V, Gavrilov-Jerković V. More than a (negative) feeling: validity of the perceived stress scale in Serbian clinical and non-clinical samples. Psihologija. 2015;48(1):5–18. 52. Hu LT, Bentler PM. Cutoff criteria for fit indexes in covariance structure analysis: conventional criteria versus new alternatives. Struct Equ Modeling. 1999;6(1):1–55. 22. Jovanović V, Gavrilov-Jerković V. More than a (negative) feeling: validity of the perceived stress scale in Serbian clinical and non-clinical samples. Psihologija. 2015;48(1):5–18. 52. Hu LT, Bentler PM. Cutoff criteria for fit indexes in covariance structure analysis: conventional criteria versus new alternatives. Struct Equ Modeling. Consent for publication Not applicable. Consent for publication Not applicable. Consent for publication Not applicable. Page 10 of 11 Page 10 of 11 Page 10 of 11 Park and Colvin BMC Psychology (2019) 7:58 Park and Colvin BMC Psychology (2019) 7:58 Park and Colvin BMC Psychology Competing interests 23. Perera MJ, Brintz CE, Birnbaum-Weitzman O, Penedo FJ, Gallo LC, Gonzalez P, Gouskova N, Isasi CR, Navas-Nacher EL, Perreira KM, Roesch SC. Factor structure of the perceived stress Scale-10 (PSS) across English and Spanish language responders in the HCHS/SOL sociocultural ancillary study. Psychol Assess. 2017;29(3):320–8. References 1999;6(1):1–55. Page 11 of 11 Park and Colvin BMC Psychology (2019) 7:58 Park and Colvin BMC Psychology (2019) 7:58 Park and Colvin BMC Psychology 53. Reckase MD. Unifactor latent trait models applied to multifactor tests: results and implications. J Edu Stat. 1979;4(3):207–30. 54. Linacre JM. A user’s guide to WINSTEPS. Chicago, IL: Winsteps.com; 2005a. 55. Wright BD, Linacre JM, Gustafson JE, Martin-Lof P. Reasonable mean-square fit values. Rasch Meas Trans. 1994;8(3):370. 56. Klein EM, Brähler E, Dreier M, Reinecke L, Müller KW, Schmutzer G, Wölfling K, Beutel ME. The German version of the perceived stress scale– psychometric characteristics in a representative German community sample. BMC Psychiatry. 2016;16(1):159. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. published maps and institutional affiliations.
W4280517384.txt	https://www.researchsquare.com/article/rs-1626115/latest.pdf	en		The antimicrobial effects of PLGA microspheres containing the antimicrobial peptide OP-145 on clinically isolated pathogens in bone infections	Research Square (Research Square)	2,022	cc-by	3,662	The antimicrobial effects of PLGA microspheres containing the antimicrobial peptide OP-145 on clinically isolated pathogens in bone infections Ye Cheng The Second Affiliated Hospital of Zhejiang Chinese Medical University Jianhua Qin Hangzhou Plastic Surgery Hospital Yuliang Huang The Second Affiliated Hospital of Zhejiang Chinese Medical University Tianyu Wang (  tianyuwang83@hotmail.com ) The Second Affiliated Hospital of Zhejiang Chinese Medical University Research Article Keywords: antimicrobial effects, PLGA, antimicrobial peptides, infection Posted Date: May 18th, 2022 DOI: https://doi.org/10.21203/rs.3.rs-1626115/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/14 Abstract Infection after fracture is a significant problem for the healing of fractures. Antimicrobial peptides combined with PLGA (poly-lactic-co-glycolic acid) microspheres can open new horizons for treating bone infections. Twenty rats in the control group were treated with physiologic saline solution after surgery, and 20 rats in the treatment group were treated with OP-145 PLGA microspheres and vancomycin after surgery. The biofilms from treatment and control groups were analyzed by fluorescence microscopy. Blood samples were collected at 12, 24, 36, 48, and 72 h. OP-145 PLGA microspheres showed significant inhibitory effects on clinically isolated strains (P < 0.05) and there were significant differences in serum CRP (P < 0.05) levels compared with control group. In conclusion, OP-145 PLGA microspheres could slowly release antimicrobial peptides and significantly reduce biofilm formation and levels of inflammatory factors. Introduction Proteomics can help determine the relationship between protein structure and function. In recent years, more and more polypeptide and protein substances have been used in the diagnosis and treatment, or as vaccines to prevent various diseases[1]. Compared with small-molecule drugs, polypeptide and protein molecular drugs are easily degraded enzymatically, with a short biological half-life[2]. In addition, their poor diffusion and distribution coefficient make it difficult for them to penetrate through biological barriers and lipid membranes. Therefore, the question is how these biological materials can be effectively delivered to the corresponding target points, making their preparation a research hotspot. At present, most biological drugs are used by injection in the clinic, necessitating frequent dosing and leading to patients’ poor compliance[3]. Small-molecule drugs combined with the biodegradable microsphere systems can effectively prevent the drug’s quick degradation in the body and help target effective parts in the body, with long-term, slow release of drugs[4]. Existing peptide and protein microspheres mainly include injection of sustained-release preparations and oral and nasal inhalers[5]. Infection after fracture is a significant problem for the healing of fractures[6]. Antimicrobial peptides combined with PLGA (poly-lactic-co-glycolic acid) microspheres can open new horizons for treating bone infections[7]. This study aimed to study the antibacterial effect of polypeptide-protein microspheres on clinically isolated pathogens in vivo and determine their cytotoxicity. Therefore, we determined bacterial counts in the biofilm and used confocal laser scanning microscopy (CLSM) to verify the effects of polypeptideprotein PLGA microspheres on clinically isolated pathogens. Meanwhile, cytotoxicity on osteoblasts was tested to verify the clinical value of polypeptide-protein PLGA microspheres. Material And Methods Strains isolation and culture conditions Page 2/14 One patient with clinical infection after fracture at the Xinhua Hospital was selected to isolate the clinical pathogenic bacteria. Bacterial specimens were obtained after washing the wound’s surface vigorously by saline solution, followed by debridement of superficial exudates. The microbiological culture was carried out under microaerophilic conditions for seven days. The bacterial specimens were tested by pyrosequencing analysis of bacterial diversity, and antimicrobial susceptibility tests were tested by the disk diffusion method[8]. Peptide OP-145 (acetyl-IGKEFKRIVERIKRFLRELVRPLR-amide) was synthesized by Shanghai Apeptide Co. Ltd. (Shanghai, China). OP-145 was puriﬁed by high performance liquid chromatography, and the identity was verified by SDS-PAGE. In addition, the purity of OP-145 (>95%) and mass were conﬁrmed by electrospray ionization mass spectrometry. Preparation of PLGA microspheres OP-145 containing PLGA microspheres was prepared by previously described methods[9]. Briefly, 200 mg of PLGA was dissolved in methylene chloride (45%, w/v). The OP-145 solution was then added to the polymer solution to form the organic phase and mixed via vortexing. The organic phase containing both the polymer and drug was then added at once to 100 mL of 0.35% (w/v) PVA solution (0.22-μm membrane filtered) to form an oil-in-water (o/w) emulsion using a homogenizer set at 14,000 rpm for 1 min. Next, leuprolide acetate microspheres with large particle sizes were prepared using a lower emulsification/size reduction force (9000 rpm for 1 min). The resultant o/w emulsion was stirred at 400 rpm for 3 h under vacuum at room temperature to allow microsphere solidification and solvent evaporation. The resultant microspheres were collected by centrifugation, washed using distilled water, and freeze-dried using a vacuum manifold. The drug release of OP-145 from PLGA microspheres was calculated by the methods of Garner J [10]. To investigate the morphology of the PLGA microparticles, the PLGA microparticles were first treated by freeze-drying, observed under a scanning electron microscope (SEM, NNS-450, FEI, USA)[11], and analyzed by x-ray photoelectron spectroscopy (XPS)[12] . Animals Sixty adult female Sprague‐Dawley (SD) rats (weighing 260–320 g) were used in this study. Twenty rats in the control group were treated with physiologic saline solution after surgery, and 20 rats in the treatment group were treated with OP-145 PLGA microspheres and vancomycin after surgery. All surgery was performed under sodium pentobarbital anesthesia, and euthanasia was accomplished with CO2 Model validation The rats were anesthetized via inhalation of 2% isoflurane. The surgical procedure was as described in the previous work with few modifications[13]. The skin was incised at the proximal end of the specimen to facilitate loading directly and longitudinally over the lateral upper forelimb. Dissection continued up to the fascia of the biceps and brachialis, which were then retracted to reveal the midshaft humerus. A Page 3/14 femoral fracture was created with an electric drill, and normal saline was not ejected after the drill stopped. An inoculum of clinically isolated bacteria (1×105 CFU/mL) in 2 mL of normal saline solution was pipetted into the femur space[14]. The surgical site was closed with Dexon 5-0 sutures. The weights of the rats were recorded at 0, 1, 2, 3, 7, and 14 d. C-reactive protein (CRP) levels were measured by enzymelinked immunosorbent assay (ELISA) at 12, 24, 36, 48, and 72 h[15]. Detection of biofilm formation The biofilms were harvested from euthanized rats on days 1, 2, and 3 to test biofilm formation in the present rat model. The specimens were prepared according to the previous methods. The specimens were fixed in 2.5% glutaraldehyde at 4°C for 1 h and stained with LIVE/DEAD staining [16]. The biofilms were observed under a Nikon 80i microscope equipped with an argon laser with an excitation wavelength of 488 nm (green fluorescence). All the images were captured and saved using Nis-Elements AR software (Nikon, Tokyo, Japan). The integrated optical density (IOD) of biofilm was evaluated using the Image-ProPlus[17]. MTT assay The cytotoxicity of OP-145 (0.5 and 1 µg/mL) to bone marrow stromal cells (BMSCs) was assessed using an MTT cell proliferation kit (Roche Applied Science). The BMSCs were incubated at 37°C under 5% CO2 for 1 and 2 days after cell inoculation as described previously. Next, a 50-mL volume of MTT working solution was added to each well, and the mixture was incubated for another 4 h. Purple crystal formazan was observed around cells at ×40 magnification under a microscope. The cell medium was carefully removed, and then 100 mL of dimethyl sulfoxide was added to each well to dissolve formazan. After 15 minutes of incubation at 37°C to completely dissolve formazan, the absorbance was measured at 490 nm on an enzyme-linked immunosorbent assay (ELISA) plate reader, and the results were expressed as optical density (OD) values. The OD values were calculated from concentration-response curves and used as a measure of cellular sensitivity. Treatment. Statistical analysis The data were analyzed using GraphPad Prism 5.0. One-way ANOVA and Tukey multiple comparison tests were used to compare different treatments. A P-value<0.05 was considered statistically significant. Results Clinically isolated strains and antimicrobial susceptibility test Page 4/14 The progress of the experiment was shown in Figure 1. Figure 2 shows the microbial diversity of the isolated strains from patients. S. aureus was the main pathogen of the infection (78.71%). Peptoniphilus and Porphyromonas were the other main bacteria in the isolated strains. In the antimicrobial susceptibility test, vancomycin, OP-145, and cefixime exhibited strong antimicrobial activity on clinically isolated strains; therefore, we used vancomycin as a positive control in the following tests. Preparation of OP-145 containing PLGA microspheres The PLGA microspheres were formed through a water-in-oil method in the microfluidic device and further cross-linked and freeze-dried to obtain a porous PLGA microsphere. Under the scanning electron microscope observation, the PLGA microspheres produced by coflow shearing of coaxial nozzles exhibited well dispersed, uniform sizes and well-maintained integrity in shapes (Figure 3.1). The XPS analysis of the carbon peak (C 286 eV) and oxygen peak (O 532.0 eV) showed that the PLGA microsphere contained C and O (Figure 3.2-4). Evaluation of in vivo anti-biofilm activity Figure 4 shows the biofilms of the three groups at 1, 2, and 3 d. The fluorescence image showed that OP145 and vancomycin exerted strong anti-biofilm effects on clinical isolates at 1, 2, and 3 d, which was instant with the IOD values (Figure 4.1-3, P<0.05). The community at the gene level showed that the variety of biofilms was reduced by the OP-145 and vancomycin. In the drug release test, the OP-145 PLGA microspheres could slowly release OP-145 at pH=3.4 and 5.3 (Figure 5.1). In the MTT test, the 0.5 concentration and 1 µg/mL of OP-145 showed no toxicity on bone marrow stromal cells (BMSCs) (Figure 5.2). The weight of the OP-145, control animals, and vancomycin animals showed no significant differences between groups overall or at any time interval according to repeated measures two-way ANOVA (Figure 5.3). The CRP levels peaked at 36 h, and the OP-145 and vancomycin groups began to exert a lower level of CRP (Figure 5.4, P<0.05). Discussion Infection is one of the most intractable problems in soft tissues, bones, and joints[18]. In addition, infections are associated with increased medical expenses or concurrent severe joint function disorders. The main pathogens in trauma and orthopedic infections are gram-positive cocci (Staphylococcus aureus and Streptococcus pyogenes)[19]. Wound bacterial biofilm is unique due to its significant resistance to antibiotics and other antibacterial agents[20]. Data show that 65–80% of wound infections are related to bacterial biofilms[21]. Bacterial biofilms have also become one of the main reasons for poor treatment outcomes, delayed healing of infected wounds, surgery, and local administration of antibiotics. At present, the methods to resolve wound bacterial biofilms include local mechanical debridement and destruction, bioengineering replacement therapy, negative pressure therapy, local drugs, etc[22]. The above methods have certain therapeutic effects; however, bacterial biofilms are still a challenge in the clinical treatment of chronic infections. Page 5/14 The main sources of bone infection include endogenous pathogens such as respiratory tract, body surface, urogenital system, and exogenous infections such as wound foreign bodies and air pollution[23]. Clinical reports show that exogenous and nosocomial infection rates can be decreased significantly through air purification[24]. Endogenous infections should be treated with antibiotics. Studies have confirmed that antibiotics in the perioperative period of orthopedic infections can significantly improve the infection, especially in aseptic surgery with large injuries and lengthy and complex surgeries[25]. Antimicrobial peptides (Amps) have been introduced in recent years. Amps is a kind of peptide with great development potential as new antibiotics. It is encoded by genes and synthesized by ribosomes. Antimicrobial peptides are small cationic peptides against infection by external pathogens, produced by the host’s innate immune defense system, and are an important effector molecule of innate immunity[26]. Natural antimicrobial peptides have a broad antibacterial spectrum, especially against clinical isolated multi-drug-resistant bacteria[27]. Moreover, the antibacterial peptide has good thermal stability and water solubility, with no toxic effects on normal cells of animals[28]. OP-145, a synthetic antimicrobial peptide developed from a screen of the human cathelicidin LL-37, exhibits strong antibacterial activity against Staphylococcus aureus[29]. As a key factor in preventing bone infection, the application method has gradually changed from previous systemic high-dose medications to the current local medications. At present, local drug use mainly adopts the methods of local implantation of antibiotics and slow-release antibiotics with various carriers[30]. PLGA, as a nano copolymer with excellent biocompatibility and biodegradability, has been approved by FDA for clinical applications[31]. In the present study, the PLGA microspheres containing the antimicrobial peptide OP-145 exerted strong anti-infection effects in vivo, indicating their potential value in controlling bone infections. In the present study, we only tested the pathogenic bacteria from only one person; however, the etiology of infection of femoral fracture may be more complicated because the bacteria in each infection are not the same. Therefore, the results require further research. In the future, we will also consider all aspects to design experiments in vivo. Conclusion OP-145 PLGA microspheres could slowly release antimicrobial peptides and significantly reduce biofilm formation and levels of inflammatory factors. These findings indicate that OP-145 PLGA microspheres may be one of the promising compounds in the control of femoral fracture infection in vivo. Declarations Ethics approval and consent to participate The current study was performed by approval of the Ethics Committee of the Xinhua Hospital of Zhejiang Province with approval number 20210123-12. In addition, the committee approved the utilization of human samples within this study. The clinical samples were taken from one patient with informed consent was applied from the participant. In this study, all animal care and use protocols were performed Page 6/14 in accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals approved by the State Council of People’s Republic of China. All animal experiments in this study were approved by the Animal Research Ethics Committee of Xinhua Hospital of Zhejiang Province (No.20212123-45). And the methods were performed in accordance with the relevant guidelines and regulations. All experimental protocols were approved by the Ethics Committee of the Xinhua Hospital of Zhejiang Province. All methods are reported in accordance with ARRIVE guidelines for the reporting of animal experiments and informed consent was obtained from all subjects. Consent for publication Not applicable. Competing interests The authors declare no conflict of interest. Availability of data and materials The authors confirm that the data supporting the findings of this study are available within the article and the datasets used during the current study are available from the corresponding author on reasonable request. Funding Statement This work was supported by Traditional Chinese Medicine Science and Technology Project of Zhejiang Province(2020ZB107), Medicine Science and Technology Project of Zhejiang Province (2020KY676) and the National Natural Science Foundation of China (82104891). Authors' contributions Tianyu Wang planned and designed the experiments. Jianhua Qin and Yuliang Huang and ZX performed the experiments. Ye Cheng analyzed the data and drafted the manuscript. All authors read and approved the manuscript. Acknowledgements The authors would like to thank Ting Zhang for her assistance with the analyses. Additionally, the authors wish to acknowledge Zhejiang University for the use of their shared equipments. References 1. Schellenberger V, Wang C-w, Geething NC, Spink BJ, Campbell A, To W, et al. A recombinant polypeptide extends the in vivo half-life of peptides and proteins in a tunable manner. Nature biotechnology. 2009;27(12):1186–90. Page 7/14 2. Gebauer M, Skerra A. Prospects of PASylation® for the design of protein and peptide therapeutics with extended half-life and enhanced action. Bioorganic & medicinal chemistry. 2018;26(10):2882–7. 3. Clair-Jones S, Prignano F, Goncalves J, Paul M, Sewerin P. Understanding and minimising injectionsite pain following subcutaneous administration of biologics: a narrative review. Rheumatology and Therapy. 2020;7(4):741–57. 4. Wang S, Liu R, Fu Y, Kao WJ. Release mechanisms and applications of drug delivery systems for extended-release. Expert Opinion on Drug Delivery. 2020;17(9):1289–304. 5. Sharma M, Dev SK, Kumar M, Shukla AK. Microspheres as suitable drug carrier in sustained release drug delivery: an overview. Asian Journal of Pharmacy And Pharmacology. 2018;4(2):102–8. 6. Metsemakers W, Kuehl R, Moriarty T, Richards R, Verhofstad M, Borens O, et al. Infection after fracture fixation: current surgical and microbiological concepts. Injury. 2018;49(3):511–22. 7. Zou F, Jiang J, Lv F, Xia X, Ma X. Preparation of antibacterial and osteoconductive 3D-printed PLGA/Cu (I)@ ZIF-8 nanocomposite scaffolds for infected bone repair. Journal of Nanobiotechnology. 2020;18(1):1–14. 8. Humphries RM, Kircher S, Ferrell A, Krause KM, Malherbe R, Hsiung A, et al. The continued value of disk diffusion for assessing antimicrobial susceptibility in clinical laboratories: report from the Clinical and Laboratory Standards Institute Methods Development and Standardization Working Group. Journal of clinical microbiology. 2018;56(8):e00437-18. 9. Andhariya JV, Jog R, Shen J, Choi S, Wang Y, Zou Y, et al. Development of Level A in vitro-in vivo correlations for peptide loaded PLGA microspheres. Journal of Controlled Release. 2019;308:1–13. 10. Garner J, Skidmore S, Park H, Park K, Choi S, Wang Y. Beyond Q1/Q2: the impact of manufacturing conditions and test methods on drug release from PLGA-based microparticle depot formulations. Journal of pharmaceutical sciences. 2018;107(1):353–61. 11. Shafiee A, Kehtari M, Zarei Z, Soleimani M, Varshochian R, Ahmadi A, et al. An in situ hydrogelforming scaffold loaded by PLGA microspheres containing carbon nanotube as a suitable niche for neural differentiation. Materials Science and Engineering: C. 2021;120:111739. 12. You G, Kim Y, Lee JH, Song J, Mok H. Exosome-modified PLGA microspheres for improved internalization into dendritic cells and macrophages. Biotechnology and Bioprocess Engineering. 2020;25(4):521–7. 13. Dong P, Gu X, Zhu G, Li M, Ma B, Zi Y. Melatonin induces osteoblastic differentiation of mesenchymal stem cells and promotes fracture healing in a rat model of femoral fracture via neuropeptide Y/neuropeptide Y receptor Y1 signaling. Pharmacology. 2018;102(5–6):272–80. 14. Pavey GJ, Qureshi AT, Hope DN, Pavlicek RL, Potter BK, Forsberg JA, et al. Bioburden increases heterotopic ossification formation in an established rat model. Clinical Orthopaedics and Related Research®. 2015;473(9):2840–7. 15. Li Z, Li M, Li F, Zhang M. based chemiluminescence enzyme-linked immunosorbent assay enhanced by biotin-streptavidin system for high-sensitivity C-reactive protein detection. Analytical Biochemistry. 2018;559:86–90. Page 8/14 16. Tawakoli P, Al-Ahmad A, Hoth-Hannig W, Hannig M, Hannig C. Comparison of different live/dead stainings for detection and quantification of adherent microorganisms in the initial oral biofilm. Clinical oral investigations. 2013;17(3):841–50. 17. Liu C, Liang G, Deng Z, Tan J, Zheng Q, Lyu F-J. The upregulation of COX2 in human degenerated nucleus pulposus: the association of inflammation with intervertebral disc degeneration. Mediators of inflammation. 2021;2021. 18. Tobudic S, Forstner C, Burgmann H, Lagler H, Steininger C, Traby L, et al. Real-world experience with dalbavancin therapy in gram-positive skin and soft tissue infection, bone and joint infection. Infection. 2019;47(6):1013–20. 19. Yang L, Feng J, Liu J, Yu L, Zhao C, Ren Y, et al. Pathogen identification in 84 Patients with posttraumatic osteomyelitis after limb fractures. Annals of Palliative Medicine. 2020;9(2):451–8. 20. Wei D, Zhu X-M, Chen Y-Y, Li X-Y, Chen Y-P, Liu H-Y, et al. Chronic wound biofilms: diagnosis and therapeutic strategies. Chinese Medical Journal. 2019;132(22):2737–44. 21. Macià MD, Del Pozo JL, Díez-Aguilar M, Guinea J. Microbiological diagnosis of biofilm-related infections. Enfermedades infecciosas y microbiologia clinica (English ed). 2018;36(6):375–81. 22. Li C, Cornel EJ, Du J. Advances and Prospects of Polymeric Particles for the Treatment of Bacterial Biofilms. ACS Applied Polymer Materials. 2021;3(5):2218–32. 23. Elashiry M, Elsayed R, Cutler CW. Exogenous and endogenous dendritic cell-derived exosomes: Lessons learned for immunotherapy and disease pathogenesis. Cells. 2021;11(1):115. 24. Boswell T, Fox P. Reduction in MRSA environmental contamination with a portable HEPA-filtration unit. Journal of Hospital Infection. 2006;63(1):47–54. 25. Hansen E, Belden K, Silibovsky R, Vogt M, Arnold WV, Bicanic G, et al. Perioperative antibiotics. The Journal of Arthroplasty. 2014;29(2):29–48. 26. Bahar AA, Ren D. Antimicrobial peptides. Pharmaceuticals. 2013;6(12):1543–75. 27. Yan Cy, Liu Yz, Xu Zh, Yang Hy, Li J. Comparison of Antibacterial Effect of Cationic Peptide LL-37 and Cefalexin on Clinical Staphylococcus aureus‐induced Infection after Femur Fracture Fixation. Orthopaedic Surgery. 2020;12(4):1313–8. 28. Lei J, Sun L, Huang S, Zhu C, Li P, He J, et al. The antimicrobial peptides and their potential clinical applications. American journal of translational research. 2019;11(7):3919. 29. Malanovic N, Leber R, Schmuck M, Kriechbaum M, Cordfunke RA, Drijfhout JW, et al. Phospholipiddriven differences determine the action of the synthetic antimicrobial peptide OP-145 on Grampositive bacterial and mammalian membrane model systems. Biochimica et Biophysica Acta (BBA)Biomembranes. 2015;1848(10):2437–47. 30. Wahl P, Guidi M, Benninger E, Rönn K, Gautier E, Buclin T, et al. The levels of vancomycin in the blood and the wound after the local treatment of bone and soft-tissue infection with antibiotic-loaded calcium sulphate as carrier material. The Bone & Joint Journal. 2017;99(11):1537–44. Page 9/14 31. He P, Zhong Q, Ge Y, Guo Z, Tian J, Zhou Y, et al. Dual drug loaded coaxial electrospun PLGA/PVP fiber for guided tissue regeneration under control of infection. Materials Science and Engineering: C. 2018;90:549–56. Figures Figure 1 OP-145 was combined with PLGA through the amine coupling reaction for on-demand infected wound healing. Page 10/14 Figure 2 1: The number of rarefaction curves in clinical isolates; 2: phylogenetic tree at the gene level; 3: the bacterial content in clinical isolates; 4: the KB paper method for common antibiotics sensitivity test. (1: vancomycin; 2: cefixime; 3: polymyxin; 4: tetracyclines; 5: OP-145; C: blank control). Page 11/14 Figure 3 1: SEM images of OP-145 PLGA; 2-4: X-ray photoelectron spectroscopy analyses of OP-145 PLGA. Page 12/14 Figure 4 1 The fluorescence images of biofilm after 1, 2, and 3 d ( 1: control groups; 2: OP-145; 3: vancomycin); 2 integrated optical density (IOD) of biofilm after 1, 2, and 3 d (1: control groups; 2: OP-145; 3: vancomycin); 3: the percentage of community abundance at the gene level after 3 d (1: control groups; 2: OP-145; 3: vancomycin). Page 13/14 Figure 5 1: The drug release of OP-145 at pH values of 5.3 and 3.4 at 0, 6, 12, 24, 48, and 72 h; 2: the cytotoxicity of OP-145 (0.5 and 1 µg/mL) to bone marrow stromal cell; 3: the weight of the OP-145, control animals, and vancomycin animals; 4: CRP level of OP-145, control animals, and vancomycin animals at 12, 24, 36, 48, and 72 h. Page 14/14
https://openalex.org/W2981636359	https://europepmc.org/articles/pmc6812834?pdf=render	English	null	E-cigarette Use, or Vaping, Practices and Characteristics Among Persons with Associated Lung Injury — Utah, April–October 2019	Morbidity and mortality weekly report	2,019	cc-by	3,699	On October 22, 2019, this report was posted as an MMWR Early Release on the MMWR website (https://www.cdc.gov/mmwr). On October 22, 2019, this report was posted as an MMWR Early Release on the MMWR website (https://www.cdc.gov/mmwr). On October 22, 2019, this report was posted as an MMWR Early Release on the MMWR website (https://www.cdc.gov/mmwr). On October 22, 2019, this report was posted as an MMWR Early Release on the MMWR website (https://www.cdc.gov/mmwr). investigated to determine symptoms, medical care history, and exposures related to the injury. Cases were classified as confirmed or probable according to established case definitions (3). Medical record abstraction was completed using a detailed form provided by CDC in September 2019. Interviews were conducted with patients, or a proxy (a spouse or parent), using an adaptation of a questionnaire developed in Illinois and Wisconsin in consultation with CDC during investigation of cases in those states (1). Medical record abstractions were conducted by UDOH staff members. Interviews were conducted by UDOH staff members or local health department staff members in-person or by telephone to assess product acquisition and use behaviors. Early Release on the MMWR website (https://www.cdc.gov/mmwr). In August 2019, the Utah Department of Health (UDOH) received reports from health care providers of several cases of lung injury in persons who reported use of electronic cigarette (e-cig arette), or vaping, products (1,2). To describe the characteristics of medical care, potentially related conditions, and exposures among 83 patients in Utah, detailed medical abstractions were completed for 79 (95%) patients. Among patients receiving chart abstractions, 70 (89%) were hospitalized, 39 (49%) required breathing assistance, and many reported preexisting respiratory and mental health conditions. Interviews were conducted by telephone or in person with 53 (64%) patients or their prox ies, and product samples from eight (15%) of the interviewed patients or proxies were tested. Among 53 interviewed patients, all of whom reported using e-cigarette, or vaping, products within 3 months of acute lung injury, 49 (92%) reported using any products containing tetrohydrocannabinol (THC), the principal psychoactive component of cannabis; 35 (66%) reported using any nicotine-containing products, and 32 (60%) reported using both. As reported in Wisconsin and Illinois (1), most THC-containing products were acquired from informal sources such as friends or illicit in-person and online dealers. THC-containing products were most commonly used one to five times per day, whereas nicotine-containing products were most commonly used >25 times per day. * The other 16 analytes are diazion, phorate, terbuphos, tetramine, paraoxon parathion, pentazocine, scopolamine, codeine, strychnine, aldrin, endrin, dichlorodiphenyltrichloroethane (DDT), fentanyl, dichlorodiphenyldichloroethylene (DDE), arecoline, pilocarpine, and morphine. US Department of Health and Human Services/Centers for Disease Control and Prevention Morbidity and Mortality Weekly Report Morbidity and Mortality Weekly Report E-cigarette Use, or Vaping, Practices and Characteristics Among Persons with Associated Lung Injury — Utah, April–October 2019 Nathaniel Lewis, PhD1,2; Keegan McCaffrey2; Kylie Sage, MS2; Chia-Jung Cheng, PhD2; Jordan Green, MPH2; Leah Goldstein2; Hillary Campbell2; Deanna Ferrell, MPH2; Nathan Malan, MPH2; Nathan LaCross, PhD2; Alejandra Maldonado, PhD2; Amy Board, PhD1; Arianna Hanchey, MPH3; Dixie Harris, MD4; Sean Callahan, MD5; Scott Aberegg, MD5; Ilene Risk, MPH6; Sarah Willardson, MPH7; Amy Carter8; Allyn Nakashima, MD2; Janae Duncan, MPA2; Cindy Burnett, MPH2; Robyn Atkinson-Dunn, PhD2; Angela Dunn, MD2 On October 22, 2019, this report was posted as an MMWR Early Release on the MMWR website (https://www.cdc.gov/mmwr). Product sample testing at the Utah Public Health Laboratory (UPHL) showed evidence of vitamin E acetate in 17 of 20 (89%) THC-containing car tridges, which were provided by six of 53 interviewed patients. The cause or causes of this outbreak is currently unknown (2); however, the predominant use among patients of e-cigarette, or vaping, products with prefilled THC-containing cartridges suggests that the substances in these products or the way in which they are heated and aerosolized play an important role in the outbreak. At present, persons should not use e-cigarette, or vaping, products that contain THC. In addition, because the specific cause or causes of lung injury are not yet known and while the investigation continues, persons should consider refraining from use of all e-cigarette, or vaping, products. D i A O b 2019 ibl f i UDOH and Utah local health departments collected e-cigarette, or vaping, products from patients for testing using gas chromatography–mass spectrometry at UPHL to identify peaks for known chemical substances (including nicotine and THC) through nontargeted testing followed by partial verification of results with targeted tests for analytes that have known chemical standards (nicotine and vitamin E acetate, along with 16 others) or known m/z values (i.e., mass) and relative retention times (myclobutanil and thiodiglycol) (4). During August 6–October 15, 2019, 83 confirmed and probable cases of EVALI were reported, primarily by clinicians and Utah Poison Control Center, to UDOH. The overall prevalence was 26 per 1,000,000 population. Most (86%) of the patients lived in Salt Lake County and surrounding urban counties (Davis, Morgan, Weber, and Utah); 14% lived in outlying counties. Abstraction of medical records was completed for 79 (95%) patients, and 53 (64%) interviews were completed. Among the 83 patients, 69 (83%) were male, and the median age was 26 years (range = 14–66 years) (Table 1). Among the 79 patients for whom medical record data were available, 70 (89%) were hospitalized during June 5–September 23 (median duration = 4 days; range = 1–17 days), including 35 (44%) who required intensive care unit (ICU) admission; nine (11%) were not hospitalized. Many patients required respiratory support; continuous or bilevel positive airway pressure was required by During August–October 2019, possible cases of e-cigarette, or vaping, product use–associated lung injury (EVALI) in Utah were MMWR / October 25, 2019 / Vol. 68 / No. 42 953 Morbidity and Mortality Weekly Report TABLE 1. On October 22, 2019, this report was posted as an MMWR Early Release on the MMWR website (https://www.cdc.gov/mmwr). Characteristics of patients with electronic cigarette (e-cigarette), or vaping, product use–associated lung injury, (N = 83) — Utah, April– October 2019 Characteristic (no. with available information) No. (%) Sex (83) Male 69 (83) Female 14 (17) Age group (yrs) (83) 14–19 11 (13) 20–29 43 (52) 30–39 23 (28) 40–66 6 (7) Required medical care/In-care diagnoses (79) Hospitalization 70 (89) ICU admission 35 (44) CPAP/BiPAP support (No intubation) 30 (38) Intubation and mechanical ventilation 9 (11) Treated with steroids 59 (75) Acute respiratory distress syndrome 20 (25) Preexisting conditions* (79) Asthma 16 (20) Chronic obstructive pulmonary disease 2 (3) Anxiety 27 (34) Depression 18 (25) Hypertension 4 (5) Heart failure 1 (1) One or more of the above 42 (53) Smoking history,† (79) Marijuana 34 (43) Tobacco 43 (54) Both marijuana and tobacco 19 (24) Abbreviations: BiPAP = bilevel positive airway pressure; CPAP = continuous positive airway pressure; ICU = intensive care unit. Denominators based on total patients with medical abstraction data available (unknowns included in denominator). † Includes current and former smokers. 30 (38%), and endotracheal intubation and mechanical venti lation was required by nine (11%). Fifty-nine (75%) patients were treated with steroids. Twenty (25%) patients received a diagnosis of acute respiratory distress syndrome. Patients reported having histories of asthma, 16 (20%); anxiety, 27 (34%); depression, 18 (23%); hypertension, four (5%); and heart failure, one (1%). Approximately half of the patients had at least one of these preexisiting conditions. Patients also reported smoking combustible marijuana (43%), tobacco (54%), or both (24%). Among the 53 patients interviewed, 49 (92%) reported use of THC-containing e-cigarette, or vaping, products during the 3 months preceding illness (Table 2); 35 (66%) reported using nicotine-containing products; and 32 (60%) reported using both THC- and nicotine-containing products. Seventeen (32%) patients reported exclusive use of THC- containing products, whereas three (6%) reported exclusive use of nicotine-containing products. Use of three brands of prefilled THC-containing cartridges was reported frequently by patients; these included Dank Vapes (21, 40%), Rove (19, 36%), and Golden Gorilla (11, 21%). Seventeen (32%) patients reported using more than one of these brands. Patients reported a total of 131 e-cigarette, or vaping, products used during the 3 months before illness and for which the method of acquisition was known; 84 of these were THC-containing products, and 47 were nicotine-containing products (Table 3). On October 22, 2019, this report was posted as an MMWR Early Release on the MMWR website (https://www.cdc.gov/mmwr). Most THC-containing products were acquired through informal sources, including friends (44%), in-person dealers (25%), and online dealers (24%). Five prod ucts were purchased at an out-of-state dispensary and one at an in-state vape shop selling these products illicitly. Among 84 THC-containing products used, frequency of use was reported for 70 of 84 (83%). Approximately two thirds (65%) of the THC-containing products were used ≤5 times per day. Among 47 nicotine-containing products used, frequency of use was reported for 29 of 47 (62%). The majority of the nicotine- containing products were used >25 times per day (55%) and were acquired primarily through in-state vape shops (49%) or convenience stores and gas stations (18%). detected in 17 (89%). Samples of nicotine-containing e-liquid, in contrast, only showed evidence of nicotine and no evidence of THC or vitamin E acetate. No other analytes were found. MMWR / October 25, 2019 / Vol. 68 / No. 42 Discussion Test results might therefore represent clusters of purchase or use by these patients rather than fully independent samples. Vitamin E acetate was identified in the majority of THC cartridge samples tested at UPHL; however, these samples only represent six patients. National data summarized recently in a news report suggested that vitamin E acetate is a now common diluent in THC cartridges (8). Quantification of vitamin E acetate in Utah’s samples is pending; however, testing of other case samples by the Food and Drug Administration and other laboratories has shown vitamin E acetate concentrations of 31%–88% and lower-than-expected THC concentrations (14%–76% versus the typically advertised 75%–95%) (8). The potential role of vitamin E acetate in lung injury remains unknown; however, the identification of vitamin E acetate among products collected from patients in Utah and elsewhere indicates that the outbreak might be associated with cutting agents or adulterants (9). Ascertaining the potential contribu tion of diluents to the current outbreak will require data from multiple states and analysis at the national level. Most patients in this analysis reported using THC-containing products (which are illegal for nonmedical use in Utah) that were sold as prefilled cartridges and obtained from informal sources. Compared with Illinois, Wisconsin, and nationally, patient use rates for prefilled THC-containing cartridges in Utah were even higher while those for nicotine-containing products were lower, reinforcing the finding that unregulated THC-containing cartridges play an important role in this outbreak (1,2). Products labeled with three different brand names, Dank Vapes, Rove, and Golden Gorilla, were each reported by a substantial proportion of patients (20%–40%), although packaging for these brands can be reproduced or purchased online. In Illinois and Wisconsin, Dank Vapes was reported far more than any other brand, Rove was reported by a few patients, and Golden Gorilla was not reported at all (1,2). Although the respective market shares of these brands are unknown, findings from the Utah investigation might reflect a distinct pattern of illicit THC supply and production in Utah or the western United States compared with that in the Midwest and other areas of the United States. The findings in this report are subject to at least five limita tions. First, because interviews were not conducted with 30 (36%) patients, nonresponse could introduce selection bias and result in inaccurate estimation of specific substances used and use patterns. US Department of Health and Human Services/Centers for Disease Control and Prevention Discussion In this study of 83 Utah residents with EVALI during August–October 2019, approximately 90% of patients were hospitalized, approximately half in ICUs, and more than half of hospitalized patients required some form of respiratory support. Three quarters were treated with steroids. It is not known why some patients have more severe illness; preexisting behaviors and conditions might play a role in injury exposure, onset, and injury progression. Whereas some patients reported preexisting respiratory problems, most were previously in good physical health, although many reported that they self- identified as current or former smokers of combustible mari juana or tobacco. Many patients reported histories of anxiety or depression, which might influence the use or patterns of e-cigarette, or vaping, product use, particularly products con taining THC (5). To date, UDOH and Utah local health departments have collected 72 products from eight (15%) of 53 patients inter viewed. Products tested at UPHL comprised 19 prefilled THC-containing cartridges from six patients and 20 nicotine- containing vaping liquids (19 bottled e-liquids and one from an atomizer) from six patients; six patients provided both THC- and nicotine-containing samples, and two provided only nicotine-containing samples). Among the 19 THC-containing cartridges, THC was detected in 19 of 19 (100%), nicotine was detected in one (5%), and evidence of vitamin E acetate was MMWR / October 25, 2019 / Vol. 68 / No. 42 US Department of Health and Human Services/Centers for Disease Control and Prevention 954 Morbidity and Mortality Weekly Report TABLE 2. Self-reported product use behaviors in the 3 months before injury onset in interviewed patients with electronic cigarette (e-cigarette), or vaping, product use–associated lung injury (N = 53) — Utah, April– October 2019 Product use and behavior No. (%) THC-containing product use Any use 49 (92) Exclusive use 17 (32) THC-containing cartridge brands used Dank Vapes 21 (40) Rove 19 (36) Golden Gorilla 11 (21) Two or more of the above 17 (32) Nicotine-containing product use Any use 35 (66) Exclusive use 3 (6) Both THC- and nicotine-containing product use 32 (60) Abbreviation: THC = tetrahydrocannabinol. TABLE 3. MMWR / October 25, 2019 / Vol. 68 / No. 42 Discussion Characteristics of tetrahydrocannabinol (THC)- or nicotine- containing products used in the 3 months preceding illness onset in patients with electronic cigarette (e-cigarette), or vaping, product use–associated lung injury (N = 131) — Utah, April–October 2019 containing products used in the 3 months preceding illness onset in patients with electronic cigarette (e-cigarette), or vaping, product use–associated lung injury (N = 131) — Utah, April–October 2019 Characteristic No. (%) THC-containing products (N = 84) Nicotine-containing products (N = 47) Method of acquisition Friend 37/84 (44) 9/47 (19) Dealer 21/84 (25) 0/47 (0) Online dealer 20/84 (24) 7/47 (15) Out-of-state dispensary 5/84 (6) 1/47 (2) In-state vape shop 1/84 (1) 23/47 (49) Convenience store/gas station 0/84 (0) 7/47 (18) Frequency of use (times per day) <1 8/70 (11) 3/29 (10) 1–5 38/70 (54) 5/29 (17) 6–25 7/70 (10) 5/29 (17) >25 17/70 (24) 16/29 (55) Testing Products tested at UPHL* 19/84 (23) 20/47 (43) Products found to contain THC 19/19 (100) 0/20 (0) Products found to contain nicotine 1/19 (5) 20/20 (100) Products found to contain vitamin E acetate 17/19 (89) 0/20 (0) Abbreviation: UPHL = Utah Public Health Laboratory. * THC-containing cartridges tested came from six patients and nicotine- containing vaping liquids came from eight patients. Test results might therefore represent clusters of purchase or use by these patients rather than fully independent samples. The median age of patients in this study was 26 years, 3 years older than the national median of 23 years; more than one third were aged ≥30 years. The older age profile in Utah suggests a need to focus on adult populations at risk in addi tion to younger persons. Utah’s rate of adult e-cigarette use (5.1%) was similar to the national rate (4.6%) in 2017 (the most recent year for which state and national data are avail able), and e-cigarette use among youths (7.6%) was lower than the national rate (13.2%) in 2017, although rates in all states increased in 2018 and 2019 (6). As of October 15, 2019, Utah’s rate of EVALI was 26 per 1 million compared with four per 1 million nationally (7). More research is needed to identify the constellation of risk factors influencing the high rate of EVALI in Utah. Abbreviation: UPHL = Utah Public Health Laboratory. * THC-containing cartridges tested came from six patients and nicotine- containing vaping liquids came from eight patients. Abbreviation: UPHL = Utah Public Health Laboratory. What is added by this report? All authors have completed and submitted the International Committee of Medical Journal Editors form for disclosure of potential conflicts of interest. No potential conflicts of interest were disclosed. Medical abstractions were completed for 79 Utah patients, 53 of whom were interviewed. Almost all patients reported using tetrahydrocannabinol (THC)-containing vaping cartridges. Most patients were hospitalized, half required breathing assistance, many reported preexisting respiratory and mental health conditions, and many identified as current or former smokers of combustible marijuana or tobacco. Most THC-containing products, acquired from six patients and, tested at Utah Public Health Laboratory, contained vitamin E acetate. Summary What is already known about this topic? An outbreak of e-cigarette, or vaping, product use–associated lung injury (EVALI) of unknown source is ongoing in the United States. Corresponding author: Nathaniel Lewis, cdceisnml@utah.gov, 801-538-9465. Discussion Second, because nonmedical THC use currently is illegal in Utah, self-reported use could be influenced by the perceived stigma of illicit substance use or fear of legal repercus sions, which might result in underreporting of use. Third, case MMWR / October 25, 2019 / Vol. 68 / No. 42 rtment of Health and Human Services/Centers for Disease Control and Prevention 955 Morbidity and Mortality Weekly Report 1Epidemic Intelligence Service, CDC; 2Utah Department of Health; 3National Center for Environmental Health, CDC; 4Intermountain Healthcare, Salt Lake City, Utah; 5University of Utah Health, Salt Lake City, Utah; 6Salt Lake County Health Department, Salt Lake City, Utah; 7Davis County Health Department, Clearfield, Utah; 8Weber-Morgan Health Department, Ogden, Utah. 1Epidemic Intelligence Service, CDC; 2Utah Department of Health; 3National Center for Environmental Health, CDC; 4Intermountain Healthcare, Salt Lake City, Utah; 5University of Utah Health, Salt Lake City, Utah; 6Salt Lake County Health Department, Salt Lake City, Utah; 7Davis County Health Department, Clearfield, Utah; 8Weber-Morgan Health Department, Ogden, Utah. What are the implications for public health practice? At present, persons should not use e-cigarette, or vaping, products containing THC. In addition, because the specific cause or causes of lung injury are not yet known and while the investigation continues, persons should consider refraining from use of all e-cigarette, or vaping, products. g 3. Schier JG, Meiman JG, Layden J, et al.; CDC 2019 Lung Injury Response Group. Severe pulmonary disease associated with electronic- cigarette-product use—interim guidance. MMWR Morb Mortal Wkly Rep 2019;68:787–90. https://doi.org/10.15585/mmwr.mm6836e2 p p g 4. Lee J, Park J, Go A, et al. Urine multi-drug screening with GC-MS or LC–MS-MS using SALLE-hybrid PPT/SPE. J Anal Toxicol 2019;42:617–24. https://doi.org/10.1093/jat/bky032 reporting in Utah relies on clinician reports, which, to date, have come largely from pulmonologists and critical care phy sicians. Consequently, there is possible reporting bias toward hospitalized patients and those with more severe respiratory symptoms. Fourth, care requirements or preexisting conditions are not always reported on medical charts, meaning that rates could be higher than reported. Finally, because laboratory analysis and coordination are currently limited, there might be factors contributing to the lung injury not yet identified. p g j y 5. Kedzior KK, Laeber LT. A positive association between anxiety disorders and cannabis use or cannabis use disorders in the general population—a meta-analysis of 31 studies. BMC Psychiatry 2014;14:136. https://doi. org/10.1186/1471-244X-14-136 g 6. CDC. State Tobacco Activities Tracking and Evaluation (STATE) system. State highlights. Atlanta, GA: US Department of Health and Human Services; CDC; 2019. https://www.cdc.gov/statesystem/statehighlights.html p g y g g 7. US Census Bureau. 2018 population estimates. Suitland, MD: US Census Bureau; 2019. https://factfinder.census.gov/faces/tableservices/ jsf/pages/productview.xhtml?src=bkmk Effective interventions to halt this outbreak might require a stronger partnership between public health and law enforce ment agencies to identify the locations of supply and distribu tion chains that are contributing to lung injuries, alongside targeted messaging to consumers. UDOH has initiated a print and social media campaign to alert the public to the potential dangers associated with use of THC-containing e-cigarette, or vaping, products. At present, persons should not use e-cigarette, or vaping, products that contain THC. In addition, because the specific cause or causes of lung injury are not yet known and while the investigation continues, persons should consider refraining from use of all e-cigarette, or vaping, products (10). j p g p 8. Sun L. Vaping lung injuries top 1,000 cases as deaths rise to 18. The Washington Post. October 3, 2019. MMWR / October 25, 2019 / Vol. 68 / No. 42 US Department of Health and Human Services/Centers for Disease Control and Prevention References 1. Layden JE, Ghinai I, Pray I, et al. Pulmonary illness related to e-cigarette use in Illinois and Wisconsin—preliminary report. New Engl J Med 2019. Epub September 6, 2019. https://www.nejm.org/ doi/full/10.1056/NEJMoa1911614 1. Layden JE, Ghinai I, Pray I, et al. Pulmonary illness related to e-cigarette use in Illinois and Wisconsin—preliminary report. New Engl J Med 2019. Epub September 6, 2019. https://www.nejm.org/ doi/full/10.1056/NEJMoa1911614 2. Perrine CG, Pickens CM, Boehmer TK, et al.; Lung Injury Response Epidemiology/Surveillance Group. Characteristics of a multistate outbreak of lung injury associated with e-cigarette use or vaping—United States, 2019. MMWR Morb Mortal Wkly Rep 2019;68:860–4. https:// doi.org/10.15585/mmwr.mm6839e1 2. Perrine CG, Pickens CM, Boehmer TK, et al.; Lung Injury Response Epidemiology/Surveillance Group. Characteristics of a multistate outbreak of lung injury associated with e-cigarette use or vaping—United States, 2019. MMWR Morb Mortal Wkly Rep 2019;68:860–4. https:// doi.org/10.15585/mmwr.mm6839e1 What are the implications for public health practice? What are the implications for public health practice? https://www.washingtonpost.com/ health/2019/10/03/vaping-lung-injuries-top-cases-deaths-rise j p g p 8. Sun L. Vaping lung injuries top 1,000 cases as deaths rise to 18. The Washington Post. October 3, 2019. https://www.washingtonpost.com/ health/2019/10/03/vaping-lung-injuries-top-cases-deaths-rise p g g j p 9. Butt YM, Smith ML, Tazelaar HD, et al. Pathology of vaping-associated lung injury. N Engl J Med 2019. Epub October 2, 2019. https://doi. org/10.1056/NEJMc1913069 p g g j p 9. Butt YM, Smith ML, Tazelaar HD, et al. Pathology of vaping-associated lung injury. N Engl J Med 2019. Epub October 2, 2019. https://doi. org/10.1056/NEJMc1913069 10. CDC. Outbreak of lung injury associated with e-cigarette use, or vaping. Atlanta, GA: US Department of Health and Human Services, CDC; 2019. https://www.cdc.gov/tobacco/basic_information/e-cigarettes/ severe-lung-disease.html MMWR / October 25, 2019 / Vol. 68 / No. 42 US Department of Health and Human Services/Centers for Disease Control and Prevention 956
https://openalex.org/W2467470679	https://europepmc.org/articles/pmc4963231?pdf=render	English	null	Treatment of Aggressive Prolactin-Secreting Pituitary Adenomas with Adjuvant Temozolomide Chemotherapy: A Review	Curēus	2,016	cc-by	5,103	Treatment of Aggressive Prolactin- Secreting Pituitary Adenomas with Adjuvant Temozolomide Chemotherapy: A Review Review Marc Moisi , Aurora S. Cruz , Tara Benkers , Steven Rostad , Frances Broyles Broyles , Kevin Yuen , Marc Mayberg 1 2 3 4 5 6 7 8 9 10 1. Seattle Science Foundation 2. Neurological Surgery, Wayne State University 3. Neurological Surgery, University of Louisville 4. Neurological Surgery, University of California, Irvine 5. Neurological Surgery, Swedish Neuroscience Institute 6. Neuro-oncology, Swedish Neuroscience Institute 7. Pathology, CellNetix 8. Endocrinology, Swedish Neuroscience Institute 9. Swedish Pituitary Center, Swedish Neuroscience Institute 10. Swedish Neuroscience Institute  Corresponding author: Marc Moisi, moisimd@aol.com Disclosures can be found in Additional Information at the end of the article Introduction And Background Pituitary adenomas are one of the more common intracranial neoplasms [1]. They tend to grow slowly without invasion and usually respond to medical therapy with dopamine agonists. However, these tumors rarely may exhibit aggressive behaviors invading local structures with multiple recurrences that do not respond to standard treatments of medical therapy, resection, and adjuvant radiation [2]. We describe a case of a patient with a prolactin-secreting adenoma with multiple recurrences over 10 years, which became progressively invasive and resistant to standard therapies, including dopamine agonists and trans-sphenoidal surgery, but responded well to adjuvant chemotherapy with temozolomide (TMZ). Received 05/22/2016 Review began 05/27/2016 Review ended 06/10/2016 Published 06/27/2016 © Copyright 2016 Moisi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License CC-BY 3.0., which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Open Access Review Article Open Access Review Article Open Access Review Article Abstract Most prolactin-secreting pituitary adenomas demonstrate slow growth and are effectively managed with medical/surgical therapy. Rarely, these tumors can behave aggressively with rapid growth and invasion of local tissues, and are refractory to medical, surgical, or radio- surgical therapies. We report a case of a prolactin-secreting adenoma in a young woman, which became progressively aggressive and refractory to usual treatment modalities, but responded to treatment with the chemotherapeutic agent temozolomide. In addition, we review the literature for treatment of refractory adenomas with temozolomide. The clinical and pathologic characteristics of aggressive prolactin-secreting adenomas are reviewed, as well as their response to dopamine agonists, surgery, radiotherapy, and chemotherapy. Categories: Diabetes & Endocrinology, Neurosurgery Keywords: aggressive pituitary tumor, prolactinoma, radiosurgery, temozolomide Introduction And Background DOI: 10.7759/cureus.658 Received 05/22/2016 Review began 05/27/2016 Review ended 06/10/2016 Published 06/27/2016 © Copyright 2016 Moisi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License CC-BY 3.0., which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. How to cite this article Moisi M, Cruz A S, Benkers T, et al. (June 27, 2016) Treatment of Aggressive Prolactin-Secreting Pituitary Adenomas with Adjuvant Temozolomide Chemotherapy: A Review. Cureus 8(6): e658. DOI 10.7759/cureus.658 Illustrative case A 37-year-old gravida-1, para-0 female presented in 2002 with secondary amenorrhea and galactorrhea. She described the onset of menses at age 13 and had normal menstrual periods through her teens. She was on birth control pills until 1999 with normal menstrual periods. After stopping birth control pills, her periods did not resume, and she noted intermittent 2016 Moisi et al. Cureus 8(6): e658. DOI 10.7759/cureus.658 galactorrhea. Evaluation revealed an elevated prolactin level of 110 ng/ml (normal <24 ng/ml), and MRI demonstrated a 10 mm pituitary lesion. She also had central hypothyroidism and was started on levothyroxine. She was placed on bromocriptine and had an excellent initial response with normalization of her prolactin and resumption of menstrual periods. Follow-up MRI showed regression of the tumor to 7 mm shown as in Figure 1. FIGURE 1: Coronal Post-Contrast MRI Sequences Following initial successful treatment with bromocriptine (arrow shows tumor within gland) FIGURE 1: Coronal Post-Contrast MRI Sequences Following initial successful treatment with bromocriptine (arrow shows tumor within gland) After six months, however, amenorrhea recurred with an elevation of prolactin to 55 ng/ml despite maximal tolerable bromocriptine therapy (15 mg daily), and in Figure 2 is an MRI that showed an increase in the tumor size to 26 mm in diameter with suprasellar extension and probable invasion to both cavernous sinuses. After six months, however, amenorrhea recurred with an elevation of prolactin to 55 ng/ml despite maximal tolerable bromocriptine therapy (15 mg daily), and in Figure 2 is an MRI that showed an increase in the tumor size to 26 mm in diameter with suprasellar extension and probable invasion to both cavernous sinuses. 2 of 17 2016 Moisi et al. Cureus 8(6): e658. DOI 10.7759/cureus.658 FIGURE 2: Coronal Post-Contrast MRI Sequences First recurrence despite maximal medical therapy, showing marked enlargement and invasion of both cavernous sinuses FIGURE 2: Coronal Post-Contrast MRI Sequences First recurrence despite maximal medical therapy, showing marked enlargement and invasion of both cavernous sinuses A trans-sphenoidal adenomectomy was performed in June 2005, with subsequent normalization of prolactin levels (11.3 ng/ml). Pathology was consistent with a pituitary adenoma showing sparse immunoreactive prolactin, with increased nuclear pleomorphism, elevated mitotic activity, and increased p53 labeling suggesting increased biologic aggressiveness and invasion as shown in Figure 3 and Figure 4. A trans-sphenoidal adenomectomy was performed in June 2005, with subsequent normalization of prolactin levels (11.3 ng/ml). Pathology was consistent with a pituitary adenoma showing sparse immunoreactive prolactin, with increased nuclear pleomorphism, elevated mitotic activity, and increased p53 labeling suggesting increased biologic aggressiveness and invasion as shown in Figure 3 and Figure 4. 3 of 17 FIGURE 3: Photomicrographs of Histologic Specimens from First Surgery (400x) Hematoxylin and eosin stain demonstrating pituitary adenoma with increased nuclear pleomorphism and elevated mitotic activity 2016 Moisi et al. Cureus 8(6): e658. DOI 10.7759/cureus.658 FIGURE 3: Photomicrographs of Histologic Specimens from First Surgery (400x) Hematoxylin and eosin stain demonstrating pituitary adenoma with increased nuclear pleomorphism and elevated mitotic activity 2016 Moisi et al. Cureus 8(6): e658. DOI 10.7759/cureus.658 2016 Moisi et al. Cureus 8(6): e658. DOI 10.7759/cureus.658 4 of 17 2016 Moisi et al. Cureus 8(6): e658. DOI 10.7759/cureus.658 FIGURE 4: Photomicrographs of Histologic Specimens from First Surgery (100x) Increased immunoreactive Ki-67 labeling index Increased immunoreactive Ki-67 labeling index Due to these findings on pathology, further treatment with stereotactic radiosurgery was recommended, but the patient declined due to concerns regarding infertility. Over the ensuing year, the tumor continued to increase in size to 17 mm diameter with further invasion of the cavernous sinuses. She subsequently developed headaches as well as right third and sixth nerve palsies, which resolved following a second trans-sphenoidal debulking surgery in June 2006. In November 2006, she underwent single-dose Cyberknife radiosurgery (marginal dose 22 Gy). However, she became amenorrheic despite normalization of her prolactin level and lack of radiographic progression for four years. Repeat MRI in 2010 showed further progression of the tumor into the right cavernous sinus, for which she was treated with Gamma Knife radiosurgery in January 2011 (20 Gy single fraction). The clinical status and MRI studies remained stable for the next three years, but in 2014, she developed progressive right third and sixth nerve palsies and MRI showed further tumor growth up to 3.4 x 3.1 x 3.8 cm. Because she was not a candidate for further radiosurgery, she underwent another trans-sphenoidal debulking surgery, and at that time, the decision was made to commence TMZ chemotherapy in October 2014. Pathology was consistent with an atypical prolactinoma with increased nuclear pleomorphism, marked elevation of the Ki-67 labeling index to 25%, and p53 labeling index to 70%. These histologic findings are shown in Figures 5-7 below. FIGURE 5: Photomicrographs of Histologic Specimens from Final Surgery Hematoxylin and eosin stain with additional nuclear pleomorphism and mitoses FIGURE 5: Photomicrographs of Histologic Specimens from Final Surgery Hematoxylin and eosin stain with additional nuclear pleomorphism and mitoses 5 of 17 2016 Moisi et al. Cureus 8(6): e658. DOI 10.7759/cureus.658 2016 Moisi et al. Cureus 8(6): e658. DOI 10.7759/cureus.658 FIGURE 6: Photomicrographs of Histologic Specimens from Final Surgery (400x) Markedly increased p53 labeling FIGURE 6: Photomicrographs of Histologic Specimens from Final Surgery (400x) Markedly increased p53 labeling FIGURE 6: Photomicrographs of Histologic Specimens from Final Surgery (400x) Markedly increased p53 labeling 6 of 17 FIGURE 7: Photomicrographs of Histologic Specimens from Final Surgery (100x) Markedly increased Ki-67 labeling index Next Generation sequencing of her tumor identified mutations in TP53, IDH2, CDH1, and FIGURE 7: Photomicrographs of Histologic Specimens from Final Surgery (100x) Markedly increased Ki-67 labeling index FIGURE 7: Photomicrographs of Histologic Specimens from Final Surgery (100x) Markedly increased Ki-67 labeling index Next Generation sequencing of her tumor identified mutations in TP53, IDH2, CDH1, and KIT. O(6)-methylguanine methyltransferase (MGMT) proliferation markers were not done on this particular case. While awaiting insurance authorization for TMZ therapy, she developed visual field deficits in her left eye as well as radiographic progression of her tumor as shown in Figure 8. Next Generation sequencing of her tumor identified mutations in TP53, IDH2, CDH1, and KIT. O(6)-methylguanine methyltransferase (MGMT) proliferation markers were not done on this particular case. While awaiting insurance authorization for TMZ therapy, she developed visual field deficits in her left eye as well as radiographic progression of her tumor as shown in Figure 8. 7 of 17 2016 Moisi et al. Cureus 8(6): e658. DOI 10.7759/cureus.658 FIGURE 8: Coronal Post-Contrast MRI Sequences Prior to craniotomy, demonstrating marked expansion with suprasellar extension and compression of optic chiasm (arrow) and intracranial invasion into right middle fossa FIGURE 8: Coronal Post-Contrast MRI Sequences Prior to craniotomy, demonstrating marked expansion with suprasellar extension and compression of optic chiasm (arrow) and intracranial invasion into right middle fossa FIGURE 8: Coronal Post-Contrast MRI Sequences Prior to craniotomy, demonstrating marked expansion with suprasellar extension and compression of optic chiasm (arrow) and intracranial invasion into right middle fossa She then underwent a right, frontotemporal craniotomy in January 2015 for decompression of the optic nerves and chiasm, with postoperative restoration of full visual fields. The postoperative MRI is shown in Figure 9. She then underwent a right, frontotemporal craniotomy in January 2015 for decompression of the optic nerves and chiasm, with postoperative restoration of full visual fields. The postoperative MRI is shown in Figure 9. 8 of 17 2016 Moisi et al. Cureus 8(6): e658. DOI 10.7759/cureus.658 FIGURE 9: Coronal Post-Contrast MRI Sequences After craniotomy at initiation of TMZ treatment, showing decompression of optic chiasm and debulking of tumor FIGURE 9: Coronal Post-Contrast MRI Sequences After craniotomy at initiation of TMZ treatment, showing decompression of optic chiasm and debulking of tumor After craniotomy at initiation of TMZ treatment, showing decompression of optic chiasm and debulking of tumor The histology was similar to the 2014 pathology. Given the rapid recurrence and intracranial invasion, she was treated with fractionated Cyberknife radiosurgery over 5 fractions (25 Gy), followed by a course of chemotherapy with 12 cycles of TMZ given on a five-day schedule every 28 days at 150 mg/m2 for the first cycle and increased to 200 mg/m2 for subsequent cycles. MRI of the brain two months following completion of radiation, in Figure 10, showed stable tumor compared to the postoperative MRI. 9 of 17 2016 Moisi et al. Cureus 8(6): e658. DOI 10.7759/cureus.658 FIGURE 10: Coronal Post-Contrast MRI Sequences After Cyberknife radiosurgery FIGURE 10: Coronal Post-Contrast MRI Sequences After Cyberknife radiosurgery FIGURE 10: Coronal Post-Contrast MRI Sequences After Cyberknife radiosurgery Following eight cycles of TMZ to date, surveillance MRI scans--demonstrated in Figures 11-12- -show continued decrease in the enhancing sellar and suprasellar tumor, with continued clinical improvement in the patient’s right third palsy, consistent with a good response to chemotherapy. Following eight cycles of TMZ to date, surveillance MRI scans--demonstrated in Figures 11-12- -show continued decrease in the enhancing sellar and suprasellar tumor, with continued clinical improvement in the patient’s right third palsy, consistent with a good response to chemotherapy. 10 of 17 2016 Moisi et al. Cureus 8(6): e658. DOI 10.7759/cureus.658 FIGURE 12: Coronal Post-Contrast MRI Sequences Sequential tumor regression over eight cycles of TMZ treatment. Note the prominent reduction in tumor volume and gadolinium enhancement. FIGURE 12: Coronal Post-Contrast MRI Sequences Sequential tumor regression over eight cycles of TMZ treatment. Note the prominent reduction in tumor volume and gadolinium enhancement. FIGURE 12: Coronal Post-Contrast MRI Sequences Sequential tumor regression over eight cycles of TMZ treatment. Note the prominent reduction in tumor volume and gadolinium enhancement. FIGURE 11: Coronal Post-Contrast MRI Sequences Sequential tumor regression over eight cycles of TMZ treatment. Note the prominent reduction in tumor volume and gadolinium enhancement. FIGURE 11: Coronal Post-Contrast MRI Sequences Sequential tumor regression over eight cycles of TMZ treatment. Note the prominent reduction in tumor volume and gadolinium enhancement. Sequential tumor regression over eight cycles of TMZ treatment. Note the prominent reduction in tumor volume and gadolinium enhancement. 11 of 17 2016 Moisi et al. Cureus 8(6): e658. DOI 10.7759/cureus.658 associated with a higher rate of inducing a worsening of hypopituitarism and higher recurrence rates [2]. Refractory prolactinoma is defined as a tumor in which dopamine agonist therapy is ineffective and where surgical resection is unsuccessful [3]. In addition to resistance to medical therapy, these tumors often demonstrate early recurrence and rapid local growth after surgery/radiotherapy [4]. Aggressive prolactinomas likely represent a further subset of refractory prolactinomas. Fortunately, aggressive pituitary adenomas are rare, comprising between 2.7%- 15% of all pituitary adenomas [3]. There is much debate and discussion regarding what constitutes an 'aggressive' adenoma and invasion [2-3]. Though there are no clear histologic features to confirm the diagnosis of an aggressive adenoma, the 2004 WHO pituitary tumor classification system defined 'atypical adenomas' that are distinct from benign adenomas but without distant or craniospinal metastatic disease, as observed with pituitary carcinomas. This histologic 'atypical adenoma' category attempts to assess aggressiveness of adenomas based on morphologic and growth-related features, including high mitotic index, increased immunohistochemical p53 staining, and an elevated Ki67 labeling index >3% [4]. The abnormal p53 staining most likely correlates with the TP53 mutation of the patient’s genetic sequencing data. Both findings are rare in adenomas and are associated with invasiveness and aggressive growth [5-6]. Although this patient’s tumor showed pathologic and clinical features corresponding with atypical adenoma, this variant is not consistent in identifying clinically aggressive adenomas, and debate remains regarding the definition and true incidence of these tumors due to the lack of consensus on diagnostic criteria [2-3]. Morphologic (hormonal) classification remains the most reliable method to predict behavior. Different pathologic criteria for atypia may need to be developed to reflect these unique tumor types. Refractory prolactinoma is defined as a tumor in which dopamine agonist therapy is ineffective and where surgical resection is unsuccessful [3]. In addition to resistance to medical therapy, In a retrospective study of 410 patients where characteristics that were predictive of aggressive behavior were defined, Trouillas, et al., proposed a classification system based on combining histochemical proliferative markers (two or more of the following: Ki67 index ≥3%; >2 mitoses per 10 HPFs; or p53 immunopositivity) and radiographic evidence of cavernous or sphenoid sinus invasion [7]. Using the Trouillas criteria, patients with Grade 2b (invasive and proliferative) adenomas had a higher probability of persistence or recurrence of 12-fold and 25- fold, respectively [7]. Review Prolactinomas, which represent 40% of all pituitary tumors, are the most common variant, with an incidence of 27 cases per million persons per year and a higher incidence in females of child- bearing age [1-2]. Lactotrophs within the tumor are inhibited by endogenous dopamine or exogenous dopamine agonists [2]. This release of prolactin stimulates milk production, inhibits gonadotropin-releasing hormone secretion, and directly impairs gonadal steroidogenesis, thereby causing the typical symptoms of hypogonadism, amenorrhea, and galactorrhea [1-2]. Prolactinomas are typically benign and remain stable or slowly increase in size, rarely causing mass effect symptoms [2]. When mass effect is present, visual symptoms related to optic chiasm compression may occur in addition to possible hypopituitarism or stalk compression [2]. As the majority of prolactinomas are benign microadenomas (<1cm), primary treatment is aimed at controlling the secretion of prolactin using dopamine agonists such as cabergoline or bromocriptine [2]. These pharmacologic approaches are effective, reducing serum prolactin levels by nearly 50% in nearly all patients and the size of the tumor by greater than 25% in the vast majority of patients [2]. In prolactinomas with mass effect or resistance to dopamine agonist medical therapy, surgery/radiosurgery may be indicated, though these therapies may be 2016 Moisi et al. Cureus 8(6): e658. DOI 10.7759/cureus.658 12 of 17 2016 Moisi et al. Cureus 8(6): e658. DOI 10.7759/cureus.658 This study also found that radiographic evidence of cavernous or sphenoid sinus invasion alone was superior to histologic markers when assessing the degree of aggressiveness of these adenomas [7]. In our case, despite only modest elevation levels in prolactin, the tumor showed progressively more aggressive characteristics, including cavernous sinus and intracranial invasion, histologic evidence of increased mitosis and markedly elevated proliferation index, and p53 positivity. In this regard, the tumor recurred multiple times despite medical therapy, multiple surgical resections, and radiosurgery. Although currently there is not a single, reliable immunohistochemical marker, imaging characteristic or universally accepted definition for aggressive adenomas; it is believed that benign adenomas may undergo genetic transformation, which may lead to invasive behavior and may eventually transform into malignant pituitary carcinomas [4, 7-8]. In our case, the tumor initially responded to medical therapy and subsequent debulking, with extended periods of clinical quiescence. However, after several years, the tumor became highly invasive and showed clinical and radiographic growth over a matter of weeks. Although there has been rapid accumulation of data on the molecular and genetic findings in pituitary adenomas, no common mutational events have been found to explain tumorigenesis [9]. Furthermore, studies have identified few markers that predict invasiveness or proliferation. However, there are retrospective studies that have demonstrated transformation of previously benign tumors into pituitary carcinoma after recurrence [4]. The challenge of treating an aggressive adenoma is inherent in its diagnosis – these tumors are resistant to medical, surgical, and radiotherapy treatments. When approaching benign 13 of 17 2016 Moisi et al. Cureus 8(6): e658. DOI 10.7759/cureus.658 micro and macroprolactinomas, initial treatment is a dopamine agonist such as cabergoline or bromocriptine, both of which have been shown to reduce tumor size, normalize serum prolactin levels, and restore gonadal function in more than 70-95% of patients [2, 5, 8]. If the tumor increases in size or hyperprolactinemia does not resolve with pharmacologic treatment, transsphenoidal surgical resection should be considered [2, 8]. As previously discussed, pharmacologically or surgically resistant tumors, especially with cavernous or sphenoid sinus invasion, should be monitored closely for recurrence [2, 7-8]. When clinical and radiographic evidence leads to the diagnosis of an aggressive adenoma, repeated surgical debulking can be helpful, though these treatments are palliative and often will lead to surgical complications including visual field deficits, infection, cerebrospinal fluid leaks, and panhypopituitarism [4]. Medically resistant and potentially aggressive prolactinomas are more commonly seen with macroprolactinomas (>1 cm), which, in one retrospective study were found to have a disappointingly low prolactin normalization rate of 36% after surgical resection, as opposed to prolactin normalization in 75% of medically refractory microprolactinomas [5]. Radiotherapy is often used in conjunction with surgery to treat aggressive or recurrent pituitary adenomas. Radiotherapy aims to slow or stop tumor growth, restore normal prolactin levels, and minimize radiation dose toxicity [6]. Radiotherapy modalities include external beam radiation therapy (EBRT) and stereotactic radiosurgery (SRS). Both have been shown to be approximately equivalent in normalizing hyperprolactinemia (34.1% for EBRT vs. 31.4% for SRS) [6]. Unfortunately, prolactinomas are the least radiosensitive pituitary tumors, with one study revealing only an 18% remission rate at four years for prolactinomas treated with Gamma Knife radiosurgery [5]. Interestingly, Cohen-Inbar et al., found that Gamma Knife radiosurgery successfully normalized prolactin levels in 50% of patients with dopamine-agonist resistant prolactinomas, although cavernous sinus invasion was a significant predictor of failure to achieve normal prolactin levels [10]. TMZ, an oral alkylating chemotherapeutic drug used primarily for the treatment of brain tumors such as glioblastoma multiforme, has been used for the past 10 years for pituitary carcinomas and aggressive pituitary adenomas [11]. TMZ primarily acts by methylating DNA bases, thereby inducing DNA fragmentation by base mismatch of the repair enzymes. TMZ was reported by Syro et al., in 2006 to successfully treat an aggressive prolactinoma and several cases have been described in the literature since [9, 12]. It is believed that expression of the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) by tumor cells can predict clinical response to TMZ therapy, with multiple studies demonstrating a strong inverse relationship between low MGMT expression and high responsiveness to TMZ and conversely, high MGMT expression with resistance to TMZ [3, 12-13]. TMZ is typically dosed at 150–200 mg/m2 for five days in 28-day cycles [11-12]. Several studies have demonstrated that lack of response after three cycles, either in tumor size or prolactin levels, is predictive of a poor outcome [12]. The literature contains prior reports of aggressive pituitary adenomas successfully treated with TMZ, most with rapid shrinkage of the tumor and reduction of serum prolactin levels demonstrated in Table 1 [13-26]. Aggressive prolactinomas appear to have the highest response rate to TMZ at 73%, followed by ACTH-secreting tumors and non-functioning adenomas [13]. TMZ, an oral alkylating chemotherapeutic drug used primarily for the treatment of brain tumors such as glioblastoma multiforme, has been used for the past 10 years for pituitary carcinomas and aggressive pituitary adenomas [11]. TMZ primarily acts by methylating DNA bases, thereby inducing DNA fragmentation by base mismatch of the repair enzymes. TMZ was reported by Syro et al., in 2006 to successfully treat an aggressive prolactinoma and several cases have been described in the literature since [9, 12]. It is believed that expression of the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) by tumor cells can predict clinical response to TMZ therapy, with multiple studies demonstrating a strong inverse relationship between low MGMT expression and high responsiveness to TMZ and conversely, high MGMT expression with resistance to TMZ [3, 12-13]. TMZ is typically dosed at 150–200 Author [reference] Year Number of Patients with PRL Secreting Adenomas Treatment Modality Follow- up Outcome 2016 Moisi et al. Cureus 8(6): e658. DOI 10.7759/cureus.658 14 of 17 2016 Moisi et al. Cureus 8(6): e658. DOI 10.7759/cureus.658 14 of 17 14 of 17 Disclosures Conflicts of interest: The authors have declared that no conflicts of interest exist. Conclusions TMZ for the treatment of aggressive pituitary adenomas, especially prolactinomas, has shown promise in controlling these invasive and highly aggressive tumors. Based upon reported anecdotal experience, TMZ may be more effective than radiotherapy as a second-line treatment after surgery. Although this patient’s tumor is an example of atypical adenoma histology and immunohistochemistry, more research is needed to better define and discover accurate biomarkers for aggressive pituitary adenomas for earlier diagnosis and prediction of treatment response. Future prospective clinical trials should be considered to evaluate the role of TMZ in the management of patients with aggressive pituitary adenomas. The relationship between MGMT and TMZ sensitivity also requires further understanding and may lead to better patient selection and tailored treatments for these invasive tumors. TABLE 1: Literature Review of Aggressive Pituitary Adenomas Successfully Treated with TMZ PRL= prolactin RT = radiotherapy TMZ = temazolamide PRL= prolactin RT = radiotherapy TMZ = temazolamide In our case, the patient had an aggressive prolactinoma resistant to dopamine agonist pharmacotherapy, with recurrence after multiple surgical resections and radiotherapy. TMZ was started as a salvage therapy for recurrent mass effect and neurological deficits in conjunction with radiosurgery. After eight cycles of TMZ, the patient responded with excellent clinical results on her MRIs demonstrating continued radiographic regression of her tumor coincident with clinical improvement. The excellent response rate of progressive pituitary adenomas treated with TMZ therapy, combined with the relatively low morbidity of this drug, raises the question as to whether TMZ should be considered as an initial component of treatment at the time tumor recurrence is detected. Bengtsson D, et al. [27] 2015 11 TMZ + resection, 8 with RT 4-91 months 7/11 responded to TMX therapy. 4/11 with progressive growth despite TMX therapy. Bruno, et al. [10] 2015 1 TMZ + resection + RT 1 month Deceased within one month of starting TMZ therapy Bush, et al. [14] 2010 1 TMZ + resection + RT unknown 80% reduction in tumor volume, clinical improvement Byrne, et al. [15] 2009 1 TMZ + resection + RT 6 months Radiographic and clinical response with stable size and prolactin levels Fadul, et al. [16] 2006 1 TMZ + resection + RT 15 months Radiographic and clinical response remaining stable at 15 month follow-up Hagen, et al. [17] 2009 2 TMZ + resection 12-34 months Radiographic and clinical response with no increase in tumor size after cessation of TMZ treatment Kovacs, et al. [18] 2006 1 TMZ + resection + RT 10 months Radiographic and clinical response Lim, et al. [19] 2006 1 TMZ + resection + RT 24 months Radiographic and clinical response with stable size and prolactin levels Losa, et al. [20] 2010 2 TMZ + resection + RT 12-21 months 1 with radiographic and clinical response and tumor control at last followup at 12 months, 1 with stable tumor size and no tumor control at last follwup at 21 months. Both patients living at last follow-up. McCormack, et al. [21] 2009 1 TMZ + resection + RT 4 months 1 patient with good radiographic and clinical response Murakami, et al. [22] 2011 1 TMZ + resection + RT 11 months Initial radiographic and clinical response with subsequent tumor growth and death Neff, et al. [24] 2007 1 TMZ + resection + RT 26 months Radiographic and clinical response with stable size and prolactin levels on chronic TMZ therapy Whitelaw, et al. [3] 2012 3 TMZ + resection 18-24 months Radiographic and clinical response Zemmoura, et al. [26] 2012 1 TMZ + resection + RT unknown Failure to respond 2016 Moisi et al. Cureus 8(6): e658. DOI 10.7759/cureus.658 15 of 17 2016 Moisi et al. Cureus 8(6): e658. DOI 10.7759/cureus.658 8. Di Ieva A, Rotondo F, Syro LV, Cusimano MD, Kovacs K: Aggressive pituitary adenomas-- diagnosis and emerging treatments. Nat Rev Endocrinol. 2014, 10:423–435. 10.1038/nrendo.2014.64 9. Syro LV, Uribe H, Penagos LC, et al.: Antitumour effects of temozolomide in a man with a large, invasive prolactin-producing pituitary neoplasm. Clin Endocrinol (Oxf). 2006, 65:552– 553. 10.1111/j.1365-2265.2006.02653.x 10. Cohen-Inbar O, Xu Z, Schlesinger D, Vance ML, Sheehan JP: Gamma Knife radiosurgery for medically and surgically refractory prolactinomas: long-term results. Pituitary. 2015, 18:820– 830. 10.1007/s11102-015-0658-1 11. Bruno OD, Juárez-Allen L, Christiansen SB et al.: Temozolomide therapy for aggressive pituitary tumors: results in a small series of patients from Argentina. Int J Endocrinol. 2015, 2015:587893. 10.1155/2015/587893 12. Ghazi AA, Rotondo F, Kovacs K, et al.: Treatment of invasive silent somatotroph pituitary adenoma with temozolomide. Report of a case and review of the literature. Endocr Pathol. 2015, 26:135–139. 10.1007/s12022-015-9361-z 13. Liu JK, Patel J, Eloy JA: The role of temozolomide in the treatment of aggressive pituitary tumors. J Clin Neurosci. 2015, 22:923–929. 10.1016/j.jocn.2014.12.007 14. Bush ZM, Longtine JA, Cunningham T, et al.: Temozolomide treatment for aggressive pituitary tumors: correlation of clinical outcome with O(6)-methylguanine methyltransferase (MGMT) promoter methylation and expression. J Clin Endocrinol Metab. 2010, 95:E280–E290. 10.1210/jc.2010-0441 15. Byrne S, Karapetis C, Vrodos N: A novel use of temozolomide in a patient with malignant prolactinoma. J Clin Neurosci. 2009, 16:1694–1696. 10.1016/j.jocn.2009.05.013 16. Fadul CE, Kominsky AL, Meyer LP, et al.: Long-term response of pituitary carcinoma to temozolomide. Report of two cases. J Neurosurg. 2006, 105:621–626. 10.3171/jns.2006.105.4.621 17. Hagen C, Schroeder HD, Hansen S, Hagen C, Andersen M: Temozolomide treatment of a pituitary carcinoma and two pituitary macroadenomas resistant to conventional therapy. Eur J Endocrinol. 2009, 161:631–637. 10.1530/eje-09-0389 18. Kovacs K, Horvath E, Syro LV, et al.: Temozolomide therapy in a man with an aggressive prolactin-secreting pituitary neoplasm: morphological findings. Hum Pathol. 2007, 38:185– 189. 10.1016/j.humpath.2006.07.014 19. Lim S, Shahinian H, Maya MM, Yong W, Heaney AP: Temozolomide: a novel treatment for pituitary carcinoma. Lancet Oncol. 2006, 7:518–520. 10.1016/s1470-2045(06)70728-8 20. Losa M, Mazza E, Terreni MR, et al.: Salvage therapy with temozolomide in patients with aggressive or metastatic pituitary adenomas: experience in six cases. Eur J Endocrinol. 2010, 163:843–851. 10.1530/eje-10-0629 21. McCormack AI, McDonald KL, Gill AJ, et al.: Low O6-methylguanine-DNA methyltransferase (MGMT) expression and response to temozolomide in aggressive pituitary tumours. Clin Endocrinol (Oxf). 2009, 71:226–233. 10.1111/j.1365-2265.2008.03487.x 22. References 1. Glezer A, Bronstein MD: Prolactinomas. Endocrinol Metab Clin North Am. 2015, 44:71–78. 10.1016/j.ecl.2014.11.003 2. Casanueva FF, Molitch ME, Schlechte JA, et al.: Guidelines of the Pituitary Society for the diagnosis and management of prolactinomas. Clin Endocrinol (Oxf). 2006, 65:265–273. 10.1111/j.1365-2265.2006.02562.x 3. Whitelaw BC, Dworakowska D, Thomas NW, et al.: Temozolomide in the management of dopamine agonist-resistant prolactinomas. Clin Endocrinol (Oxf). 2012, 76:877–886. 10.1111/j.1365-2265.2012.04373.x 4. Chatzellis E, Alexandraki KI, Androulakis II, Kaltsas G: Aggressive pituitary tumors. Neuroendocrinology. 2015, 101:87–104. 10.1159/000371806 5. Oh MC, Aghi MK: Dopamine agonist-resistant prolactinomas. J Neurosurg. 2011, 114:1369– 1379. 10.3171/2010.11.jns101369 6. Sheplan Olsen LJ, Robles Irizarry L, Chao ST, et al.: Radiotherapy for prolactin-secreting pituitary tumors. Pituitary. 2012, 15:135–145. 10.1007/s11102-011-0348-6 7. Trouillas J, Roy P, Sturm N, et al.: A new prognostic clinicopathological classification of pituitary adenomas: a multicentric case-control study of 410 patients with 8 years post- operative follow-up. Acta neuropathol. 2013, 126:123–135. 10.1007/s00401-013-1084-y 2016 Moisi et al. Cureus 8(6): e658. DOI 10.7759/cureus.658 16 of 17 2016 Moisi et al. Cureus 8(6): e658. DOI 10.7759/cureus.658 Murakami M, Mizutani A, Asano S, et al.: A mechanism of acquiring temozolomide resistance during transformation of atypical prolactinoma into prolactin-producing pituitary carcinoma: case report. Neurosurgery. 2011, 68:E1761–E1767. 10.1227/NEU.0b013e318217161a 23. Neff LM, Weil M, Cole A, et al.: Temozolomide in the treatment of an invasive prolactinoma resistant to dopamine agonists. Pituitary. 2007, 10:81–86. 10.1007/s11102-007-0014-1 23. Neff LM, Weil M, Cole A, et al.: Temozolomide in the treatment of an invasive prolactinoma resistant to dopamine agonists. Pituitary. 2007, 10:81–86. 10.1007/s11102-007-0014-1 24. Syro LV, Ortiz LD, Scheithauer BW, et al.: Treatment of pituitary neoplasms with temozolomide: a review. Cancer. 2011, 117:454–462. 10.1002/cncr.25413 24. Syro LV, Ortiz LD, Scheithauer BW, et al.: Treatment of pituitary neoplasms with temozolomide: a review. Cancer. 2011, 117:454–462. 10.1002/cncr.25413 25. Zemmoura I, Wierinckx A, Vasiljevic A, Jan M, Trouillas J, François P: Aggressive and malignant prolactin pituitary tumors: pathological diagnosis and patient management. Pituitary. 2013, 16:515–522. 10.1007/s11102-012-0448-y 26. Bengtsson D, Schrøder HD, Andersen M, et al.: Long-term outcome and MGMT as a predictive marker in 24 patients with atypical pituitary adenomas and pituitary carcinomas given treatment with temozolomide. J Clin Endocrinol Metab. 2015, 100:1689–1698. 10.1210/jc.2014-4350 2016 Moisi et al. Cureus 8(6): e658. DOI 10.7759/cureus.658 17 of 17
https://openalex.org/W3186114942	https://cronfa.swan.ac.uk/Record/cronfa57450/Download/57450__20678__adec15fb855d42fea66ae045ff2e8ce1.pdf	English	null	Scaling of axial muscle architecture in juvenile <i>Alligator mississippiensis</i> reveals an enhanced performance capacity of accessory breathing mechanisms	Journal of anatomy	2,021	cc-by	11,561	O R I G I N A L A R T I C L E O R I G I N A L A R T I C L E Kayleigh A. R. Rose1 \| Peter G. Tickle2 \| Ruth M. Elsey3 \| William I. Sellers4 \| Dane A. Crossley II5 \| Jonathan R. Codd6 1Department of Biosciences, College of Science, Swansea University, Wales, UK 2School of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds, Leeds, UK 3Louisiana Department of Wildlife and Fisheries, Rockefeller Wildlife Refuge, Grand Chenier, LA, USA 4Department of Earth and Environmental Sciences, Faculty of Science and Engineering, University of Manchester, Manchester, UK 5Department of Biological Sciences, University of North Texas, Denton, TX, USA 6Faculty of Biology, Medicine and Health, University of Manchester, Manchester, UK Correspondence Jonathan R Codd, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, UK. Email: jonathan.codd@manchester.ac.uk Funding information Biotechnology and Biological Sciences Research Council, Grant/Award Number: BB/I021116/1; National Science Foundation, Grant/Award Number: 17565187 1Department of Biosciences, College of Science, Swansea University, Wales, UK 2School of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds, Leeds, UK 3Louisiana Department of Wildlife and Fisheries, Rockefeller Wildlife Refuge, Grand Chenier, LA, USA 4Department of Earth and Environmental Sciences, Faculty of Science and Engineering, University of Manchester, Manchester, UK 5Department of Biological Sciences, University of North Texas, Denton, TX, USA 6Faculty of Biology, Medicine and Health, University of Manchester, Manchester, UK Correspondence Jonathan R Codd, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, UK. Email: jonathan.codd@manchester.ac.uk Funding information Biotechnology and Biological Sciences Research Council, Grant/Award Number: BB/I021116/1; National Science Foundation, Grant/Award Number: 17565187 This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2021 The Authors. Journal of Anatomy published by John Wiley & Sons Ltd on behalf of Anatomical Society. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2021 The Authors Journal of Anatomy published by John Wiley & Sons Ltd on behalf of Anatomical Society Journal of Anatomy. 2021;00:1–14. Received: 16 November 2020 \| Revised: 8 July 2021 \| Accepted: 12 July 2021 DOI: 10.1111/joa.13523 Received: 16 November 2020 \| Revised: 8 July 2021 \| Accepted: 12 July 2021 DOI: 10.1111/joa.13523 Received: 16 November 2020 \| Revised: 8 July 2021 \| Accepted: 12 July 2021 Received: 16 November 2020 \| Revised: 8 July 2021 \| Accepted: 12 July 2021 DOI: 10 1111/joa 13523 DOI: 10.1111/joa.13523 Scaling of axial muscle architecture in juvenile Alligator mississippiensis reveals an enhanced performance capacity of accessory breathing mechanisms Kayleigh A. R. Rose1 \| Peter G. Tickle2 \| Ruth M. Elsey3 \| William I. Sellers4 \| Dane A. Crossley II5 \| Jonathan R. Codd6 O R I G I N A L A R T I C L E lished by John Wiley & Sons Ltd on behalf of Anatomical Society. he terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, y cited der the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium perly cited. 1 \| INTRODUCTION intermittently correlated, or even uncorrelated with, the gait cycle (Farmer & Carrier, 2000a,b). Breathing flexibility in crocodilians is facilitated by the more derived components of the respiratory system which are also the principal muscles controlling dive performance. It remains conten- tious, however, as to which function evolved first (Uriona & Farmer, 2006). The accessory diaphragmaticus is a thin muscle that encases the viscera with straps originating from the cranial aspect of the is- chia and caudal-most gastralia within the rectus abdominis (Boelaert, 1942; Farmer & Carrier, 2000a; Gans & Clark, 1976; Munns et al., 2012) or epipubis (Fechner & Schwarz-Wings, 2013; Gans & Clark, 1976). Contraction of the diaphragmaticus pulls the liver caudad and increases thoracic volume facilitating inspiration (Farmer & Carrier, 2000a; Gans & Clark, 1976; Naifeh et al., 1970). Inspiration can also be facilitated by pubic muscles (ischiopubis, ischiotruncus and trun- cocaudalis) that rotate the pubic plates ventrocaudally to increase abdominal volume (Claessens, 2009). In expiration, the rectus ab- dominis may also be recruited to displace the gastralia cranially, push the viscera craniodorsally and rotate the pubic plates craniodorsally to expel air (Claessens, 2009; Gans & Clark, 1976). Furthermore, in forced expiration, it was recently demonstrated that the epaxial ilio- costalis can be recruited (Codd et al., 2019). The iliocostalis lies with a myotomal arrangement across and between the vertebral rib ele- ments embedding uncinate processes, which are accessory breath- ing structures ancestral to archosaurs (Codd et al., 2019). While costal aspiration and diaphragmatic visceral translation are the principal mechanisms for the control of tidal volume in crocodilians (Claessens, 2009; Gans & Clark, 1976; Munns et al., 2012; Uriona & Farmer, 2008), their contributions can vary greatly, and the con- straints underlying this remain poorly studied. Energetic demand, body temperature, digestive state and being partially or fully sub- merged in water all influence muscle recruitment. However, these factors often covary in studies, making it difficult to understand their independent effects upon breathing (Codd et al., 2019; Gans & Clark, 1976; Munns et al., 2012; Uriona & Farmer, 2006). Vertebrate trunk muscles can have multifaceted functions in loco- motion, body support and respiration (Carrier, 1987; Codd et al., 2005; Farmer & Carrier, 2000a; O'Reilly et al., 2000; Schilling, 2011). 1 \| INTRODUCTION Understanding the inter- and intra-specific variations in the functional anatomy of the trunk can reveal adaptations that facilitate simultaneous breathing and locomotion (Brainerd & Owerkowicz, 2006; Carrier, 1991; Klein & Codd, 2010; Lambertz & Perry, 2015). Crocodilians are an interesting group in which to examine these adaptations as they have diverse breathing mechanics and locomotor modes (Codd et al., 2005; Codd et al., 2019; Farmer & Carrier, 2000a; Gans & Clark, 1976). However, the plasticity of these systems is little understood and there is currently no quantitative data on the functional anatomy of the crocodilian trunk. Lung ventilation in crocodilians is facilitated via contractions of trunk muscles that control costal rotation, visceral displacement, pelvic rotation, vertebral flexion and translation of gastralia on the ventral surface (Claessens, 2009; Codd et al., 2019; Farmer & Carrier, 2000a; Gans & Clark, 1976; Naifeh et al., 1970). Two components of this system are ancestral. One, the sub-costal transversus abdominis, is an expiratory pump, basal to tetrapods (Brainerd, 1999). It con- tracts bilaterally to push the liver–lung complex anteriorly and ro- tate the mobile and elongated pubic plates craniodorsally causing expulsion of air (Gans & Clark, 1976). The other, the costal aspiration pump, is ancestral to amniotes (Brainerd, 1999) and involves bilateral intercostal muscle contraction to rotate the tripartite ribs cranio- laterally or caudomedially in inspiration or expiration, respectively (Brocklehurst et al., 2017; Gans & Clark, 1976). In the fish ancestors of amniotes, both the transversus and costal pumps functioned orig- inally in lateral bending of the trunk for propulsion. With the evolu- tion of the respiratory roles of these pumps came a biomechanical constraint on simultaneous breathing and locomotion for early am- niotes, Carrier's constraint (Carrier, 1987, 1991). However, most ex- tant amniote groups have evolved accessory breathing mechanisms to overcome this constraint (Brainerd & Owerkowicz, 2006; Codd & Klein, 2010; Klein & Codd, 2010). In crocodilians, breathing and locomotion are decoupled by their upright gait, derived accessory breathing muscles and transverse processes on the vertebrae that function as attachment sites for epaxial muscles, thereby reduc- ing lateral trunk bending (Farmer & Carrier, 2000b). Crocodilians can, therefore, fine-tune ventilatory rate with activity that is only Another variable which confounds experimental results on phys- iological performance is body size which can constrain functional anatomy (Schmidt-Nielsen, 1984). K E Y W O R D S K E Y W O R D S allometry, archosaur, axial anatomy, breathing, crocodilian, flexibility, locomotion, muscle architecture, ventilatory mechanics mechanics as they increase in size. Furthermore, the functional specialisation of the diaphragmaticus and compliance of the body wall in the lumbar region against which it works may contribute to low-cost breathing in crocodilians. Abstract Abstract Quantitative functional anatomy of amniote thoracic and abdominal regions is cru- cial to understanding constraints on and adaptations for facilitating simultaneous breathing and locomotion. Crocodilians have diverse locomotor modes and variable breathing mechanics facilitated by basal and derived (accessory) muscles. However, the inherent flexibility of these systems is not well studied, and the functional spe- cialisation of the crocodilian trunk is yet to be investigated. Increases in body size and trunk stiffness would be expected to cause a disproportionate increase in mus- cle force demands and therefore constrain the basal costal aspiration mechanism, necessitating changes in respiratory mechanics. Here, we describe the anatomy of the trunk muscles, their properties that determine muscle performance (mass, length and physiological cross-sectional area [PCSA]) and investigate their scaling in juvenile Alligator mississippiensis spanning an order of magnitude in body mass (359 g–5.5 kg). Comparatively, the expiratory muscles (transversus abdominis, rectus abdominis, ili- ocostalis), which compress the trunk, have greater relative PCSA being specialised for greater force-generating capacity, while the inspiratory muscles (diaphragmaticus, truncocaudalis ischiotruncus, ischiopubis), which create negative internal pressure, have greater relative fascicle lengths, being adapted for greater working range and contrac- tion velocity. Fascicle lengths of the accessory diaphragmaticus scaled with positive allometry in the alligators examined, enhancing contractile capacity, in line with this muscle's ability to modulate both tidal volume and breathing frequency in response to energetic demand during terrestrial locomotion. The iliocostalis, an accessory expira- tory muscle, also demonstrated positive allometry in fascicle lengths and mass. All accessory muscles of the infrapubic abdominal wall demonstrated positive allometry in PCSA, which would enhance their force-generating capacity. Conversely, the basal tetrapod expiratory pump (transversus abdominis) scaled isometrically, which may in- dicate a decreased reliance on this muscle with ontogeny. Collectively, these find- ings would support existing anecdotal evidence that crocodilians shift their breathing 3Louisiana Department of Wildlife and Fisheries, Rockefeller Wildlife Refuge, Grand Chenier, LA, USA Funding information Biotechnology and Biological Sciences Research Council, Grant/Award Number: BB/I021116/1; National Science Foundation, Grant/Award Number: 17565187 \| 1 wileyonlinelibrary.com/journal/joa Journal of Anatomy. 2021;00:1–14. 2 \| 2 \| 2 \| ROSE et al. \| 3 ROSE et al. 3 TA B LE 1 Specimen body size metrics and other measurements taken size and stiffening of the trunk, muscle force demands increase dis- proportionately, and the basal costal aspiration pump is expected to be constrained. It was hypothesised by Munns et al (2012) that larger crocodilians, in response, may rely more upon diaphragmatic breathing. Investigating the functional specialisation of the trunk muscles and any changes during early ontogeny may shed more light on this (Fechner & Schwarz-Wings, 2013; Gans, 1976; Munns et al., 2012). Muscle architecture (arrangement and geometric properties of the fascicles) is directly linked to performance allowing indirect inference of changes in functional output potential (Bodine et al., 1982; Gans, 1982; Gans & Bock, 1965; Lieber & Friden, 2000; Lieber & Ward, 2011; Roy et al., 1984; Sacks & Roy, 1982). For example, maximum force generation is proportional to effective physiological cross-sectional area (Roy et al., 1984), and maximum working range and contraction velocity are proportional to fascicle length (Bodine et al., 1982; Winters et al., 2011). However, there is an inherent trade-off between muscle strength and contractility when it comes to muscle design, which worsens with increasing body size. Muscles, therefore, tend to be specialised either to have more fibres in se- ries (for strength) or more length to their fascicles (for contractility/ power) and often demonstrate allometric scaling of their properties to meet functional demands. 2.2 \| Dissection and measurement protocols The dermis was cut at the sternum and peeled around the trunk to expose the musculoskeletal anatomy. Muscles of the left side of the trunk were then examined and dissected following descriptions of Maurer (1896), Gasc (1981), Frey (1988), Murakami et al., (1991), Farmer and Carrier (2000a), and Tsuihiji (2007). Any details of the anatomy not found in the literature were recorded. Muscles were dissected sequentially: (a) truncocaudalis, ischiotruncus, ischiopubis (ventral aspect); (b) obliquus externus superficialis, obliquus externus profundus, iliocostalis (lateral aspect); (c) rectus abdominis (ventral aspect); (d) diaphragmaticus; (e) obliquus internus (lateral aspect); (f) transversus abdominis (medial aspect). In order to inspect the trans- versus abdominis and any intercostal muscles remaining on the ribs from the medial aspect, the ribs were removed from the trunk by cutting along their vertebral articulations. \| 3 2.2 \| Dissection and measurement protocols taken ID Mb (kg) SVL (m) TL (m) Muscle architecture Mass of gastralia 11 0.359 0.240 0.495 Y 109 0.592 0.280 0.593 Y 1 0.575 0.281 0.535 Y 110 0.810 0.288 0.625 Y 4 0.763 0.300 0.625 Y 105 1.125 0.320 0.695 Y 106 0.889 0.320 0.675 Y 104 0.889 0.323 0.704 Y 108 0.920 0.330 0.710 Y 103 1.042 0.336 0.723 Y 2 1.019 0.342 0.723 Y 102 1.125 0.350 0.753 Y 107 0.986 0.350 0.757 Y 3 1.200 0.355 0.745 Y 111 1.386 0.367 0.773 Y 101 1.533 0.370 0.790 Y 6 1.765 0.396 0.833 Y 112 1.840 0.407 0.848 Y 5 2.456 0.420 0.903 Y 10 2.817 0.450 0.950 Y 8 2.995 0.450 0.960 Y 9 3.611 0.470 0.995 Y 7 3.658 0.480 1.020 Y L2_16 3.575 0.508 1.048 Y L1_9 4.552 0.514 1.092 Y L3_150 5.497 0.546 1.207 Y 158 7.257 0.591 1.225 Y 58 7.484 0.597 1.270 Y 19 7.700 0.616 1.270 Y Our principal objective here was to investigate the functional specialization of the trunk muscles in juvenile American alligators, Alligator mississippiensis and scaling of muscle properties in speci- mens spanning an order of magnitude in body mass (359 g–.5 kg). Similar work on the appendicular system has revealed a general trend towards a reduction in musculoskeletal capacity for terres- trial performance in the Alligatoridea, Gavialidae and Crocodylidae (Allen et al., 2010; Allen et al., 2014; Dodson, 1975; Farlow et al., 2005; Iijima & Kubo, 2019; Livingston et al., 2009; Meers, 2002). Given the decreasing functional capacity of the appendicular system as development proceeds in crocodilians and the continued need to support breathing, swimming and diving with the trunk, we hypoth- esised that anatomical properties of the axial system will demon- strate positive allometric growth as muscle performance keeps pace with functional demand. Any differences in scaling between different components of the system may be indicative of changes in respiratory mechanics with increasing body size. We also provide functional muscle descriptions and discuss our findings in relation to empirical evidence on muscle functions in breathing and locomotion. 1 \| INTRODUCTION The need for detailed quanti- tative functional anatomy of the trunk in crocodilians of different body sizes has long been noted (Gans, 1976; Munns et al., 2012). Crocodilians undergo changes in body size of several orders of mag- nitude in growing continuously from hatching (~30 g, 15–20 cm) and through adulthood (200–400 kg, 3m+). With increasing body \| 3 -0.45 -0.30 -0.15 0.00 0.15 0.30 0.45 0.60 0.75 log body mass (kg) -0.70 -0.65 -0.60 -0.55 -0.50 -0.45 -0.40 -0.35 -0.30 -0.25 log SVL (m) R2 = 0.976 logSVL = 0.30logM - 0.48 Muscle mass (g, ±0.001), belly length (mm) and five fascicle lengths (mm) were recorded from each muscle using scales (Toledo XS204; Columbus, OH) and a measuring rule. Photographs were taken of the iliocostalis and rectus abdominis next to a 1 cm scale to quantify fascicle lengths using ImageJ software. Effective physiological cross-sectional area (PCSA) was calculated for each muscle using Equation 1: (1) PCSA ( cm2) = muscle mass (g) 휌(1.06 g cm −1) × average fascicle length (cm) (1) PCSA ( cm2) = muscle mass (g) 휌(1.06 g cm −1) × average fascicle length (cm) where ρ is the muscle density (Sacks & Roy, 1982). A muscle density of 1.06 g cm-3 was assumed as found in mammals (Mendez & Keys, 1960) and birds (Paxton et al., 2010) and has previously been used for croc- odilians (Allen et al., 2010; Allen et al., 2014) and monitor lizards (Cieri et al., 2020; Dick & Clemente, 2016). log body mass (kg) The rectus abdominis has two portions (the outer, embedding gas- tralia, and the inner, without gastralia (Bhullar, 2009; Farmer & Carrier, 2000a; Fechner & Schwarz-Wings, 2013)). There was no obvious sep- aration of the muscle into two parts and so it was dissected as one unit. Gastralia were not removed from the rectus abdominis before muscle mass was measured. Gastralia masses measured from three specimens of a similar snout-vent length (SVL) (Table 1) were used to estimate gastralial masses for all other specimens under the assumption that their masses scale isometrically. Whether or not gastralial mass was considered, conclusions from the results of subsequent analyses on scaling and relative muscle functional specialization were not affected and so we do not represent each graphically. FI G U R E 1 Scaling of snout-vent length with body mass in specimens ranging 0.359–5.497 kg 1–8. The vertebral rib elements are associated with three muscle lay- ers: (a) superficially and in the intercostal spaces, the IC (Figure 2b,c); (b) in the intercostal spaces, the IED (Figure 2d) and (c) beneath and dis- tally, crossing the joint between the vertebral and intermediate rib ele- ments, the ICID (Figure 2e,f). The intermediate and sternal rib elements are surrounded by five hypaxial layers. 2.1 \| Specimens Twenty-six female juvenile A. mississipiensis cadaveric specimens (Table 1, body mass: 0.359–5.497 kg; snout-vent length: 0.240– 0.546 m; total length: 0.496–1.297 m) were acquired after the completion of unrelated studies at the University of North Texas between 2016 and 2019. Dissections were carried out on the day of euthanasia. ROSE et al. FI G U R E 1 Scaling of snout-vent length with body mass in specimens ranging 0.359–5.497 kg -0.45 -0.30 -0.15 0.00 0.15 0.30 0.45 0.60 0.75 log body mass (kg) -0.70 -0.65 -0.60 -0.55 -0.50 -0.45 -0.40 -0.35 -0.30 -0.25 log SVL (m) R2 = 0.976 logSVL = 0.30logM - 0.48 2.3 \| Scaling analyses Scaling of absolute muscle architectural properties with body mass was investigated using a model II regression technique (reduced major axis) in R (v 3.6.1) via the ‘lmodel2 (1.7-3)’ package (R Core Team, 2019). In every case, log(muscle property) was regressed on log(body mass). For isometric scaling of an object, that is for an object to scale geometrically, all lengths would increase ∝ body mass(0.33), all cross-sectional areas ∝ body mass(0.66) and all volumes ∝ body mass(1.00). Scaling exponents and 95% CIs higher, lower or straddling these exponent values were considered representative of positive allometry, negative allometry and isometry, respectively. Body mass was chosen as the covariate rather than SVL, as SVL scaled with body mass0.30 (R2 = 0.98, p < 0.001, 95% CIs: 0.29–0.31), with negative allometry, not isometrically (Figure 1). 3.1.1 \| Obliquus externus superficialis The OES (Figure 2b) is immediately ventrad to the IC. The OES ex- tends along the lateral body wall in line with the intermediate and sternal rib elements from the first thoracic rib to the end of the lum- bar region. The pectoralis and TC attach superficially onto the OES at its cranial and caudal ends, respectively. A thick fat pad was stored between the pectoralis and EOS in all specimens. Deep to the pec- toralis, the OES is connected to the mesosternum by aponeurosis. Fascicles of the OES run only slightly obliquely, dorsocranial to ven- trocaudal. In the thoracic region, the OES attaches onto the lateral surface of the ventral IC and the medial surface of the dorsal fascia. The dorsal fascia terminates over the dorsal projections of the OES. In the lumbar region, the OES strongly attaches to the ventral aspect of the IC. The ventral margin of the OES is connected to the lateral edge of the outer portion of the RA. From superficial to deep, these include: (a) OES (Figure 2b); (b) OEP (Figure 2c); (c) between adjacent in- termediate rib elements only, the IEV (Figure 2d); (d) between adjacent intermediate and sternal ribs in the thoracic region, II (Figure 2e), and in the lumbar region, OI (Figure 2e) and (e) the TA and ICID (Figure 2f). On the ventral body, walls are the RA (Figure 2b,c, Figure 3a) and three muscles ventral to the pubic plates which, from superficial to deep, are the TC, ISCHU and ISP (Figure 3a,b). The DI (Figure 3c), TC, ISCHU and ISP have previously been described in detail for A. mississippiensis and are not described here (Boelaert, 1942; Farmer & Carrier, 2000a; Gans & Clark, 1976; Naifeh et al., 1971). 4 \| ROSE et al. 3.1 \| Functional muscle descriptions The tripartite ribs, including sternal, intermediate and vertebral ele- ments with uncinate processes, and muscle names and their abbrevia- tions are summarised in Figure 2. Uncinate processes were cartilaginous in these juvenile specimens and occurred on the vertebral rib elements \| 5 ROSE et al. FI G U R E 2 Lateral view of the anatomy of the trunk in Alligator mississippiensis. Anterior is to the left. a) The tripartite ribs 1–8 including the sternal (ventral), intermediate (lateral) and vertebral (dorsal) rib elements and their uncinate processes. Subsequent panels illustrate muscle layers from superficial to deep. b) The iliocostalis (IC) is covered by a thick fascia (the dorsal fascia) in the thoracic region which was only visible in specimens with SVL >0.40 m and body mass >1.77 kg. The obliquus externus superficialis (OES) attaches the ventrolateral IC, medial to the dorsal fascia; the rectus abdominis (RA) and the truncocaudalis (TC) attach to the ventrolateral rim of the OES and make up the ventral body wall. c) Following the removal of the dorsal fascia and OES, the IC lies with a myotomal arrangement across and between the verterbral ribs, while the obliquus externus profundus (OEP) is connected strongly to the ventral rim of the IC, dorsal lateral rims of the RA and TC. d) The intercostales externi dorsales and ventrales (IED and IEV) occupy the vertebral and intermediate intercostal spaces, respectively. The obliquus internus (OI) attaches to the ventral rim of the IC in the lumbar region (dotted line). e) The intercostales interni (II) occupy the spaces between both the sternal and the intermediate rib elements. The OI is continuous from the II. f) The intercostales internalis dorsalis longus (ICID) and transversus abdominis (TA). Notice how the TA extends dorsally only as far as the intermediate rib elements and the ICID occupy only the distal ends of the vertebral rib elements. 3.1 \| Functional muscle descriptions The transversus abdominis (TA) is medial to the OI and also attached to the ventral rim of the IC as well as the mediolateral edge of the RA together with the eighth and floating sternal rib (a) (d) (b) (e) (c) (f) dorsal fascia iliocostalis (IC) obliquus externus superficialis (OES) obliquus externus profundus (OEP) rectus abdominis (RA) truncocaudalis (TC) intercostales externi dorsales (IED) intercostales externi ventrales (IEV) intercostales interni (II) obliquus internus (OI) intercostales internales dorsales longi (ICID) transversus abdominis (TA) vertebral rib uncinate process intermediate rib sternal rib 1 8 ROSE et al. (a) ( (b) ( dorsal fascia obliquus externus superficialis (OES) rectus abdominis (RA) truncocaudalis (TC) vertebral rib uncinate process intermediate rib sternal rib 1 8 (d) (a) vertebral rib obliquus internus (OI) (e) (b) obliquus externus superficialis (OES) (c) (f iliocostalis (IC) obliquus externus profundus (OEP) (f) (c) iliocostalis (IC) obliquus externus profundus (OEP) (c) iliocostalis (IC) transversus abdominis (TA) obliquus externus profundus (OEP) FI G U R E 2 Lateral view of the anatomy of the trunk in Alligator mississippiensis. Anterior is to the left. a) The tripartite ribs 1–8 including the sternal (ventral), intermediate (lateral) and vertebral (dorsal) rib elements and their uncinate processes. Subsequent panels illustrate muscle layers from superficial to deep. b) The iliocostalis (IC) is covered by a thick fascia (the dorsal fascia) in the thoracic region which was only visible in specimens with SVL >0.40 m and body mass >1.77 kg. The obliquus externus superficialis (OES) attaches the ventrolateral IC, medial to the dorsal fascia; the rectus abdominis (RA) and the truncocaudalis (TC) attach to the ventrolateral rim of the OES and make up the ventral body wall. c) Following the removal of the dorsal fascia and OES, the IC lies with a myotomal arrangement across and between the verterbral ribs, while the obliquus externus profundus (OEP) is connected strongly to the ventral rim of the IC, dorsal lateral rims of the RA and TC. d) The intercostales externi dorsales and ventrales (IED and IEV) occupy the vertebral and intermediate intercostal spaces, respectively. The obliquus internus (OI) attaches to the ventral rim of the IC in the lumbar region (dotted line). e) The intercostales interni (II) occupy the spaces between both the sternal and the intermediate rib elements. The OI is continuous from the II. f) The intercostales internalis dorsalis longus (ICID) and transversus abdominis (TA). 3.1 \| Functional muscle descriptions Notice how the TA extends dorsally only as far as the intermediate rib elements and the ICID occupy only the distal ends of the vertebral rib elements. The transversus abdominis (TA) is medial to the OI and also attached to the ventral rim of the IC as well as the mediolateral edge of the RA together with the eighth and floating sternal rib Iliocostalis The OEP (Figure 2c) is located immediately medial to the OES. The dorsal cranial aspect of the OEP extends only as far as the second set of thoracic ribs, and ventrally, it exposes the first to five thoracic sternal rib portions. Fascicles of the OEP attach to the ventral rim of the IC so that the two portions of the external obliques effectively sandwich the ventrolateral rim of the IC. The OEP attaches strongly to the lateral aspect of the inner portion of the RA and the cranial rim of the TC. Fascicles of the OEP appear longer and run more obliquely, dorsocranial to ventrocaudal, than those belonging to the superficial portion and are continuous with those of the TC. Syn: iliocostalis dorsalis (Maurer, 1896; Tsuiji, 2007); iliocostalis dorsali (Frey, 1988). The epaxial IC (Figure 2b,c) lies across and between the thoracic vertebral ribs with a myotomal arrangement and extends through the lumbar region to the cranial aspect of the ilium. Each myoseptum of the IC originates from the transverse processes of the vertebrae and extends caudolaterally from the ribs. Fascicles of the IC run craniocaudally between myosepta. In the smallest speci- mens, the myotomal arrangement of the IC was not visible and the muscle appeared thin and continuous with the hypaxial musculature. In specimens with SVL >0.40 m and body mass >1.77 kg, the IC was covered by a thick fibrous fascia (the dorsal fascia) throughout the Syn: iliocostalis dorsalis (Maurer, 1896; Tsuiji, 2007); iliocostalis dorsali (Frey, 1988). The epaxial IC (Figure 2b,c) lies across and between the thoracic vertebral ribs with a myotomal arrangement and extends through the lumbar region to the cranial aspect of the ilium. Each myoseptum of the IC originates from the transverse processes of the vertebrae and extends caudolaterally from the ribs. Fascicles of the IC run craniocaudally between myosepta. In the smallest speci- mens, the myotomal arrangement of the IC was not visible and the muscle appeared thin and continuous with the hypaxial musculature. In specimens with SVL >0.40 m and body mass >1.77 kg, the IC was covered by a thick fibrous fascia (the dorsal fascia) throughout the 6 \| ROSE et al. FI G U R E 3 Ventral view of the muscles of the infrapubic abdominal wall in Alligator mississippiensis. a) The truncocaudalis (TC), ischiotruncus (ISCHU) and ischiopubis (ISP). 3.1.9 \| Intercostales internales dorsales longi Syn: transversus dorsalis (Murakami et al., 1991). The ICID (Figure 2f) are the deepest subcostal muscles located medially to the distal- vertebral and proximal-intermediate rib elements. The ICID are diamond shaped, roughly the size of one intercostal space, with fas- cicles running obliquely (cranioventral to caudodorsal). An aponeu- rotic sheath extends from either end. Ventrally, this sheath connects with the dorsal rim of the TA. Iliocostalis The ISP was always darker in colouration and a thick fat pad always lay beneath it. b) Following removal of the TC and ISCHU. The ISP and fat pad have also been removed from the specimen's left side, revealing the pubio-ischio femoralis (PIF). c) Origin of the diaphragmaticus on the last gastralial set 6 \| ROSE et ROSE et al. FI G U R E 3 Ventral view of the muscles of the infrapubic abdominal wall in Alligator mississippiensis. a) The truncocaudalis (TC), ischiotruncus (ISCHU) and ischiopubis (ISP). The ISP was always darker in colouration and a thick fat pad always lay beneath it. b) Following removal of the TC and ISCHU. The ISP and fat pad have also been removed from the specimen's left side, revealing the pubio-ischio femoralis (PIF). c) Origin of the diaphragmaticus on the last gastralial set 3.1.4 \| Rectus abdominis The RA (Figure 2b,c, Figure 3a), on the ventral body wall, is inter- rupted by multiple sets of gastralia. Fascicles of the RA run cranio- caudally between gastralial sets. The muscle is connected to the mesosternum, OES, OEP, TA, II, caudal sternal rib elements 7 and 8, OI, ISCHU, TC and pubic plates via the last gastralial set as is the DI. Both the OES and OEP are attached to the lateral aspects of the RA (outer and inner portions, respectively). The TC inserts onto the ventral surface of the RA about three sets of gastralia cranially, and the ISCHU approximately two sets cranially. The last gastralial set is attached to the pubis via a cartilaginous/ligamen- tous separation. The OI (Figure 2d,e) is immediately deep to the external obliques oc- cupying only the lumbar region. It is a thin muscle, continuous with the II, interrupted by only the 9th floating thoracic vertebral rib. The fascicle arrangement of the OI is very similar to that of the II and runs perpendicular to that of the external obliques. Dorsally, fascicles at- tach onto the ventral rim of the IC. Ventrally, the OI and the embed- ded sternal rib elements of thoracic ribs 7 and 8 are also connected to the inner portion of the RA at its lateral edges. 3.1.5 \| Intercostales externi dorsales Syn: intercostales externus proprius (Frey, 1988). Immediately medial to the IC lie the dorsal portions of the external intercostals, IED (Figure 2d). Fascicles of the IED also run craniocaudally between vertebral rib elements. Intercostales interni thoracic region and the myotomes of the IC beneath were visibly conspicuous. Cartilaginous uncinate processes were embedded within the myosepta of the IC on the eight thoracic vertebral ribs and their presence and size varied between specimens. The myosepta and uncinate processes became visibly broader with increasing SVL. The II (Figure 2e) occupies the intercostal spaces between the inter- mediate and sternal rib portions with fascicles running cranioventral to caudodorsal. 4.1 \| Axial musculature and the hepatic piston model of breathing 3.2.1 \| Functional specialisation of inspiratory and expiratory muscles 3.2.2 \| Scaling of muscle architectural properties Scaling exponents for muscle architectural properties versus body mass is shown in Figure 5 and statistical outputs in Table 2. Muscle mass scaled with positive allometry in only the IC, RA, TC and ISCHU and scaled isometrically in the OES, OEP, TA, DI and ISP. PCSA scaled with positive allometry in the RA, TC, ISCHU and ISP, indicating a potential for increasing force generation over develop- ment. PCSA scaled isometrically in the IC, OES, TA and DI. Muscle fascicle lengths scaled with positive allometry in the IC OES, and DI, indicating potential for an increased length range over which the muscles can generate force, and capacity for speed of contraction. By comparison, fascicle lengths scaled isometrically in the EOP, TA and RA, TC, ISCHU and ISP. Therefore, positive allometric scaling was characteristic of at least one architectural property of each of the accessory breathing muscles, and only one muscle, the TA scaled isometrically in all architectural properties. Specialisations of the axial muscles identified in our study are therefore consistent with the hepatic piston model. There were striking differences in specialisation between expiratory and inspi- ratory muscles. Compared to inspiratory muscles, the expiratory muscles (transversus abdominis, rectus abdominis, iliocostalis) showed adaptation for increased force generation, with greater relative PCSA. The expiratory muscles are, therefore, specialised for put- ting the cylinder under compression to speed up the expulsion of air when necessary. Here, expiratory work is divided between the myotomes of the iliocostalis, gastralial rows in the rectus abdominis and spread through the trunk in the transversus abdominis, meaning greater overall forces can be exerted. It should also be noted that the force-generating potential of the transversus abdominis is far ex- ceeded by that of both the iliocostalis and rectus abdominis that have assumed accessory roles in forced expiration. 3.2.1 \| Functional specialisation of inspiratory and expiratory muscles In the hepatic piston model of crocodilian breathing proposed by Gans and Clark (1976) and updated by Claessens (2009), the body walls are represented as a cylinder, which is expandable in the dor- soventral and mediolateral axes and compartmentalized into the thoracic and abdominal cavities. During inspiration, the intercostals rotate the ribs craniolaterally, the diaphragmaticus pulls the liver cau- dally and the infrapubic muscles rotate the pubic plates caudoven- trally, which together draw air into the lungs and increase the volume of the cylinder (Claessens, 2009; Farmer & Carrier, 2000a; Gans & Clark, 1976). During the non-ventilatory phase (when an inhalation is held and no axial muscles are active), the glottis is closed preventing passive exhalation via collapse of the thoracic walls (Naifeh et al., 1971) and the viscera are no longer being retracted by the diaphrag- maticus but are kept slightly caudad and compressed by the posi- tive pressure caused by the inspiratory air volume (Claessens, 2009). Relaxation of the glottal sphincter allows partial exhalation through passive elastic recoil of the thoracic walls and viscera (Claessens, 2009; Naifeh et al., 1971). However, experimental work suggests that the roles of the intercostals change according to surrounding media, rotating the ribs in the terrestrial environment and stabilising the ribs when submerged in water, allowing diaphragmatic breathing to take over. Furthermore, it seems that under stress, during exer- cise and possibly even while resting terrestrially in the absence of external hydrostatic pressure on the body, extra muscular force may be required from the abdominal walls (transversus abdominis and rec- tus abdominis) and dorsal epaxial musculature (iliocostalis) in order to speed up the caudomedial rotation of the ribs and dorsovental flat- tening of the cylinder to expel air (Codd et al., 2019; Gans & Clark, 1976; Uriona & Farmer, 2008). Relative muscle masses are given in Figure 4a. Plots of size- normalised PCSA versus size-normalised fascicle length (Figure 4b– d) demonstrated that, comparatively, muscles involved in expiration (TA, RA, IC) had the greatest relative effective PCSA thereby indi- cating greater force-generating capacity while inspiratory muscles (DI, TC, ISCHU, ISP) had greater relative fascicle lengths, indicative of enhanced working range and speed of contraction. The physio- logical functions of the EOS, EOP and IO are unknown. The external obliques are intermediate in relative mass, PCSA and fascicle lengths compared to muscles with known respiratory function indicating in- termediate capacities. \| 7 lumbar region, however, the TA also attaches to the ventral rim of the IC, immediately medial to the OI. A thin fascia separates the TA from the medial surface of the OI. lumbar region, however, the TA also attaches to the ventral rim of the IC, immediately medial to the OI. A thin fascia separates the TA from the medial surface of the OI. empirical evidence (partially summarised in Table 3) on muscle ac- tivity during breathing and locomotion and how this is affected by energetic demand, prandial status and body size to illustrate bio- mechanical constraints. 3.1.10 \| Transversus abdominis The IEV (Figure 2d) lies immediately deep to the EOP and immedi- ately ventrad of the IC. The IEV is external to the intercostal spaces between only the intermediate ribs and their fascicles run obliquely, dorsocranial to ventrocaudal, in parallel with those of the OEP and TC and perpendicular to those of the ICID. The TA (Figure 2f) is the deepest subcostal muscle (along with ICID). TA fascicles run along the dorsoventral axis. The TA is attached to the lateral edges of the inner RA and terminates one-quarter of the way up the intermediate rib elements where it attaches onto an aponeurotic sheath which connects it to the ICID muscles. In the \| 7 \| 7 ROSE et al. 4 \| DISCUSSION This study is the first investigation into the functional special- isation of the crocodilian trunk musculature. Regardless of body size, inspiratory and expiratory muscles were differentially special- ised for greater relative contractile and force-generating capaci- ties, respectively. As hypothesised, some axial muscle properties scaled with positive allometry. In most cases, this was true for only one or two muscle properties, with muscles prioritising specialisa- tion towards force-generating capacity at the cost of contractility or vice versa. We place our data in context by comparison with 8 \| ROSE et al. FI G U R E 4 Size-normalised trunk muscle architectural properties. a) Mean ± SD relative muscle masses. Masses of the pectoralis (PEC) and serratus (SER) are also included for comparison. b) Function space plot of axial muscles. Normalised PCSA (PCSA/body mass(0.67)) is plotted against normalised fascicle length (Fascicle length/body mass(0.33)) with one data point per muscle per individual. c) Rectus abdominis (RA) and iliocostalis (IC). d) Transversus abdominis (TA), obliquus externus superficialis (OES), obliquus externus profundus (OEP), ischiopubis (ISP), ischiotruncus (ISCHU), truncocaudalis (TC) and diaphragmaticus (DI). Data points for expiratory and inspiratory muscles are denoted by a ‘+’ and ‘−’, respectively. Circles represent muscles whose potential roles in breathing have not yet been investigated 8 \| ROSE et al. 8 FI G U R E 4 Size-normalised trunk muscle architectural properties. a) Mean ± SD relative muscle masses. Masses of the pectoralis (PEC) and serratus (SER) are also included for comparison. b) Function space plot of axial muscles. Normalised PCSA (PCSA/body mass(0.67)) is plotted against normalised fascicle length (Fascicle length/body mass(0.33)) with one data point per muscle per individual. c) Rectus abdominis (RA) and iliocostalis (IC). d) Transversus abdominis (TA), obliquus externus superficialis (OES), obliquus externus profundus (OEP), ischiopubis (ISP), ischiotruncus (ISCHU), truncocaudalis (TC) and diaphragmaticus (DI). Data points for expiratory and inspiratory muscles are denoted by a ‘+’ and ‘−’, respectively. Circles represent muscles whose potential roles in breathing have not yet been investigated capacity (Uriona & Farmer, 2006). The diaphragmaticus also plays an important role when respiratory demand increases by controlling in- creases the respiratory frequency and tidal volume (Farmer & Carrier, 2000a). For example, after transection of the diaphragmaticus, the length of time required to complete inspiration increased (Uriona & Farmer, 2006), and exercise-induced increases in respiratory rate and tidal volume were reduced (Munns et al., 2012). \| 9 ROSE et al. FI G U R E 5 Scaling exponents for muscle architectural properties versus body mass. Boxes indicate the exponent and 95% CIs. Horizonal lines indicate exponent value for isometry in length (yellow, 0.33) PCSA (blue, 0.67) and mass (red, 1) the body wall (Perry and Dunker 1978; Milsom & Vitalis, 1984). In many amniotes, the elastic work of breathing is inversely related to breathing frequency. In lizards, geckos and turtles, for example, high frequency, low volume breaths are the most economic breathing pattern and modulating respiratory frequency, as opposed to tidal volume is more economical in controlling ventilation rate (Milsom & Vitalis, 1984; Vitalis & Milsom, 1986). However, in studies on A. mississippiensis, where changes in breathing frequency and/or tidal volume were induced to increase ventilation rate, the opposite was found, where the metabolic cost of breathing increased with fre- quency, but not volume (Skovgaard et al., 2016; Wang & Warburton, 1995). Similarly, following vagotomy the duration of inspiration in- creases, leading to a lower cost breathing (Skovgaard & Wang, 2007). An increased inspiratory time has been hypothesised to reduce flow resistance and therefore allow for an elevated tidal volume without higher energetic cost (Skovgaard & Wang, 2007). One al- ternative/additional explanation for the greater metabolic cost of modulating breathing frequency as opposed to tidal volume in crocodilians might relate to muscle architectural specialisation. The diaphragmaticus has the potential to contribute towards a high pro- portion of tidal volume (Claessens, 2009) as it is specialised for de- veloping length change economically rather than force; for any given ventilation rate, it would be more economical for this muscle to con- tract over a greater distance than to contract faster and more often. Every contraction of the muscle, even if smaller, would require the muscle to be active along the full length of each fascicle involved, and the cost of muscle contraction is expected to be proportional to active muscle volume (Biewener, 2016). The diaphragmaticus may, therefore, assist with low-cost breathing via both its archi- tectural specialisation and ability to modulate contraction distance and speed for large volume and low-frequency breathing (Farmer & Carrier, 2000b). 4.3 \| Changes in breathing mechanics with increasing body size Recruitment of accessory breathing structures has been important in mitigating the locomotor constraints on breathing (Brocklehurst et al 2020, Codd et al 2019). With increasing body size and stiff- ening of the trunk, the subsequent constraints on the basal costal aspiration pump are expected to necessitate a change in breathing mechanics. Anecdotal evidence suggests a reduction in costal aspi- ration and increases in breathing facilitated by the diaphragmaticus (Gans, 1976; Munns et al., 2012). The present data provide evi- dence in support of the hypothesis that crocodilians become more reliant on accessory breathing mechanisms as the trunk stiffens, \| 9 Another potential explanation, also relating to the diaphragmaticus, is that this muscle does not appear vary the volume of the chest area greatly and seems to have more of an effect on the volume of the ventral lumbar region, where viscera are getting com- pressed and released. Therefore, the diaphragmaticus is not having to overcome elastic and non-elastic forces in expanding the body wall as great as the costal aspiration pump in the thoracic region. An increased inspiratory time has been hypothesised to reduce flow resistance and therefore allow for an elevated tidal volume without higher energetic cost (Skovgaard & Wang, 2007). One al- ternative/additional explanation for the greater metabolic cost of modulating breathing frequency as opposed to tidal volume in crocodilians might relate to muscle architectural specialisation. The diaphragmaticus has the potential to contribute towards a high pro- portion of tidal volume (Claessens, 2009) as it is specialised for de- veloping length change economically rather than force; for any given ventilation rate, it would be more economical for this muscle to con- tract over a greater distance than to contract faster and more often. FI G U R E 5 Scaling exponents for muscle architectural properties versus body mass. Boxes indicate the exponent and 95% CIs. Horizonal lines indicate exponent value for isometry in length (yellow, 0.33) PCSA (blue, 0.67) and mass (red, 1) respiratory demands at rest but the diaphramaticus is recruited to enhance respiratory effort as metabolic demand increases oxygen demand (Munns et al., 2012). Both external oblique muscles demonstrated intermediate masses and regional specialisations, but comparatively, the super- ficial portion, prioritising force generation over working range, and the deep portion, prioritising working range over force generation. We speculate that a respiratory role for these muscles seems likely given that they are directly connected to muscles of the costal, ab- dominal and pelvic components of the ventilatory system. 4 \| DISCUSSION Conversely, for resting animals, transection of the diaphragmaticus does not af- fect any breathing parameters (Munns et al., 2012). Together these findings support the hypothesis that costal ventilation can meet Compared to the expiratory muscles, inspiratory muscles (dia- phragmaticus, truncocaudalis, ischiotruncus, ischiopubis) were special- ised for a greater range of length change and speed of contraction, having longer relative fascicle lengths. A greater working range is important in the diaphragmaticus for the caudal displacement of the viscera in controlling up to 60% of tidal volume (Claessens, 2009). A greater working range may also be more effective for creating a neg- ative pressure within the cylinder, allowing large volumes of air to be drawn into the thorax. The distance over which the diaphragmat- icus can contract is also important in controlling post-prandial vital \| 9 TA B LE 2 Ontogenetic scaling exponents for logged muscle properties regressed on logged body mass (0.359–5.497 kg). Expected exponents for isometric scaling are (1) in brackets. Positive allometric growth is denoted as ‘+’ and isometric growth as ‘iso’. Subtracting estimated masses of the gastralia from the measured masses of the RA did not change the outcome of the results. All p-values are two-tailed g g p Muscle Property Slope Lower Upper N R2 p Scaling DI Mass 0.92 (1) 0.81 1.03 21 0.939 <0.001 iso PCSA 0.73 (0.67) 0.52 1.05 19 0.501 <0.001 iso Fascicle length 0.56 (0.33) 0.38 0.81 19 0.429 0.002 + TA Mass 1.04 (1) 0.88 1.23 15 0.922 <0.001 iso PCSA 0.77 (0.67) 0.59 0.99 15 0.805 <0.001 iso Fascicle length 0.41 (0.33) 0.28 0.61 15 0.560 0.001 iso TC Mass 1.23 (1) 1.14 1.33 26 0.969 <0.001 + PCSA 0.90 (0.67) 0.80 1.03 26 0.910 <0.001 + Fascicle length 0.38 (0.33) 0.32 0.44 26 0.864 <0.001 iso ISCHU Mass 1.14 (1) 1.01 1.28 25 0.922 <0.001 + PCSA 0.98 (0.67) 0.79 1.23 23 0.759 <0.001 + Fascicle length 0.43 (0.33) 0.29 0.63 23 0.226 0.022 iso ISP Mass 1.13 (1) 0.96 1.32 26 0.857 <0.001 + PCSA 0.89 (0.67) 0.72 1.11 26 0.728 <0.001 + Fascicle length 0.43 (0.33) 0.32 0.59 26 0.416 <0.001 iso IC Mass 1.13 (1) 1.00 1.27 25 0.920 <0.001 + PCSA 0.76 (0.67) 0.60 0.95 20 0.792 <0.001 iso Fascicle length 0.40 (0.33) 0.33 0.48 21 0.847 <0.001 + EOS Mass 1.09 (1) 0.97 1.23 25 0.97 <0.001 iso PCSA 0.81 (0.67) 0.64 1.03 23 0.715 <0.001 iso Fascicle length 0.45 (0.33) 0.34 0.61 23 0.577 <0.001 + EOP Mass 1.08 (1) 0.98 1.20 25 0.947 <0.001 + PCSA 0.85 (0.67) 0.69 1.05 23 0.778 <0.001 + Fascicle length 0.44 (0.33) 0.32 0.62 23 0.426 <0.001 iso RA Mass 1.12 (1) 1.02 1.23 23 0.959 <0.001 + PCSA 3.30 (0.67) 2.67 4.07 22 0.791 <0.001 + PCSAcorrected 3.95 (0.67) 3.38 4.62 22 8.886 <0.001 + Fascicle length 0.30 (0.33) 0.21 0.43 22 0.614 <0.001 iso demonstrating that the functional capacities of the muscles are enhanced. All accessory breathing muscles demonstrated positive allometric scaling of a least one muscle architectural property, spe- cialising force-generating capacity over contractility or vice versa. 4.2 \| Muscle architecture and the metabolic cost of breathing The metabolic cost of breathing (ml O2 min−1 kg−1) is reported to be low in crocodilians (~5% resting metabolic rate), on a par with birds and mammals (Skovgaard et al., 2016; Wang & Warburton, 1995). It is often considered in relation to the forces that must be generated to overcome elastic recoil of the lung, resistive forces in compress- ing the body wall and elastic and non-elastic forces in expanding 10 ROSE et al. 10 \| 10 \| 11 ROSE et al. TA B LE 3 Conditions under which different muscles have been observed to be active in breathing and loco TA B LE 3 Conditions under which different muscles have been observed to be active in breathing and locomotion in crocodilians N.B. temperature, body size and species are not considered here. Information on muscle activity was taken from Gans and Clark (1976), Farmer and Carrier (2000a, b), Uriona and Farmer (2006, 2008), Uriona et al., (2009), Munns et al., (2012), Codd et al., (2019) and personal observations. ‘X’ muscle not active. TA B LE 3 Conditions under which different muscles have been observed to be active in breathing and locomotion in crocodilians N.B. temperature, body size and species are not considered here. Information on muscle activity was taken from Gans and Clark (1976), Farmer and Carrier (2000a, b), Uriona and Farmer (2006, 2008), Uriona et al., (2009), Munns et al., (2012), Codd et al., (2019) and personal observations. ‘X’ muscle not active. N.B. temperature, body size and species are not considered here. Information on muscle activity was taken from Gans and Clark (1976), Farmer and Carrier (2000a, b), Uriona and Farmer (2006, 2008), Uriona et al., (2009), Munns et al., (2012), Codd et al., (2019) and personal observations. ‘X’ muscle not active. N.B. temperature, body size and species are not considered here. Information on muscle activity was taken from Gans and Clark (1976), Farmer and Carrier (2000a, b), Uriona and Farmer (2006, 2008), Uriona et al., (2009), Munns et al., (2012), Codd et al., (2019) and personal observations. ‘X’ l t ti ‘$’ breathing can persist if the muscle is transected. $ g p ‘&’ low intermittent activity correlated with gait cycle while principal function is in respiration, ‘+’ very high activity correlated with gait cycle. ‘?’ has not been investigated or cannot be discerned from the literature due to differences in nomenclature and potential differences between taxa. ‘&’ low intermittent activity correlated with gait cycle while principal function is in respiration, ‘+’ very high activity correlated with gait cycle. ‘?’ has not been investigated or cannot be discerned from the literature due to differences in nomenclature and potential differences between taxa. has a principal role in expiration during terrestrial locomotion and elevated metabolic demand (Farmer & Carrier, 2000a). Muscle mass and PCSA scaled with positive allometry in the rectus abdominis, enhancing the force-generating potential of this muscle. Comparatively, all architectural properties of the transversus ab- dominis scaled isometrically indicating no increase in functional ca- pacity to support the increased demands of a greater body mass. The function of the transversus abdominis in expiration is basal to tet- rapods and is coupled in breathing and locomotion for some species (Brainerd, 1999). In A. mississippiensis, the transversus abdominis has low intermittent activity that is correlated with the terrestrial gait cycle (Farmer & Carrier, 2000a). The lack of change in this muscle's specialisation may indicate a reduction in its functional role com- pared to other breathing muscles over ontogeny. Active expiration is important in crocodilians, however. The roles of the iliocostalis and rectus abdominis are expected to become integral in meeting respira- tory demands during terrestrial locomotion as body size increases. Large volumes of exhalation would not be able to exit the glottis fast enough by passive means. Importantly, the activity of the expiratory rectus abdominis is decoupled from locomotion, being uncorrelated with the terrestrial gait cycle allowing high respiratory rates (Uriona et al 2009). In the accessory diaphragmaticus fascicle lengths scaled with positive allometry while mass and PCSA scaled isometrically. The shift in specialization towards greater contractile capacity at the cost of force generation in the diaphragmaticus would allow it to provide a greater contribution to tidal volume as the costal aspiration pump becomes constrained by heavier bony elements and lower chest wall compliance. Cinematographic evidence demonstrates that the muscle displaces viscera relatively further caudad during inspiration in larger individuals which would allow for greater relative tidal vol- umes (Claessens, 2009). These functional changes further support the accessory role of the diaphragmaticus as well as the hypothesis that larger individuals are more dependent on a respiratory role of \| 11 ROSE et al. 11 5 \| CONCLUSIONS 5 Here we demonstrate that the functional specialisation of the croco- dilian trunk changes to meet changing biomechanical demands and constraints. We propose that ontogenetic changes in muscle archi- tectural specialisation facilitate fine control over inspiratory volume and frequency to minimise the cost of breathing and allow sustained simultaneous breathing and locomotion. We suggest that isometric scaling of muscle properties of the transversus abdominis indicates that, with increasing body size, the basal tetrapod expiratory pump becomes less important compared to the derived components of the ventilatory apparatus which scale with positive allometry in some of their geometrical properties for improved contractile or force- generating capacities. Future research directly linking anatomical changes in muscle architecture to EMG studies of muscle activity patterns across the full-size range of crocodilians would allow us to test these ideas. Allometric scaling of trunk muscle architectural properties with increasing body size would also affect performance in aquatic lo- comotion. Accessory breathing muscles of crocodilians also have key roles in diving (Uriona et al., 2009). The diaphragmaticus, for example contracts bilaterally to shift the centre of buoyancy rela- tive to the centre of gravity in order to control body pitch and change depth in the water column. During diving, when loads are added to the tail to counteract forward pitch, diaphragmaticus ac- tivity increases (Munns et al., 2012). Large increases in SVL and body mass may also require the diaphragmaticus to shift the vis- cera further caudad corresponding to our finding of an increase in relative fascicle length. If the diaphragmaticus is transected, dive duration decreases, indicating the muscle's role in increas- ing the duration of dives (Uriona et al., 2009). Submerged lung volume (which would influence total mass-specific oxygen stores) has a scaling exponent greater than that for resting metabolism in crocodilians (Wright & Kirshner, 1987), which has been inter- preted as indicating that the duration of dives could increase with body size (Cott, 1961). The positive allometry in fascicle length of the diaphragmaticus would also support increased dive durations with increasing body size. ORCID William I. Sellers https://orcid.org/0000-0002-2913-5406 Jonathan R. Codd https://orcid.org/0000-0003-0211-1786 William I. Sellers https://orcid.org/0000-0002-2913-5406 Jonathan R. Codd https://orcid.org/0000-0003-0211-1786 There is a paucity of evidence for exactly which trunk mus- cles are recruited during crocodilian swimming, which is principally powered by the tail. However, the overall mechanical efficiency of swimming in crocodilians decreases with ontogeny (Seebacher et al., 2003). If the transversus abdominis plays an important role in trunk bending and stabilization as it does in the salamander (Bennett et al., 2001) isometric scaling of both fascicle lengths and PCSA may pose a constraint on function. Furthermore, positive al- lometric scaling was more prevalent for muscle PCSA than for mus- cle fascicle length across the trunk muscles examined. Prioritising muscle force over contractility may be a biomechanical explana- tion for why crocodilians are relatively slow swimmers compared to semi-aquatic mammals as was hypothesised by Seebacher at al (2003). ACKNOWLEDGEMENTS We would like to thank Steve Perry for the many illuminating dis- cussions and his lifetime of research into respiratory biology. We also thank Janna Crossley, Derek Nelson, Justin Connor and Brandt Smith for assistance in the lab. This research was supported by fund- ing from the National Science Foundation (NSF-17565187) to DAC and the Biotechnology and Biological Sciences Research Councils (BBSRC–BB/I021116/1) to JRC. The ischiopubis is also used in controlling dive pitch. Relative force-generating capacity of the ischiopubis increases with body mass, which is not consistent with its specialization to rotate the pubic plates through large moment arms; however, it is not known how pubic plate morphometrics change over ontogeny and this may affect the muscle moment arms which could mitigate this architec- tural change. The increase in relative force-generating capacity may also be necessary to work against forces exerted by other trunk muscles during locomotion as body size increases. \| 11 ROSE et al. Similarly, truncocaudalis increased in both relative PCSA and mass with ontog- eny, but not fascicle length, enhancing capacity to generate force to rotate the pubic plates. Increases in mass in the rectus abdominis and truncocaudalis were likely due to proliferation and/or hyperplasia of muscle fascicles as the muscle became more specialised for force generation. The ischiotruncus and ischiopubis each become relatively thicker as they become more specialised for force generation at the expense of working range. The ischiopubis was always observed to be a deeper red colour than the other two pubic muscles (Figure 3a), indicating that this derived part of the system probably has an im- portant aerobic role in breathing and/or locomotion although fibre typing would be required to confirm this. the diaphragmaticus (Munns et al., 2012). Furthermore, the negative allometry of the viscera (Eme et al., 2019) indicates that the visceral mass (free of food) that the diaphragmaticus displaces becomes rela- tively lower over ontogeny. If the relative force that the diaphragmat- icus has to apply becomes lower, the efficiency of the hepatic piston mechanism likely increases with body size. In the accessory iliocostalis (Codd et al., 2019) muscle mass and fascicle lengths scaled with positive allometry indicative of enhanc- ing contractile capacity. Although PCSA scaled isometrically in this muscle, force-generating capacity might also be enhanced with de- velopment for two reasons. Firstly, the myosepta of the iliocostalis originate from the transverse processes of the vertebrae which con- tinue to scale with positive allometry following sexual maturation (Ikejiri, 2015). Secondly, there is progressive broadening and ossi- fication of the uncinate processes embedded within its myosepta. Therefore, the relative surface area onto which the iliocostalis can attach and the strength of that surface area against which it works are both expected to increase. Increases in relative fascicle length and relative PCSA were also found in the superficial and deep obliques, respectively, indicating the sustained importance of their function over ontogeny; however, it is not known whether they assist breathing. All accessory muscles of the infrapubic abdominal wall demon- strated positive allometric scaling in PCSA. The rectus abdominis AUTHOR CONTRIBUTIONS JRC and PGT conceived the initial idea and performed the pilot data studies. KARR, WIS, PGT, JRC and DAC designed the study. RME facilitated alligator egg collection. KARR was responsible for data collection, analysis and interpretation and drafted the manuscript. All authors contributed to the critical revision of the manuscript. The rectus abdominis is active bilaterally in controlling pitch and unilaterally in body rolling during dives. The enhanced force- generating potential of this muscle would facilitate pitch and roll of the heavier and longer body. It may become particularly important for “death rolling” while holding heavier prey with the snout during feeding (Fish et al., 2007). DATA AVAILABILITY STATEMENT all data are included in the manuscript 12 \| 4.4 \| Changes in locomotor performance with increasing body size ROSE et al. 12 (1976) Studies on ventilation of Caiman crocodilus (Crocodilia: Reptilia). Respiration Physiology, 26, 285–301. https:// doi.org/10.1016/0034-5687(76)90001-3. Carrier, D.R. (1991) Conflict in the hypaxial musculoskeletal system - documenting an evolutionary constraint. American Zoologist, 31, 644–654. Gasc, J.P. (1981) Axial musculature. In: Gans, C. & Parsons, T.S. (Eds.) Biology of the reptilia. London: Academic Press, pp. 355–435. Cieri, R.L., Dick, T.J.M. & Clemente, C.J. (2020) Monitoring muscle over three orders of magnitude: Widespread positive allometry among locomotor and body support musculature in the pectoral girdle of varanid lizards (Varanidae). Journal of Anatomy, 237, 1114–1135. https://doi.org/10.1111/joa.13273. Iijima, M. & Kubo, T. (2019) Allometric growth of limb and body propor- tions in crocodylians. Journal of Zoology, 309, 200–211. https://doi. org/10.1111/jzo.12714. Ikejiri, T. (2015) Modes of ontogenetic allometric shifts in crocodylian vertebrae. Biological Journal of the Linnean Society, 116, 649–670. https://doi.org/10.1111/bij.12607. Claessens, L.P. (2009) A cineradiographic study of lung ventilation in Alligator mississippiensis. Journal of Experimental Zoology Part A: Ecological Genetics and Physiology, 311, 563–585. https://doi. org/10.1002/jez.530. Klein, W. & Codd, J.R. (2010) Breathing and locomotion: Comparative anatomy, morphology and function. Respiratory Physiology & Neurobiology, 173, S26–S32. https://doi.org/10.1016/j. resp.2010.04.019. Codd, J.R., Boggs, D.F., Perry, S.F. & Carrier, D.R. (2005) Activity of three muscles associated with the uncinate processes of the giant Canada goose Branta canadensis maximus. Journal of Experimental Biology, 208, 849–857. https://doi.org/10.1242/jeb.01489. Lambertz, M. & Perry, S.F. (2015) Remarks on the evolution of the avian sternum, dinosaur gastralia, and their functional significance for the respiratory apparatus. Zoologischer Anzeiger - A Journal of Comparative Zoology, 255, 80–84. https://doi.org/10.1016/j. jcz.2015.02.008. Codd, J.R. & Klein, W. (2010) Breathing, locomotion and everything in- between Preface. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 156, 301–302. https://doi. org/10.1016/j.cbpa.2010.03.010. Lieber, R.L. & Friden, J. (2000) Functional and clinical significance of skel- etal muscle architecture. Muscle and Nerve, 23, 1647–1666. Codd, J.R., Rose, K.A.R., Tickle, P.G., Sellers, W.I., Brocklehurst, R.J., Elsey, R.M. et al. (2019) A novel accessory respiratory muscle in the American alligator (Alligator mississippiensis). Biology Letters, 15, 20190354. https://doi.org/10.1098/rsbl.2019.0354. Lieber, R.L. & Ward, S.R. (2011) Skeletal muscle design to meet func- tional demands. Philosophical Transactions of the Royal Society of London. Series B, Biological sciences, 366, 1466–1476. https://doi. org/10.1098/rstb.2010.0316. Cott, H.B. (1961) Scientific results of an inquiry into the ecology and eco- nomic status of the Nile Crocodile (Crocodilus niloticus) in Uganda and Northern Rhodesia. Transactions of the Zoological Society London, 29, 211–356. mississippiensis: Estimating the size of extinct mesoeucrocodyl- ians. Journal of Vertebrate Paleontology, 25, 354–369. Bhullar, B.A. (2009) A reevaluation of the unusual abdominal muscula- ture of squamate reptiles (Reptilia: Squamata). Anatomical Record (Hoboken), 292, 1154–1161. https://doi.org/10.1002/ar.20955. mississippiensis: Estimating the size of extinct mesoeucrocodyl- ians. Journal of Vertebrate Paleontology, 25, 354–369. Farmer, C.G. & Carrier, D.R. (2000a) Pelvic aspiration in the American alligator (Alligator mississippiensis). Journal of Experimental Biology, 203, 1679–1687. Biewener, A.A. (2016) Locomotion as an emergent property of muscle contractile dynamics. Journal of Experimental Biology, 219, 285–294. https://doi.org/10.1242/jeb.123935. Farmer, C.G. & Carrier, D.R. (2000b) Ventilation and gas exchange during treadmill locomotion in the American alligator (Alligator mississip- piensis). Journal of Experimental Biology, 203, 1671–1678. Bodine, S.c., Roy, R.r., Meadows, D.a., Zernicke, R.f., Sacks, R.d., Fournier, M. & et al. (1982) Architectural, histochemical, and contractile char- acteristics of a unique biarticular muscle: the cat semitendinosus. Journal of Neurophysiology, 48, 192–201. https://doi.org/10.1152/ jn.1982.48.1.192. Fechner, R. & Schwarz-Wings, D. (2013) The muscles of the infrapubic abdominal wall of a 6-month-old Crocodylus niloticus (Reptilia: Crocodylia). Anatomia Histologia and Embryologia, 42, 175–182. https://doi.org/10.1111/ahe.12000. Boelaert, R. (1942) Sur la physiologie de la respiration de l’ Alligator mis- sissippiensis. Archives Internationales de Physiologie, 52, 57–72. Fish, F.E., Bostic, S.A., Nicastro, A.J. & Beneski, J.T. (2007) Death roll of the alligator: mechanics of twist feeding in water. Journal of Experimental Biology, 210, 2811–2818. https://doi.org/10.1242/ jeb.004267. Brainerd, E.L. (1999) New perspectives on the evolution of lung venti- lation mechanisms in vertebrates. Experimental Biology Online, 4, 1–28. Brainerd, E.L. & Owerkowicz, T. (2006) Functional morphology and evo- lution of aspiration breathing in tetrapods. Resp Physiol Neurobi, 154, 73–88. https://doi.org/10.1016/j.resp.2006.06.003. Frey, E. (1988) Anatomie des Korperstammes von Alligator mississippien- sis Daudin. Stuttgarter Beiträge zur Naturkunde Series A, 424, 1–106. Gans, C. (1976) Questions in crocodilian physiology. Zoologica Africana, 11, 241–250. https://doi.org/10.1080/00445096.1976.11447531. Brocklehurst, R.J., Moritz, S., Codd, J., Sellers, W.I. & Brainerd, E.L. (2017) Rib kinematics during lung ventilation in the American alli- gator (Alligator mississippiensis): An XROMM analysis. Journal of Experimental Biology, 220, 3181–3190. https://doi.org/10.1242/ jeb.156166. Gans, C. (1982) Fiber architecture and muscle function. Exercise and Sport Sciences Reviews, 10, 160–207. Gans, C. & Bock, W.J. (1965) The functional significance of muscle ar- chitecture–a theoretical analysis. Ergeb Anat Entwicklungsgesch, 38, 115–142. Carrier, D.R. (1987) Lung ventilation during walking and running in 4 spe- cies of lizards. Exp Biol, 47, 33–42. Gans, C. & Clark, B. REFERENCES Allen, V., Elsey, R.M., Jones, N., Wright, J. & Hutchinson, J.R. (2010) Functional specialization and ontogenetic scaling of limb anatomy in Alligator mississippiensis. Journal of Anatomy, 216, 423–445. https://doi.org/10.1111/j.1469-7580.2009.01202.x. Allen, V., Molnar, J., Parker, W., Pollard, A., Nolan, G. & Hutchinson, J.R. (2014) Comparative architectural properties of limb muscles in Crocodylidae and Alligatoridae and their relevance to divergent use of asymmetrical gaits in extant Crocodylia. Journal of Anatomy, 225, 569–582. https://doi.org/10.1111/joa.12245. Bennett, W.O., Simons, R.S. & Brainerd, E.L. (2001) Twisting and bend- ing: the functional role of salamander lateral hypaxial musculature during locomotion. Journal of Experimental Biology, 204, 1979–1989. 13 ROSE et al. 13 Skovgaard, N. & Wang, T. (2007) Low cost of ventilation in the vag- otomised alligator (Alligator mississippiensis). Respiratory Physiology & Neurobiology, 159, 28–33. https://doi.org/10.1016/j. resp.2007.04.010. Munns, S.L., Owerkowicz, T., Andrewartha, S.J. & Frappell, P.B. (2012) The accessory role of the diaphragmaticus muscle in lung ven- tilation in the estuarine crocodile Crocodylus porosus. Journal of Experimental Biology, 215, 845–852. https://doi.org/10.1242/ jeb.061952. Tsuihiji, T. (2007) Homologies of the longissimus, iliocostalis, and hy- paxial muscles in the anterior presacral region of extant diapsida. Journal of Morphology, 268, 986–1020. https://doi.org/10.1002/ jmor.10565. Murakami, G., Akita, K. & Sato, T. (1991) Arrangement and innerva- tion of the iliocostalis and longissimus muscles of the brown caiman (Caiman-Crocodilus Fuscus - Alligatoridae, Crocodilia). The American Journal of Anatomy, 192, 241–256. https://doi. org/10.1002/aja.1001920304. Uriona, T.J. & Farmer, C.G. (2006) Contribution of the diaphragmaticus muscle to vital capacity in fasting and post-prandial American alli- gators (Alligator mississippiensis). Journal of Experimental Biology, 209, 4313–4318. https://doi.org/10.1242/jeb.02523. Naifeh, K.H., Huggins, S.E. & Hoff, H.E. (1971) The nature of the nonven- tilatory period in crocodillan respiration. Respiration Physiology, 11, 178–185. https://doi.org/10.1016/0034-5687(71)90022-3. 209, 4313–4318. https://doi.org/10.1242/jeb.02523. Uriona, T.J. & Farmer, C.G. (2008) Recruitment of the diaphragmaticus, is- chiopubis and other respiratory muscles to control pitch and roll in the American alligator (Alligator mississippiensis). Journal of Experimental Biology, 211, 1141–1147. https://doi.org/10.1242/jeb.015339. Naifeh, K.H., Huggins, S.E., Hoff, H.E., Hugg, T.W. & Norton, R.E. (1970) Respiratory patterns in crocodilian reptiles. Respiration Physiology, 9, 31–42. https://doi.org/10.1016/0034-5687(70)90003-4. O'Reilly, J.C., Summers, A.P. & Ritter, D.A. (2000) The evolution of the functional role of trunk muscles during locomotion in adult amphib- ians. American Zoologist, 40, 123–135. Uriona, T.J., Lyon, M. & Farmer, C.G. (2009) The importance of the M. diaphragmaticus to the duration of dives in the American alligator (Alligator mississippiensis). Zoology (Jena), 112, 263–269. https:// doi.org/10.1016/j.zool.2008.10.002. Paxton, H., Anthony, N.B., Corr, S.A. & Hutchinson, J.R. (2010) The ef- fects of selective breeding on the architectural properties of the pelvic limb in broiler chickens: a comparative study across mod- ern and ancestral populations. Journal of Anatomy, 217, 153–166. https://doi.org/10.1111/j.1469-7580.2010.01251.x. Vitalis, T.Z. & Milsom, W.K. (1986) Pulmonary mechanics and the work of breathing in the semiaquatic turtle, pseudemys-scripta. Journal of Experimental Biology, 125, 137–155. Wang, T. & Warburton, S.J. (1995) Breathing pattern and cost of venti- lation in the american alligator. Respiration Physiology, 102, 29–37. https://doi.org/10.1016/0034-5687(95)00043-D. Roy, R.R., Powell, P.L., Kanim, P. & Simpson, D.R. Livingston, V.J., Bonnan, M.F., Elsey, R.M., Sandrik, J.L. & Wilhite, D.R. (2009) Differential limb scaling in the american alligator (Alligator mississippiensis) and its implications for archosaur locomotor evo- lution. Anatomical Record (Hoboken), 292, 787–797. https://doi. org/10.1002/ar.20912. Dick, T.J.M. & Clemente, C.J. (2016) How to build your dragon: scaling of muscle architecture from the world's smallest to the world's largest monitor lizard. Frontiers in Zoology, 13, ARTN 8. Maurer, F. (1896) Die ventrale Rumpfmuskulatur einiger Reptilien. Leipzig: Engelmann. monitor lizard. Frontiers in Zoology, 13, ARTN 8. Dodson, P. (1975) Functional and ecological significance of relative growth in alligator. Journal of Zoology, 175, 315–355. Meers, M.B. (2002) Cross-sectional geometric properties of the croco- dylian humerus: an exception to Wolff's Law? Journal of Zoology, 258, 405–418. https://doi.org/10.1017/S0952836902001553. Eme, J., Cooper, C.J., Alvo, A., Vasquez, J., Muhtaseb, S., Rayman, S. et al. (2019) Scaling of major organs in hatchling female American alli- gators (Alligator mississippiensis). Journal of Experimental Zoology Part A: Ecological and Integrative Physiology, 331, 38–51. https://doi. org/10.1002/jez.2236. Mendez, J. & Keys, A. (1960) Density and composition of mammalian muscle. Metabolism, 9, 184–188. Milsom, W.K. & Vitalis, T.Z. (1984) Pulmonary mechanics and the work of breathing in the lizard, Gekko-Gecko. Journal of Experimental Biology, 113, 187-+. Farlow, J.O., Hurlburt, G.R., Elsey, R.M., Britton, A.R.C. & Langston, W. (2005) Femoral dimensions and body size of Alligator 14 \| 14 14 ROSE et al. 14 \| (1984) Architectural and histochemical analysis of the semitendinosus muscle in mice, rats, guinea pigs, and rabbits. Journal of Morphology, 181, 155–160. https://doi.org/10.1002/jmor.1051810204. Winters, T.M., Takahashi, M., Lieber, R.L. & Ward, S.R. (2011) Whole mus- cle length-tension relationships are accurately modeled as scaled sarcomeres in rabbit hindlimb muscles. Journal of Biomechanics, 44, 109–115. https://doi.org/10.1016/j.jbiomech.2010.08.033. Sacks, R.D. & Roy, R.R. (1982) Architecture of the hindlimb muscles of cats - functional-significance. Journal of Morphology, 173, 185–195. https://doi.org/10.1002/jmor.1051730206. Wright, J.C. & Kirshner, D.S. (1987) Allometry of lung-volume during voluntary submergence in the saltwater crocodile Crocodylus- Porosus. Journal of Experimental Biology, 130, 433–436. Schilling, N. (2011) Evolution of the axial system in craniates: morphol- ogy and function of the perivertebral musculature. Frontiers in Zoology, 8, Artn 8. Schmidt-Nielsen, K. (1984) Scaling, why is animal size so important?. New York, NY: Cambridge University Press, Cambridge. How to cite this article: Rose, K.A.R., Tickle, P.G., Elsey, R.M., Sellers, W.I., Crossley, D.A. II & Codd, J.R. (2021) Scaling of axial muscle architecture in juvenile Alligator mississippiensis reveals an enhanced performance capacity of accessory breathing mechanisms. Journal of Anatomy, 00, 1–14. https:// doi.org/10.1111/joa.13523 How to cite this article: Rose, K.A.R., Tickle, P.G., Elsey, R.M., Sellers, W.I., Crossley, D.A. II & Codd, J.R. (2021) Scaling of axial muscle architecture in juvenile Alligator mississippiensis reveals an enhanced performance capacity of accessory breathing mechanisms. Journal of Anatomy, 00, 1–14. https:// doi.org/10.1111/joa.13523 How to cite this article: Rose, K.A.R., Tickle, P.G., Elsey, R.M., Sellers, W.I., Crossley, D.A. II & Codd, J.R. (2021) Scaling of axial muscle architecture in juvenile Alligator mississippiensis reveals an enhanced performance capacity of accessory breathing mechanisms. Journal of Anatomy, 00, 1–14. https:// doi.org/10.1111/joa.13523 Seebacher, F., Elsworth, P.G. & Franklin, C.E. (2003) Ontogenetic changes of swimming kinematics in a semi-aquatic reptile (Crocodylus porosus). Australian Journal of Zoology, 51, 15–24. https://doi. org/10.1071/Zo02036. Skovgaard, N., Crossley, D.A. & Wang, T. (2016) Low cost of pulmonary ventilation in American alligators (Alligator mississippiensis) stimu- lated with doxapram. Journal of Experimental Biology, 219, 933–936. https://doi.org/10.1242/jeb.135871.
https://openalex.org/W4311307922	https://www.research-collection.ethz.ch/bitstream/20.500.11850/588950/1/Determinantstotheadoptionofenergy-efficientretrofitsandtheroleofpolicymeasures.pdf	English	null	Determinants to the adoption of energy-efficient retrofits and the role of policy measures	Applied economics letters	2,022	cc-by	5,398	ETH Library ETH Library CONTACT Nilkanth Kumar nkumar@ethz.ch Center of Economic Research (CER-ETH),Zürichbergstrasse 18, Zürich 8092, Switzerland Supplemental data for this article can be accessed online at https://doi.org/10.1080/13504851.2022.2156456. 1The EU, as well as Switzerland, have undertaken initiatives to boost the energy-efficiency retrofits. For example, see European Commission (2021) and Swiss National Science Foundation (2021). 2For example, see European Energy Efficiency Platform (2021) and Energy Star (2021). © 2022 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. Journal Article This page was generated automatically upon download from the ETH Zurich Research Collection. For more information, please consult the Terms of use. This page was generated automatically upon download from the ETH Zurich Research Collection. For more information, please consult the Terms of use. APPLIED ECONOMICS LETTERS 2024, VOL. 31, NO. 10, 885–892 https://doi.org/10.1080/13504851.2022.2156456 ABSTRACT Buildings are responsible for around 45% of total energy consumption and one-third of CO2 emissions annually in Switzerland. Policymakers have argued that an increased rate of energy- saving retrofits in existing buildings will play a critical role in meeting the energy and climate policy goals. This article examines the determinants for the households’ investment decisions to under take energy-efficient retrofits and investigates the role of policy measures. We estimate random effects probit models using a rich data for 1663 owned single-family Swiss households for retrofits undertaken during 2010 to 2014. Results suggest that while the building vintage appears to be a relevant determinant; energy-related attitudes of decision makers and policy instruments are also likely to play an important role. In particular, we find a suggestive evidence of tax deduction policy in supporting households’ decisions to undertake energy-saving retrofits as well as the intensity of renovation. Direct monetary policies such as tax deductions should be focused particularly upon poorly insulated old buildings and those that rely on non-renewable energy sources for heating purposes. Determinants to the adoption of energy-efficient retrofits and the role of policy measures Massimo Filippini a,b and Nilkanth Kumar a aCenter of Economic Research (CER-ETH), ETH Zürich, Zürich, Switzerland; bUniversità della Svizzera italiana, Lugan KEYWORDS Renovation; tax deduction; energy efficiency; Switzerland; residential sector KEYWORDS Renovation; tax deduction; energy efficiency; Switzerland; residential sector JEL CLASSIFICATION C23; D04; H23; H71; Q48 I. Introduction as energy-related knowledge and energy-saving behaviour may also be important. Given that the upfront costs of retrofits are likely to be high, and the (private) financial benefits on these invest ments – in term of reduced energy costs and increased property value – maybe realized only after few years, policy instruments become attrac tive and already play an important role in EU as well as in the US.2 In Switzerland too, the govern ment promotes energy-saving renovations primar ily with three types of policy instruments: subsidy programs, availability of tax deductions for energy- efficiency investments, and support for energy- related audit and consulting. Our main objective in this study is to analyse the determinants to the adoption of energy-efficient retrofits (EERs) by Swiss households, and we are particularly inter ested in examining the role of policy instruments as potential drivers. We put particular emphasis on the tax deduction policy because i) a tax policy is generally more salient and owners are asked every year about it, e.g. when filing the tax returns, Buildings account for around 40% of total energy consumption and CO2 emissions in European countries, primarily due to poor insulation in exist ing buildings as well as continued dependence on fossil fuels for heating purposes. In Switzerland too, the buildings stock are responsible for approxi mately 45% of the total end energy demand and one-third of the overall CO2 emissions (Energieschweiz 2022). Even though technological solutions exist to achieve substantial improve ments, the current rate of energy-efficient renova tions for countries in the European Union (EU), and in Switzerland, is only about 1–1.5% per year (Jakob et al. 2013) and an important increase is necessary to meet the energy and climate policy goals.1 For home owners, several factors determine whether or not they undertake new energy- efficiency related investments. These include cru cial attributes such as age of the building and level of income. Behavioural and cognitive factors such M. FILIPPINI AND N. KUMAR 886 cognitive factors, in addition to socio-demographics and dwelling attributes. The article contributes to the relatively scarce literature that examines empiri cal evidence on the impact of policy measures, such as tax deductions and subsidies, on energy-saving renovation decisions. 3For an extensive review of different retrofit policy instruments for existing residential buildings across several countries, please refer to Zhang et al. (2021). The authors categorize around 165 retrofit policies into 4 groups of measures: direction and command, assessment and disclosure, research and service, and financial incentives, and summarize their implementation, success, and obstacles. They point out that even though buildings are ageing and several policies are available, the EER rates still remain very low across the world. They go on to identify several hurdles in successful uptake of policies, such as awareness and information issues, technical issues, financial issues, and management and other issues. 4Our survey captured information on different types of EERs, namely, energy-saving retrofits of windows or doors, roof, façade, space heating system, water heating system, and other measures performed during the last five years. 5For a discussion on the concept of energy literacy and its link with financial literacy and bounded rationality, see (Blasch et al. 2017; Blasch, Filippini, and Kumar 2019). I. Introduction Notably, this is also a rather new study that uses a large panel of owned, single- family households and their investment decisions in energy-efficient retrofits – one that is likely to have resemblance to similar settings across many coun tries within Europe. ii) tax deductions are also easier to obtain than a subsidy for an energy saving retrofitting – in fact, for a retrofitting subsidy, the owner of a single-family house generally has to request a form, fill-it in before starting the retrofitting work, and send all documents to an administrative unit that will evaluate the request, and iii) there are ongoing political discussion on introducing more tax credits for energy-efficient retrofits. Few studies have looked into the effectiveness of policies that provide incentives for residential energy-efficiency renovations and have found mixed results. Tax incentives for energy efficiency appear to have existed in the US since the 1970s, but some of the first empirical studies did not find a positive effect on energy-efficiency investments (Walsh 1989; Dubin and Henson 1988). Hassett and Metcalf (1995), on the other hand, highlighted the importance of controlling for unobserved het erogeneity and exploit panel data in order to mea sure the impact of tax-based incentive policies for making energy conservation investments by US households. They find a positive and significant impact of tax-related policy for such investments. Alberini, Bigano, and Boeri (2014), who examine free-riding behaviour on energy efficiency incen tives among Italian homeowners, also argue that when it comes to household responsiveness to the incentive amount, ‘the evidence is mixed and inconclusive’.3 Empirical model The primary outcome of interest in our analysis is a dichotomous variable that captures whether or not a household has undertaken an EER. This variable is equal to one if a household performed one or more EER, and is zero otherwise.4 For this main outcome, we estimate a probit model of the following form: We examine the determinants of i) investment decisions to undertake an EER, and ii) the renova tion intensity, in owned single-family dwellings using a panel data from 2010 to 2014 for a sample of 1663 Swiss households. The results provide sug gestive evidence that the tax deduction policy has a significant positive effect on households’ decisions to undertake energy-saving retrofits. Our methodo logical framework relates to that of Kok, McGraw, and Quigley (2011) and Banfi, Filippini, and Ramseier (2011), but we use richer data that also allows us to account for the role of behavioural and Here, EER i is a latent variable for EERi, β terms represent coefficients to be estimated, and i denotes a stochastic error term assumed to be independent and identically distributed across the households. The model accounts for several explanatory vari ables: SEC denotes socio-economic characteristics, e.g. household income, household size; DW repre sents dwelling characteristics, e.g. building period, floor area; LIT denotes energy-related literacy and financial literacy of respondents5; BEH is an indica tor of the energy-saving behaviour of respondents; and POL denotes policy instruments. APPLIED ECONOMICS LETTERS 887 policy instruments to promote EER by households and several of these policies have been implemen ted at the cantonal level. Primarily, there are three types of measures: a) Tax deduction – availability of tax credit or deduction at the cantonal level; b) Subsidies – in the form of energy subsidy programs at the cantonal level (there is also an important federal program that promote energy saving reno vation); and c) Informational measures – such as support for energy-related audit and consulting either at home or at a central location (such as the utility). Information on these policy measures were collected using external sources, and merged with the survey data at the canton and year level.7 We use the number of renovations to create a secondary outcome variable to identify house holds that differ in terms of the intensity of renova tion. 6Figure A.1 in the supplementary material shows the overall number of renovations carried out by households within our sample. As basic checks, we also estimated panel count models using both Poisson regression and negative binomial approach for the number of EERs undertaken. The overall essence of the main results, important determinants etc. are similar to reported findings. 7Switzerland is a federal state composed of 26 cantons that are relatively independent in defining and implementing policy measures that aim to promote energy efficient renovations. This heterogeneity in the energy policy measures can be exploited by researchers in empirical analysis. In the supplementary material, we summarize the policy information and the data sources collected as part of this exercise. While exploring external data sources, we found that there is a general lack of a systematic collection, storage and availability of information on policy measures (both current as well as historical) across different regions and cantons. 8This variable also encompasses personnel and material expenses for cantonal energy policy measures, but do not include expenses towards renovation of cantonal buildings or budget for subsidy programs. Information on the three policy measures were collected from annual reports ‘Stand der Klima- und Energiepolitik der Kantone’ published by the BFE for the years 2010–2018. Table A.1 in the supplementary material presents an overview of the three policy variables across all Swiss cantons from 2010 to 2014. 9We followed several propensity score matching based strategies as robustness check for the impact of the tax deduction policy. The result supports a positive role of the cantonal tax deduction policy towards EER investments. In order to restrict this analysis to more homogeneous households, we considered another specification with two adjacent cantons – one with the policy and the other without. The positive and significant role of the cantonal tax deduction policy remained robust. These results are reported in Table A.3 in the supplementary materials. However, we keep the panel data random effects model used in the main analysis as we are also able to consider time-invariant unobserved heterogeneity. 10Different subsets of the dataset have been used in research articles focusing on some other topics related to energy efficiency and household energy use (Blasch et al. 2017; Blasch, Filippini, and Kumar 2019; Blasch et al. 2021). 11Table A.2 in the supplementary material reports summary statistics for the full CEPE 2015 dataset for reference. Empirical model FILIPPINI AND N. KUMAR 888 population statistics for the Swiss population and argue that the sample resembles more to the urban and sub-urban households of Switzerland. However, it would be somewhat difficult to com ment on the representativeness of our data given this unique target sample and a lack of a direct reference representative sample to compare it to. For brevity, Table 1 provides an overall summary of the cross-sectional variables whereas Table 2 presents statistics for the panel variables for the sample of households used in the empirical analy sis. We note that the respondents in owned SFHs are typically older than 40 years, fall in the middle or high-income group, and about two out of five also have a university degree. In Table 2, overall around 9% households have undertaken one or more EERs and there also seems to be some varia tion across the years in terms of renovation inten sity (EER Intensity). In terms of policy measures, most households seem to have the possibility to benefit from the tax deduction policy and there again seems to some variation across the years for the other two policy measures related to subsidy and consulting measures. variables and perform additional analysis and robustness checks.9 Endogeneity-related concerns, such as reverse causality with policy variables may also exist. This is less likely to be a concern with the tax deduction policy, but cannot be ruled-out in the context of the other policies. Alberini, Bigano, and Boeri (2014) point out that such programs could disproportionately attract some people who would undertake such renovations anyway. Furthermore, following Hassett and Metcalf (1995), we minimize potential omitted variable biases by specifying a rich model and by including Mundlak adjustment terms that account for unobserved heterogeneity using group means for the time-varying controls (Mundlak 1978). Empirical model We create three groups: households with no renovation (No EER), with 1–2 types of renova tions (Some EERs), and those with three or more types of renovations (Many EERs).6 Given the ordi nal nature of this variable, we estimate an ordered probit model of the following form: EERInt i is a latent continuous variable denoting the renovation intensity by household i and α terms are coefficients to be estimated (notation for explanatory variables and i is same). The prob ability that household i has reached the renovation intensity groupj, where j can vary from 1 (No EER) to 3 (Many EERs), is given by: y y We examine three policy variables: the first vari able on tax deduction is dichotomous and repre sents whether or not a tax relief for energy- efficiency investments was available to households within a canton during a year. The second policy variable captures the per capita annual expenses of energy-related subsidy programs that includes the subsidies paid out by the cantons (including global contributions from the federal government). The third policy captures the per capita annual cantonal expenses towards provisions related to other mea sures, such as informational campaigns and energy-related consulting.8 In terms of correla tions, while correlation between tax deduction and subsidy policies was very low (−0.04), we find only a weak positive correlation between the sub sidy and consulting policies (0.23) and between tax deduction and consulting policies (0.15). Here, EERIntis are the ordinal values of the reno vation intensity as defined above, and kjs are the threshold parameters. For both outcomes, we estimate first a panel data random effects model and another variation where we include Mundlak’s adjustment terms. All mod els are estimated using maximum-likelihood approach and control for the year fixed effects using year dummies. One limitation with our dataset is that the tax deduction policy for EERs does not vary within a canton over the study period, i.e. each of the nine canton in our data either always had a tax deduction policy in place, or did not have it at all. While we cannot change this natural experiment framework, we include a rich set of observable characteristics at the household level as explanatory We are especially interested to examine whether government policies aimed to promote energy- efficiency investments are influencing the invest ment renovation decisions. Like many European countries, Switzerland has also adopted some M. Data The dataset comes from a large survey conducted by the Centre for Energy Policy and Economics (CEPE) during 2015–2016 as part of a research project focused on energy usage by Swiss house holds. This dataset (hereafter, referred to as CEPE 2015 dataset) consisted of 8’378 households living primarily in urban and suburban regions of nine Swiss cantons. Comprehensive details of the survey and the data are provided in Blasch et al. (2018).10 In addition to disaggregate household level infor mation on socio-demographics and dwelling char acteristics, the CEPE 2015 dataset contains information on investments in EERs over the last five years resembling the period 2010–2014. We restrict the sample to owned, single-family house holds as these are likely to have more autonomy in undertaking EERs. For the final estimations, we have 1663 households that have declared that they undertook none, or some, EERs over the last five years.11 About half of owned single-family house holds (SFHs) in the sample were built before 1970 and the average living space is 180 m2 with three inhabitants. Blasch et al. (2018) compared the over all sample in the CEPE 2015 dataset to available III. Empirical results Table 3 reports the average marginal effects corre sponding to four panel data models – Model (RE- 1): probit random effects for the main outcome; Model (REM-1): probit random effects with Mundlak adjustment terms for the main outcome; Model (RE-2): ordered probit random effects for the second outcome; and Model (REM-2): ordered probit random effects with Mundlak adjustment terms for the second outcome. While we note that the estimated coefficients are quite similar across the models, for the remaining discussions, we refer to results reported in REM models (unless other wise stated). The coefficients on all three policy variables are positive and coefficients on the tax deduction APPLIED ECONOMICS LETTERS 889 889 Table 1. Overview of the dataset and underlying variables. Variable Mean Std. Dev. Min. Max. Obs. Number of EER (over last 5 years) 0.77 1.26 0 6 1663 Household size 2.98 1.25 1 6 1663 Size of dwelling (in sqm) 179.15 63.24 100 400 1663 Number of rooms 5.05 0.97 0 6 1663 Building built before 1940 0.26 0.44 0 1 1663 Building built in 1940–1970 0.24 0.43 0 1 1663 Building built in 1970–2000 0.35 0.48 0 1 1663 Building built in 2000–2015 0.14 0.35 0 1 1663 Household has kids/teens 0.37 0.48 0 1 1663 Household has elderly 0.34 0.47 0 1 1663 Respondent’s age < 40 years 0.07 0.25 0 1 1663 Respondent’s age between 40–60 years 0.47 0.5 0 1 1663 Respondent’s age > 60 years 0.46 0.5 0 1 1663 Household Income < 6k 0.17 0.37 0 1 1663 Household Income in 6k-12k 0.54 0.5 0 1 1663 Household Income > 12k 0.29 0.45 0 1 1663 Respondent has university degree 0.42 0.49 0 1 1663 Respondent’s partner has univ. degree 0.23 0.42 0 1 1663 Respondent’s investment literacy 0.67 0.47 0 1 1663 Respondent’s energy literacy 4.55 3.16 0 11 1663 Respondent’s energy-saving behavior 2.53 1.02 0 4 1663 Morally obliged to reduce consumption 0.73 0.44 0 1 1663 Concerned about free-riding by others 0.08 0.28 0 1 1663 Notes: This table reports the summary statistics for the sample of 1663 owned single-family households in the CEPE 2015 dataset. Table 1. Overview of the dataset and underlying variables. Table 2. Overview of the dataset and underlying variables. III. Empirical results Variable (panel) 2010 2011 2012 2013 2014 2015 Overall Atleast one EER 0.0898 0.0702 0.0971 0.120 0.0950 0.136 0.0951 (0.286) (0.256) (0.296) (0.325) (0.293) (0.344) (0.293) EER Intensity: “No EER” 0.910 0.930 0.903 0.880 0.905 0.864 0.905 (0.286) (0.256) (0.296) (0.325) (0.293) (0.344) (0.293) EER Intensity: “Some EER” 0.077 0.062 0.083 0.105 0.082 0.107 0.082 (0.266) (0.242) (0.276) (0.307) (0.275) (0.310) (0.275) EER Intensity: “Many EER” 0.013 0.008 0.014 0.015 0.013 0.030 0.013 (0.114) (0.089) (0.119) (0.121) (0.112) (0.167) (0.113) Tax deduction policy 0.878 0.884 0.885 0.883 0.883 1 0.884 (0.328) (0.320) (0.320) (0.321) (0.322) (0) (0.320) Subsidy expenses per capita 11.59 12.12 15.30 11.53 12.53 21.12 12.74 (5.252) (4.599) (6.778) (8.543) (6.001) (0) (6.575) Consulting expenses per capita 4.373 4.456 4.555 2.579 2.366 1.387 3.600 (4.729) (4.444) (4.369) (1.296) (1.263) (0) (3.660) Household size 3.089 3.024 2.969 2.920 2.907 2.738 2.976 (1.276) (1.256) (1.236) (1.228) (1.234) (1.120) (1.246) # Observations 1437 1510 1534 1552 1663 103 7799 Notes: This table reports the mean, standard deviation (in parentheses) and count for the panel variables used in the empirical analysis. The annual data covers the period from 2010 to 2014 except for one region, for which data is available from 2011 to 2015. Table 2. Overview of the dataset and underlying variables. Notes: This table reports the mean, standard deviation (in parentheses) and count for the panel variables used in the empirical analysis. The annual data covers the period from 2010 to 2014 except for one region, for which data is available from 2011 to 2015. on investment in energy-efficient vehicles, Cerruti, Daminato, and Filippini (2019) find evidence of limited awareness of energy-efficiency policies among the Swiss population. Moreover, as dis cussed previously, sometimes the request of sub sidies can be cumbersome and time-taking. policy are significant at 95% level in both REM-1 and REM-2. The subsidy-related variable has smal ler coefficients and is insignificant in all models. The third policy variable is also positive but insig nificant (but has a weaker significance in RE-2). It is worth noting that each of the policy considered can have different impact due to salience, public awareness, transaction costs and perceived benefit by home-owners. For example, a tax deduction policy is generally more salient as it is asked every year when filing tax returns. Whereas, sub sidy expenses and consulting expenses could be linked to limited awareness, e.g. III. Empirical results in a recent study We observe some expected findings with respect to dwelling and socio-economic variables. Compared to the buildings built before 1940 (reference category), those built in 1940–1970 are associated with a higher likelihood to undergo more retrofits whereas newly constructed buildings (after 2000) are naturally asso ciated with a much lower likelihood. The coefficient M. FILIPPINI AND N. KUMAR 890 Table 3. Marginal effects for probit and ordered probit models for EER (CEPE 2015 dataset). Probit Ordered Probit RE-1 REM-1 RE-2 REM-2 Tax deduction policy 0.0219* 0.0232 0.0180 0.0188** (0.0113) (0.0116) (0.0089) (0.0091) Subsidy expenses per capita 0.0007 0.0006 0.0007 0.0006 (0.0005) (0.0006) (0.0004) (0.0005) Consulting expenses per capita 0.0016 0.0017 0.0013* 0.0015 (0.0010) (0.0013) (0.0008) (0.0011) Household size 0.0042 −0.0068 0.0042 −0.0040 (0.0037) (0.0093) (0.0029) (0.0075) Size of dwelling (in sqm) −0.0001* −0.0001* −0.0000 −0.0000 (0.0001) (0.0001) (0.0000) (0.0000) Number of rooms 0.0058 0.0054 0.0026 0.0023 (0.0041) (0.0041) (0.0032) (0.0032) Building built in 1940–1970 0.0280* 0.0278* 0.0202* 0.0201* (0.0088) (0.0089) (0.0069) (0.0070) Building built in 1970–2000 −0.0050 −0.0051 −0.0075 −0.0078 (0.0085) (0.0086) (0.0067) (0.0068) Building built in 2000–2015 −0.1455* −0.1455* −0.1148* −0.1150* (0.0179) (0.0179) (0.0138) (0.0139) Household has kids/teens −0.0103 −0.0127 −0.0082 −0.0099 (0.0100) (0.0102) (0.0079) (0.0080) Household has elderly −0.0108 −0.0104 −0.0085 −0.0083 (0.0092) (0.0092) (0.0073) (0.0073) Respondent’s age < 40 years 0.0167 0.0178 0.0143 0.0150 (0.0144) (0.0144) (0.0113) (0.0113) Respondent’s age > 60 years −0.0134 −0.0126 −0.0116 −0.0111 (0.0098) (0.0098) (0.0077) (0.0077) Household Income in 6k-12k −0.0026 −0.0027 −0.0011 −0.0011 (0.0096) (0.0096) (0.0076) (0.0076) Household Income > 12k 0.0036 0.0035 0.0035 0.0035 (0.0117) (0.0118) (0.0093) (0.0093) Respondent has university degree −0.0017 −0.0014 −0.0012 −0.0009 (0.0079) (0.0079) (0.0062) (0.0062) Respondent’s partner has univ. degree 0.0170* 0.0166* 0.0150 0.0148 (0.0088) (0.0088) (0.0069) (0.0070) Std(investment literacy) −0.0020 −0.0018 −0.0013 −0.0012 (0.0038) (0.0039) (0.0030) (0.0031) Std(energy literacy) 0.0027 0.0027 0.0026 0.0025 (0.0035) (0.0035) (0.0028) (0.0028) Std(energy-saving behavior) 0.0046 0.0047 0.0032 0.0033 (0.0036) (0.0036) (0.0028) (0.0028) Morally obliged to reduce consumption 0.0232* 0.0232* 0.0193* 0.0190* (0.0079) (0.0080) (0.0063) (0.0063) Concerned about free-riding by others 0.0259 0.0260 0.0205 0.0204 (0.0118) (0.0119) (0.0093) (0.0094) Observations 7799 7799 7799 7799 Notes: This table reports the average marginal effects (AME) obtained for the panel data models estimated on the CEPE 2015 dataset. The main outcome variable is a dichotomous variable for whether or not any EER was undertaken (RE-1 and REM-1). 12The dichotomous variables related to moral obligation and concern for free-riding are equal to one for respondents who agreed, or strongly agreed, to the two statements: ‘I feel morally obliged to reduce my energy consumption’. and ‘I am not willing to reduce my energy consumption if others don’t do the same’.. III. Empirical results The secondary outcome variable represents renovation intensity as three ordered groups (RE-2 and REM-2). For RE-2 and REM-2, the AME have been calculated using margins in Stata v13.1 and represent probability of a specified outcome level (Some EER). Coefficients on the year fixed effects and on the Mundlak terms have not been reported. Std() refers to standardized form (z-scores, with zero mean and unit standard deviation) for the three indices investment literacy, energy literacy, and energy-saving behaviour. Robust standard errors in parentheses. , * and *** respectively denote significance at 10%, 5% and 1% levels. Marginal effects for probit and ordered probit models for EER (CEPE 2015 dataset). Notes: This table reports the average marginal effects (AME) obtained for the panel data models estimated on the CEPE 2015 dataset. The main outcome variable is a dichotomous variable for whether or not any EER was undertaken (RE-1 and REM-1). The secondary outcome variable represents renovation intensity as three ordered groups (RE-2 and REM-2). For RE-2 and REM-2, the AME have been calculated using margins in Stata v13.1 and represent probability of a specified outcome level (Some EER). Coefficients on the year fixed effects and on the Mundlak terms have not been reported. Std() refers to standardized form (z-scores, with zero mean and unit standard deviation) for the three indices investment literacy, energy literacy, and energy-saving behaviour. Robust standard errors in parentheses. , * and *** respectively denote significance at 10%, 5% and 1% levels. on the highest level of household income is also posi tive but insignificant. While the coefficients on the literacy and energy-saving behaviour of the respon dent are not significant, interestingly, level of educa tion of the survey respondent’s partner living in the same household appears to also show an important association. Respondents who feel morally obliged to reduce their energy consumption appear to be posi tively associated to undertake (more) EERs. Interestingly, the same is also true for respondents who are concerned about potential free-riding beha viour by others.12 Overall, we conclude that while all policy mea sures seem to be positively associated with house APPLIED ECONOMICS LETTERS 891 holds undertaking EER decisions (on both types of outcomes), the role of the tax deductions seems to be more noticeable and robust. A tax deduction policy is generally more salient to the public. III. Empirical results Owners of single-family houses belong to a high- income group and are likely to pay higher taxes. The tax deduction could be particularly interesting for home owners undertaking EERs to reduce recurring energy costs and increase their property value, and to also save on higher taxes. While we obtain similar results to Hassett and Metcalf (1995) on the positive role of tax deduction policy, the role of other policy measures appear to be somewhat inconclusive – similar to that of Alberini, Bigano, and Boeri (2014) and even to some early empirical literature (Walsh 1989; Dubin and Henson 1988). systematic and comprehensive data related to pol icy measures at disaggregate level in order to facil itate future empirical research in this direction. IV. Conclusion Increased rate of energy-saving retrofits in exist ing buildings will play a crucial rule in meeting energy and climate policy objectives. It is hence important to examine the determinants of household renovation decisions. While the building vintage is a relevant factor; energy- related attitudes of decision makers, as well as policy instruments are also likely to play a very important role. In particular, we find that a tax deduction policy is associated with a significant positive impact on households’ investment deci sions related to energy-saving renovations, both on whether or not to undertake an energy- saving renovation, but also on the renovation intensity. We cannot completely rule out endo geneity-related concerns, so our results on the impact of the policies should be considered as suggestive evidence. Disclosure statement Disclosure statement No potential conflict of interest was reported by the authors. Massimo Filippini http://orcid.org/0000-0002-5972-4439 Nilkanth Kumar http://orcid.org/0000-0002-5055-5798 Massimo Filippini http://orcid.org/0000-0002-5972-4439 Nilkanth Kumar http://orcid.org/0000-0002-5055-5798 Funding The work was supported by the Bundesamt für Energie [SI/ 501886-01]. Acknowledgements This research has been funded by the Swiss Federal Office of Energy (FOEN) under contract number SI/501886-01. Neither FOEN nor any other partners were responsible for the survey design, analysis and interpretation of the data. We would also like to thank Björn Lalin for assistance in data collection on policy measures, two anonymous reviewers for their suggestions, and researchers at CEPE, ETH Zurich for their feedback during internal discussions. All omissions and remaining errors are our responsibility. References Alberini, A., A. Bigano, and M. Boeri. 2014. “Looking for Free Riding: Energy Efficiency Incentives and Italian Homeowners.” Energy Efficiency 7 (4): 571–590. doi: https://doi.org/10.1007/s12053-013-9241-7 . Banfi, S., M. Filippini, and C. Ramseier (2011). Erneuerung von Einfamilienhäusern: Eine mikroökonomische Analyse für ausgewählte Schweizer Kantone. Final report for Swiss Federal Office of Energy (SFOE). From a policy perspective, the federal and can tonal bodies should continue to promote these policies to attract consumers to undertake more energy-efficiency retrofits. Given the potential for larger reductions in energy-requirement, direct monetary policies such as tax deductions should be particularly focused on poorly insulated old buildings and those that rely on non-renewable energy sources for heating purposes. Lastly, con certed efforts from both public and private stake holders may be necessary towards capturing Blasch, J., N. Boogen, M. Filippini, and N. Kumar. 2017. “Explaining Electricity Demand and the Role of Energy and Investment Literacy on End-Use Efficiency of Swiss Households.” Energy Economics 68 (S1): 89–102. doi:10. 1016/j.eneco.2017.12.004. Blasch, J., N. Boogen, M. Filippini, and N. Kumar (2018). Energy Efficiency, Bounded Rationality and Energy-Related Financial Literacy in the Swiss Household Sector. Final report. Swiss Federal Office of Energy (SFOE). Bern. Blasch, J., M. Filippini, and N. Kumar. 2019. “Boundedly Rational Consumers, Energy and Investment Literacy, and the Display of Information on Household Appliances.” M. FILIPPINI AND N. KUMAR 892 Resource and Energy Economics 56: 39–58. doi:10.1016/j. reseneeco.2017.06.001. jrc.ec.europa.eu/articles/financing-energy-renovations- european-building-stock jrc.ec.europa.eu/articles/financing-energy-renovations- european-building-stock Hassett, K. A., and G. E. Metcalf. 1995. “Energy Tax Credits and Residential Conservation Investment: Evidence from Panel Data.” Journal of Public Economics 57 (2): 201–217. doi:10.1016/0047-2727(94)01452-T. Blasch, J., M. Filippini, N. Kumar, and A. L. Martinez-Cruz. 2021. “Boosting the Choice of Energy-Efficient Home Appliances: The Effectiveness of Two Types of Decision Support.” Applied Economics. Forthcoming. doi:10.1080/ 00036846.2021.2014395. Jakob, M., W. Ott, H. Berleth, R. Bolliger, S. Bade, A. Karlegger, and A. Jaberg. 2013. Erneuerungstätigkeit und Erneuerungsmotive bei Wohn-und Bürobauten. Zürich: Im Auftrag Energieforschung Stadt Zürich. Cerruti, D., C. Daminato, and M. Filippini. 2019. “The Impact of Policy Awareness: Evidence from Vehicle Choices Response to Fiscal Incentives.” Economics Working Paper Series 19: 316. doi:10.2139/ssrn.3358407. Kok, N., M. McGraw, and J. M. Quigley. 2011. “The Diffusion of Energy Efficiency in Building.” The American Economic Review 101 (3): 77–82. doi:10.1257/aer.101.3.77. Dubin, J. A., and S. E. Henson. 1988. “The Distributional Effects of the Federal Energy Tax Act.” Resources and Energy 10 (3): 191–212. doi:10.1016/0165-0572(88)90020-5. Mundlak, Y. 1978. References “On the Pooling of Time Series and Cross Section Data.” Econometrica 46 (1): 69–85. doi:https://doi. org/10.2307/1913646 . Energieschweiz. 2022. “Energieeffiziente Gebäude.” accessed 06 November 2022. https://www.energieschweiz.ch/ gebaeude/ Swiss National Science Foundation (2021). Buildings and Settlements. NRP 70 and 71. accessed 30 June 2021. Swiss National Science Foundation (2021). Buildings and Settlements. NRP 70 and 71. accessed 30 June 2021. https://nfp-energie.ch/en/key-themes/197/synthese Energy Star (2021). Federal Income Tax Credits and Other Incentives for Energy Efficiency. accessed 28 May 2021. https://www.energystar.gov/about/federal_tax_credits https://nfp-energie.ch/en/key-themes/197/synthese Walsh, M. J. 1989. “Energy Tax Credits and Housing Improvement.” Energy Economics 11 (4): 275–284. doi:10. 1016/0140-9883(89)90043-1. European Commission (2021). Renovation Wave. Department: Energy. accessed 25 June 2021. https://ec. europa.eu/energy/topics/energy-efficiency/energy-efficient -buildings/renovation-wave_en Zhang, H., K. Hewage, H. Karunathilake, H. Feng, and R. Sadiq. 2021. “Research on Policy Strategies for Implementing Energy Retrofits in the Residential Buildings.” Journal of Building Engineering 43: 103161. doi:10.1016/j.jobe.2021.103161. European Energy Efficiency Platform (2021). Financing Energy Renovations in the European Building Stock. European Commission. accessed 02 July 2021. https://e3p.
https://openalex.org/W4210813575	https://nhess.copernicus.org/articles/22/3571/2022/nhess-22-3571-2022.pdf	English	null	Comment on nhess-2021-351	null	2,022	cc-by	8,480	Probabilistic fault displacement hazard analysis for the north Tabriz fault Mohamadreza Hosseini and Habib Rahimi Department of Earth Physics, Institute of Geophysics, University of Tehran, Tehran, Iran Received: 19 November 2021 – Discussion started: 7 December 2021 Revised: 28 July 2022 – Accepted: 30 July 2022 – Published: 2 November 2022 sible to predict the extent of fault slip on or near the active fault (Baize et al., 2020). Abstract. The probabilistic fault displacement hazard anal- ysis is one of the newest methods of estimating the amount of probabilistic displacement in fault surface rupture areas. Considering the strike-slip mechanism of the north Tabriz fault, Iran, using the earthquake method and historical earth- quakes in 1721 and 1780, the surface displacement of the north Tabriz fault has been investigated, and the probabilis- tic displacement in different scenarios has been estimated. The north Tabriz fault’s 50–60 km long section was selected as the source of possible surface rupture from its historical data. Two scenarios were considered according to probabilis- tic displacements, return periods, and magnitudes according to paleoseismic studies of the north Tabriz fault. For both scenarios, the probabilistic displacements for the exceedance rate of 5 % in 50, 475, and 2475 years for the probabilis- tic principle displacements (on the fault) of the north Tabriz fault have been estimated. A way to reduce the effects of fault rupture hazards on a structure is to study the probability of fault displacement. This approach can consider the exceedance rate of differ- ent displacement levels of the event under construction with a displacement hazard curve (Youngs et al., 2003). So far, fault displacement data have been collected and analyzed by several researchers to evaluate the fault rupture properties. Investigation of fault displacement and extraction of experi- mental relationships are reported by Wells and Coppersmith (1994) and reviewed by Petersen and Wesnousky (1994). Each earthquake causes a superﬁcial shaking at the site, but each earthquake does not cause a surface rupture in the area. Therefore, only the data of earthquakes that have caused the rupture in the region are used to obtain the attenuation rela- tionships (Youngs et al., 2003). A method for estimating the probabilistic fault displace- ment hazard for strike-slip faults in the world has been pre- sented and mapped due to the impact of fault displacement hazard on the fault trace type and the complexity of this effect and hazard of fault displacement for strike-slip faults studied by Petersen et al. (2011). Probabilistic fault displacement hazard analysis for the north Tabriz fault Mohamadreza Hosseini and Habib Rahimi Department of Earth Physics, Institute of Geophysics, University of Tehran, Tehran, Iran Principal displacements are consid- ered primary ruptures that occur on or within a few meters of the active fault. Distributed displacements outside the fault are causative and usually appear as discontinuous ruptures or shears at distances of several meters to several hundred kilo- meters from the fault trace. The principal and distributed dis- placements are introduced as net displacements derived from horizontal and vertical displacements (Petersen et al., 2011). Nat. Hazards Earth Syst. Sci., 22, 3571–3583, 2022 https://doi.org/10.5194/nhess-22-3571-2022 © Author(s) 2022. This work is distributed under the Creative Commons Attribution 4.0 License. 1 Introduction Earthquakes are a serious threat to many human activities, not only because of earth-shaking but also because of sur- face ruptures. Reducing earthquake losses and damage re- quires predicting the amplitude and location of ground move- ments and possible surface displacements in the future. Fault displacement hazard assessments are based on empirical re- lationships obtained using historical seismic rupture data. These relationships evaluate the probability of co-seismic surface slip of ruptures on the fault (primary) and outside the fault (distributed) for different magnitudes and distances to the causal fault. In addition, these relationships make it pos- To estimate the probabilistic fault displacement hazard, we used the Petersen et al. (2011) method, but recently some studies have been conducted with this approach. Katona et 2 Seismotectonic (2003) on the north Tabriz fault, a section with a length of 50–60 km was considered a source of possi- ble rupture in the future. Sites at distances of 50 m from each other and cells with dimensions of 25×25 m on the fault trace were considered to estimate probability displacement. Also, according to the study by Petersen et al. (2011), the trace of the north Tabriz fault was considered a simple trace due to the absence of large instrumental earthquakes associated with surface rupture. Many studies have been done on the historical displacements of the north Tabriz fault. According to the paleoseismic studies reported by Hessami et al. (2003) and Ghassemi et al. (2016), the probabilistic displacement is between 0 to 4.5 m and 0 to 7.1 m, respectively. The magni- tude and return period of large earthquakes (Mw ∼7.7 within 645 years and Mw ∼7.3 within 300 years), according to Mousavi-Bafrouei et al. (2014) and Djamour et al. (2011), respectively, are considered. The focal mechanism of earthquakes in northwestern Iran and southeastern Turkey shows that the convergence between the Saudi and Eurasian plates becomes depreciable dur- ing right-lateral strike-slip faults. The strike-slip fault is the southeast continuation of the north Anatolian fault into Iran, consisting of discontinuous fault sections with a northwest– southeast extension (Jackson, 1992). Some of these fault fragments have been ruptured and left deformed from the earthquakes in 1930, 1966, and 1976 (Hessami et al., 2003). Nevertheless, the north Tabriz fault is one of the com- ponents of this right-lateral strike-slip system, which has not had a major earthquake during the last two centuries. Among the many historical earthquakes in the Tabriz region, only two devastating earthquakes in 1042 and 1721 with a magnitude of Ms ∼7.3 and one in 1780 with a magnitude of Ms ∼7.4 were associated with a surface rupture along the north Tabriz fault (Hessami et al., 2003). The 1721 and 1780 AD earthquakes had at least 50 and 60 km of surface rupture (about 40 km overlap), respectively. Berberian (1997) believe that large earthquakes along the north Tabriz fault are concentrated at speciﬁc times and spatially related. 2 Seismotectonic With over 2 million people and an area of 167 km2 Tabriz, in northwestern Iran, is one of the most populated cities in the country that has experienced devastating earthquakes throughout history. One of the main problems of the city of Tabriz is the proximity of the city to the north Tabriz fault and the expansion of construction around it. Based on the reported historical earthquakes by Berberian and Ar- shadi (1976), since 858 CE, this city and the surrounding area have experienced several large and medium destructive earth- quakes. Numerical values are obtained from Petersen et al. (2011) codes, and this method has been performed in a case study in northwestern Iran. In this study, several input parameters such as maximum magnitude, return period, faulting mecha- nism, surface rupture length, mapping accuracy, sites located on trace, cell size, regression, and displacement models, ac- cording to Petersen et al. (2011), have been used to esti- mate the probabilistic displacement and exceedance rate of the north Tabriz fault, Iran, in different scenarios. q The focal mechanism of earthquakes in northwestern Iran and southeastern Turkey shows that the convergence between the Saudi and Eurasian plates becomes depreciable dur- ing right-lateral strike-slip faults. The strike-slip fault is the southeast continuation of the north Anatolian fault into Iran, consisting of discontinuous fault sections with a northwest– southeast extension (Jackson, 1992). Some of these fault fragments have been ruptured and left deformed from the earthquakes in 1930, 1966, and 1976 (Hessami et al., 2003). Nevertheless, the north Tabriz fault is one of the com- ponents of this right-lateral strike-slip system, which has not had a major earthquake during the last two centuries. Among the many historical earthquakes in the Tabriz region, only two devastating earthquakes in 1042 and 1721 with a magnitude of Ms ∼7.3 and one in 1780 with a magnitude of Ms ∼7.4 were associated with a surface rupture along the north Tabriz fault (Hessami et al., 2003). The 1721 and 1780 AD earthquakes had at least 50 and 60 km of surface rupture (about 40 km overlap), respectively. Berberian (1997) believe that large earthquakes along the north Tabriz fault are concentrated at speciﬁc times and spatially related. Based on the results of a paleoseismic study reported by Hessami et al. M. Hosseini and H. Rahimi: PFDHA for the north Tabriz fault M. Hosseini and H. Rahimi: PFDHA for the north Tabriz fault al. (2021) investigated the hazard of surface displacement due to faults in the design of nuclear power plants. Nurmi- nen et al. (2020) concentrate on off-fault rupturing and de- veloping an original probability model for the occurrence of distributed ruptures from 15 historical reverse earthquakes. Goda (2021) proposed an alternative approach based on stochastic source modeling and fault displacement analysis using Okada equations, and the developed method was ap- plied to the Hector Mine earthquake (1999). the north Tabriz fault, due to the devastating large histori- cal earthquakes and the possible rupture hazard of the north Tabriz fault in the future, using the probabilistic fault dis- placement hazard analysis (PFDHA) method is essential. Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. 3572 2 Seismotectonic p y In the ﬁrst step, probabilistic fault displacement and the annual exceedance rate of displacement for two given sce- narios (Mw ∼7.7 within 645 years and Mw ∼7.3 within 300 years) by considering 5 % in 50, 475, and 2475 years at the site with geographical coordinates 38.096◦N, 46.349◦E have been obtained. In the second step, due to the passage of the north Tabriz fault through the city of Tabriz (Fig. 1), considering a 2 km long section from the north Tabriz fault, the probabilistic displacement has been estimated, and the probabilistic displacement 2D map is explored. Due to the Tabriz fault having a very high level of hazard in the fu- ture and due to the lack of instrumental data on this fault, there is increased uncertainty in the numerical calculations of this fault. The north Tabriz fault has a high level of dan- ger because it passes through the ﬁfth district of the city of Tabriz, and in the case of possible surface rupture, it might lead to much damage in this residential area. With 150 000 people and at 32 000 km2, this region has essential regions such as Baghmisheh, Elahieh, Rashidieh, etc. (Fig. 1). For A more detailed study of the temporal distribution of earthquakes in Tabriz by Berberian and Yates (1999) also shows the cluster distribution of earthquakes over time. Due to the absence of seismic events for more than 200 years in the Tabriz area (decluttering period), the study area has passed the ﬁnal stages of stress storage, and it is ready to re- lease the stored energy. Therefore, Hessami et al. (2003) in- vestigated the spatial-temporal concentration of earthquakes associated with the north Tabriz fault. Based on paleontolog- ical seismic studies on the western part of the north Tabriz fault, Hessami et al. (2003) introduced four earthquakes that occurred continuously on the western part of the north Tabriz fault. The return periods of these earthquakes were suggested to be 821±176 years. During each seismic event of the north Tabriz fault, the amount of right-lateral strike-slip displace- ment has been estimated at 3.5 to 4.5 m. In addition, Berbe- https://doi.org/10.5194/nhess-22-3571-2022 Nat. Hazards Earth Syst. Sci., 22, 3571–3583, 2022 M. Hosseini and H. Rahimi: PFDHA for the north Tabriz fault 357 Figure 1. 2 Seismotectonic The north Tabriz fault and its vicinity to the populated city of Tabriz with a part of this fault passing through the town. Th probabilistic displacement area in the city of Tabriz is shown, assuming a simple fault trace (due to the lack of sufﬁcient instrument data these possible displacements can be seen up to a distance of ±150 m from the fault trace in the future. Figure 1 is generated using Googl Earth with Digital Globe imagery (© Google Earth 2021). M. Hosseini and H. Rahimi: PFDHA for the north Tabriz fault M. Hosseini and H. Rahimi: PFDHA for the north Tabriz fault 3573 Figure 1. The north Tabriz fault and its vicinity to the populated city of Tabriz with a part of this fault passing through the town. The probabilistic displacement area in the city of Tabriz is shown, assuming a simple fault trace (due to the lack of sufﬁcient instrument data); these possible displacements can be seen up to a distance of ±150 m from the fault trace in the future. Figure 1 is generated using Google Earth with Digital Globe imagery (© Google Earth 2021). rian (1997) considered the possibility of all parts of the north Tabriz fault fracturing at once and mentioned it as one of the critical issues in the earthquake hazard for the city of Tabriz in the northwestern region of Iran. sess shaking hazards and establish seismic design parameters (Cornell, 1968). A method for analyzing the hazard of proba- bilistic fault displacement was introduced in two approaches of earthquake and displacement (Youngs et al., 2003). This method was ﬁrst proposed to estimate the displacement of Yucca Mountain faults, which were the landﬁll of nuclear waste (Stepp et al., 2001). Then, the probabilistic fault dis- placement hazard analysis method was introduced for an en- vironment with normal faults. The probability distributions obtained for each type of fault in the world can be used in areas with similar tectonics (Youngs et al., 2003). M. Hosseini and H. Rahimi: PFDHA for the north Tabriz fault M. Hosseini and H. Rahimi: PFDHA for the north Tabriz fault 3574 Hosseini and H. Rahimi: PFDHA for the north Tabriz fault Figure 2. Deﬁnition of the variables used in fault rupture analysis: x and y are site coordinates, z dimensions of the area intended to calculate the probability of fault rupture at the site (for example, dimensions of the building foundation), r the distance from the site to the fault trace, ratio l/L the distance from the fault so that l is the measured distance from the nearest point on the rupture to the nearest end of the rupture, L the total length of the rupture, and s the distance from the end of the rupture to the end of the fault (Petersen et al., 2011). In the displacement approach, without examining the rupture mechanism, the displacement characteristics of the fault ob- served at the site are used to determine the hazard in that area. The exceedance rate of displacements and the distribution of fault displacements are obtained directly from the fault characteristics of geological features (Youngs et al., 2003). To calculate the exceedance rate in the earthquake approach, relationships similar to probabilistic seismic hazard analysis were used. The rate of exceedance, vk(z), is calculated ac- cording to the Cornell relationship (Cornell, 1968) as follows (Youngs et al., 2003): vk(z) = X nαn(m0) Z m un m0 fn(m) Z ∞ 0 fkn(r\|m) · P ∗(Z > z\|m,r)dr dm , (1) Figure 2. Deﬁnition of the variables used in fault rupture analysis: x and y are site coordinates, z dimensions of the area intended to calculate the probability of fault rupture at the site (for example, dimensions of the building foundation), r the distance from the site to the fault trace, ratio l/L the distance from the fault so that l is the measured distance from the nearest point on the rupture to the nearest end of the rupture, L the total length of the rupture, and s the distance from the end of the rupture to the end of the fault (Petersen et al., 2011). (1) in which the ground motion parameter (Z) (maximum ground acceleration, maximum response spectral accelera- tion) exceeds the speciﬁed level (z) at the site (k). Consider- ing Eq. M. Hosseini and H. Rahimi: PFDHA for the north Tabriz fault (1) and calculating the exceedance rate of displace- ment (D) from a speciﬁc value (d), the displacement param- eter replaces the parameters of ground motion (Youngs et al., 2003): vk(d) = X nαn(m0) Z m un m0 fn(m) Z ∞ 0 fkn (r\|m) · P ∗(D > d\|m,r)dr dm . (2) λ(D ≥D0)xyz = α(m) Z m,s fM,S (m,s)P [sr ̸= 0\|m] × Z r P [D ̸= 0\|z,sr ̸= 0] × P[D ≥D0\|l/L,m,D ̸= 0] fR(r)drdmds . ( (2) The expression P (D > d\|m,r) is the “attenuation func- tion” of the fault displacement at or near the earth’s surface. The expression P (D > d\|m,r) is the “attenuation func- tion” of the fault displacement at or near the earth’s surface. This displacement attenuation function is different from the usual ground motion attenuation function and includes the multiplication of the following two probabilities (Youngs et al., 2003): × P[D ≥D0\|l/L,m,D ̸= 0] This displacement attenuation function is different from the usual ground motion attenuation function and includes the multiplication of the following two probabilities (Youngs et al., 2003): fR(r)drdmds . (4) (4) The magnitude of the earthquake is indicated by m in Eq. (4), and to assess the displacement hazard due to fault rupture, these probability density functions describe dis- placement potential due to earthquakes on or near a rupture (Petersen et al., 2011). In the following, each of the parame- ters for estimating probabilistic fault displacement hazard is described. P ∗ kn(D > d\|m,r) = Pkn (Slip\|m,r) Pkn (D > d\|m,r,Slip) , (3) (3) Pkn (D > d\|m,r,Slip) , (3) where D and d are the displacements on the fault (principal fault) and displacement on the outside of the fault (distributed fault), respectively, and (x,y) are considered as coordinates of the site. r, I, L, and s are the vertical distance from the fault area, the distance of the site on the fault rupture to the nearest rupture, the total length of the fault surface rupture, and the rupture distance to the end of the fault, respectively. The deﬁnitions of these variables are shown in Fig. 2. 3 Methodology of probabilistic fault displacement hazard analysis In this study, the method introduced by Petersen et al. (2011) has been used to estimate the probabilistic fault displacement hazard caused by the north Tabriz fault. Details of the men- tioned method are provided in Petersen et al. (2011), and a summary of this approach is provided here. The earthquake approach is similar to analyzing proba- bilistic seismic hazards related to displacement, features such as faults, partial shear, fracture, or unbroken ground at or near the ground surface. the attenuation relationships of the fault displacement replace the ground-shaking relationships. Probabilistic seismic hazard analysis has been used since its development in the late 1960s and early 1970s to as- https://doi.org/10.5194/nhess-22-3571-2022 Nat. Hazards Earth Syst. Sci., 22, 3571–3583, 2022 M. Hosseini and H. Rahimi: PFDHA for the north Tabriz fault Table 1. Probability of distributed rupture for different cell sizes (Petersen et al., 2011). 3.5 Surveying accuracy The accuracy of fault location is a function of geological and geomorphic conditions that play an essential role in diagnos- ing and interpreting a geologist in converting this spatial in- formation into geological maps and fault geographic infor- mation systems. A fault map is generated using aerial pho- tography imagery, the interpretation of fault patterns from geomorphology, and the conversion of fault locations into a base map. In many cases, identifying the location and trace of the fault may be difﬁcult because sediments and erosion may obscure or cover the fault surface, leading to more un- certainty in identifying the actual location of the fault. There- fore, trace-mapped faults are divided into four categories – accurate, approximate, inferred, and concealed – based on how clearly and precisely they are located (Petersen et al., 2011). 3.2 Probabilities Probability P [SR ̸= 0\|M] is the ratio of cells with a rupture on the principal fault to the total number of cells considered. Therefore, the probability of surface rupture P[SR ̸= 0\|M] is considered due to a certain magnitude M due to faulting. According to Wells and Coppersmith’s (1993) studies, due to empirical relationships between different fault parameters, probability has been obtained for different faults in the world, such as strike-slip, normal, and reverse. Therefore, in hazard analysis of fault displacement, it is necessary to investigate the possibility of surface rupture with magnitude (M) on the ground; so as a result, Eq. (5) introduced by Wells and Cop- persmith (1993) can be used. According to this relation, the coefﬁcients a and b are constant, and strike-slip faults with −12.51 and 2.553 have been reported. This relationship has a 10 % probability for the size of Mw ∼5 and a 95 % proba- bility of surface rupture for a magnitude of Mw ∼7.5 (Paul C. Rizzo Associates, Inc., 2013). No. Cell size a(z) b(z) Standard (m2) deviation (σ) 1 25 × 25 −1.1470 2.1046 1.2508 2 50 × 50 −0.9000 0.9866 1.1470 3 100 × 100 −1.0114 2.5572 1.0917 4 150 × 150 −1.0934 3.5526 1.0188 5 200 × 200 −1.1538 4.2342 1.0177 3.4 Cell size In calculating the hazard of principal fault displacements, as shown in Eq. (4), the hazard level will not change by changing the cells’ size. This parameter can be examined by the availability of principal displacement data in the study area. In calculating the hazard of distributed rupture (dis- tributed displacement), considering the method of Youngs et al. (2003), the probability of surface rupture was investi- gated by modeling secondary displacements up to a distance of 12 km from the fault. According to studies by Petersen et al. (2011), the relationship between the calculations of the probability of rupture of the principal faults, Eq. (5), in cal- culating the probability of rupture of the distributed faults became the following relationship (Petersen et al., 2011): P [sr ̸= 0\|m] = ea+bm 1 + ea+bm (5) (5) This rupture probability was used to estimate the exceedance displacement rate because of earthquakes such as Loma Pri- eta in 1989 with a magnitude of Mw ∼6.9 and Alaska in 2002 with a magnitude of Mw ∼6.7. These earthquakes did not cause a rupture to reach the earth’s surface. There- fore, these two earthquakes did not cause surface deforma- tion and are considered non-tectonic phenomena (Petersen et al., 2011). The expression P [D ̸= 0\|z,sr ̸= 0] indicates the probability of non-zero displacement at a distance r from the rupture in an area of size z2 and the magnitude event m associated with the surface rupture. The probability P [D ≥ D0\|l/L,m,D ̸= 0] for displacements more signiﬁcant than or equal to the value given at this site is intended for the prin- cipal displacement (on the fault). This probability is obtained by integrating around a log-normal distribution (Petersen et al., 2011). Ln(p) = a(z)ln(r) + b(z) . (6) (6) The values of the coefﬁcients used for the cell sizes of 25× 25 m to 200 × 200 m in the above relationship are given in Table 1 (Petersen et al., 2011). 3.1 Probability density function The probability density function fM,s(m,s) determines the magnitude of the earthquake and the location of the ruptures on a fault. Since the magnitude and the rupture position on the causal fault are correlated, a probabilistic distribution is used to calculate these parameters. In the next step, the vari- ability in the rupture location is considered. A probability density function fR(r) is considered to deﬁne the area of perpendicular distances (r) to the site to different potential ruptures (Petersen et al., 2011). g The following equation has been used to obtain the ex- ceedance rate of probabilistic displacement due to the princi- pal fault (on the fault) (Petersen et al., 2011): Nat. Hazards Earth Syst. Sci., 22, 3571–3583, 2022 https://doi.org/10.5194/nhess-22-3571-2022 3575 M. Hosseini and H. Rahimi: PFDHA for the north Tabriz fault M. Hosseini and H. Rahimi: PFDHA for the north Tabriz fault 3576 Table 2. Summary of mapping accuracy: the measured distance from the mapped fault trace to the observed surface rupture (Pe- tersen et al., 2011). signiﬁcant displacement was observed near the end of the fault surface rupture (Petersen et al., 2011). Many collected surface rupture data behave asymmetrically ruptured (Wes- nousky, 2008). Mapping Mean One-sided Two-sided standard accuracy (m) standard deviation on deviation (m) the fault (m) All 30.64 43.14 52.92 Accurate 18.47 19.54 26.89 Approximate 25.15 35.89 43.82 Concealed 39.35 52.39 65.52 Inferred 45.12 56.99 72.69 However, there is no way to determine surface rupture ar- eas with larger displacements. Thus, the distribution of asym- metric displacements along the length of a fault will deﬁne more considerable uncertainties, especially near the end of the fault rupture (Petersen et al., 2011). In studying the distri- bution and principle displacement, two different approaches are introduced by Petersen et al. (2011). In the ﬁrst approach, the best-ﬁt equations using the least-squares method related to the natural logarithm of magnitude and distance displace- ment ratio were developed in a multivariate analysis (Paul C. Rizzo Associates, Inc., 2013). has a complex or straightforward trace, construction within a distance of 150 and 300 m, respectively, from the fault should be avoided. Table 2 summarizes the standard devi- ations for the displacements observed in strike-slip earth- quakes for different classiﬁcations of mapping accuracy (Pe- tersen et al., 2011). The mean displacement will be obtained according to the exponential values obtained from these ﬁt- ting equations. The following equation has been used to ob- tain the mean displacement (Petersen et al., 2011): In the second approach, the average displacement normal- izes the displacement data as a distance function. In normal- ized analysis, magnitude is not directly considered but inﬂu- ences calculations through the presence of magnitude in the mean displacement, calculated through Wells and Copper- smith’s (1994) studies. Three models (bilinear, elliptical, and quadratic) were considered to provide the principal fault dis- placement in multivariate and normalized analysis (Petersen et al., 2011). Dmean = eµ+σ 2/2 . (7) (7) However, in multivariate analysis, the three models intro- duced have the same aleatory uncertainty, and there is no clear basis for preferring one model to the other models. M. Hosseini and H. Rahimi: PFDHA for the north Tabriz fault As a result, in the probabilistic displacement hazard analysis, all three models with the same weights were used according to Table 3. The results obtained from the multivariate analysis were preferred over the normalized analysis because, in the normalized analysis, the stochastic uncertainty of calculat- ing the mean displacement from the Wells and Coppersmith (1994) study is added to the stochastic uncertainty of the re- sults of the Petersen attenuation relationships (Paul C. Rizzo Associates, Inc., 2013). 3.6 Epistemic and aleatory uncertainty There are uncertainties about the quality of mapping and the complexity of the fault trace that lead to epistemic uncer- tainty at the site of future faults. The probability density func- tion for r includes both epistemic and aleatory components. Displacements on and off the principal fault can include epis- temic uncertainty and random variability. Epistemic uncer- tainty is related to displacement measurement errors along the fault rupture. Random variability is related to the nat- ural variability in fault displacements between earthquakes. However, the measured rupture variability involves epistemic mapping uncertainties because there are currently no data to separate these uncertainties. In addition, epistemic uncer- tainty of location is introduced due to limitations in the accu- racy of basic maps or images and the accuracy of the equip- ment used to transfer this information to the map or database (Petersen et al., 2011). In this study, multivariate analysis and probabilistic dis- placement estimation have been used in the three models mentioned. The equation of the three models is obtained in the multivariate method as shown in Table 3, and 5 % un- certainty was considered in the modeling of the strike-slip displacement data (Petersen et al., 2011). 3.3 Rate parameter α(m) When the potential magnitude of an earthquake of a certain magnitude is modeled, it is possible to estimate how often these ruptures occur. The α(m) rate parameter describes the frequency of repetition of these earthquakes in this model. This parameter is a function of magnitude and can only func- tion as a single rupture function or a function of cumula- tive earthquakes above the magnitude of the minimum im- portance in engineering projects (Youngs et al., 2003). This parameter is usually based on slip rate, paleoseismic rate of large earthquakes, or historical fault rate earthquakes and is described in earthquake units per year. By removing the α(m) parameter from Eq. (4), the deterministic fault displacement hazard can be estimated (Petersen et al., 2011). A practical example shows that for an active fault with large earthquakes repeated over several hundred years, fault rupture hazard analysis should be a critical topic for design- ing structures or pipelines close to this fault. If the fault https://doi.org/10.5194/nhess-22-3571-2022 Nat. Hazards Earth Syst. Sci., 22, 3571–3583, 2022 3.7 Attenuation relationship of strike-slip faults Furthermore, considering the re- ported method by Petersen et al. (2011), the probabilistic displacements for an exceedance rate of 5 % in 50, 475, and 2475 years for the principal probabilistic displacements (on the fault) of the north Tabriz fault have been explored. The obtained results in this study can be summarized as follows. and b, although the estimated maximum displacement values are equal at some distances, at farther distances perpendicu- lar to the assumed site, these numerical values are different from each other. one of the sites located on the Tabriz fault trace related to the entire segment as shown in Fig. 1. Also, for each scenario, two values of displacement (0 to 4.5 m and 0 to 7.1 m) were considered according to Hessami et al. (2003) and Ghassemi et al. (2016), respectively. Furthermore, considering the re- ported method by Petersen et al. (2011), the probabilistic displacements for an exceedance rate of 5 % in 50, 475, and 2475 years for the principal probabilistic displacements (on the fault) of the north Tabriz fault have been explored. The obtained results in this study can be summarized as follows. For both scenarios (Mw ∼7.7, 645 years, and Mw ∼7.3, 300 years), maximum displacements for 5 % in 475 years were observed to a distance of 60 m perpendicular to the assumed site. For the ﬁrst scenario (Mw ∼7.7, 645 years), maximum displacements for 5 % in 2475 years using proba- bility displacements of 0 to 4.5 m and 0 to 7.1 m were calcu- lated up to 100 and 80 m perpendicular to the assumed site, respectively. For the second scenario (Mw ∼7.3, 300 years), the maximum displacement for 5 % in 2475 years using prob- ability displacement of 0 to 4.5 m and 0 to 7.1 m were ob- served to 80 m and 40 m perpendicular to the assumed site, respectively. In the displacement of 4.5 m, maximum displacements for the ﬁrst scenario (Mw ∼7.7, 645 years) for 5 % in 50, 475, and 2475 years were estimated at 186, 469, and 469 cm. For the second scenario (Mw ∼7.3, 300 years), the maxi- mum displacements were calculated at 230, 469, and 469 cm, respectively, as shown in Fig. 3a. 3.7 Attenuation relationship of strike-slip faults In this study, to estimate the probabilistic displacement of the north Tabriz fault, the attenuation relationship of Petersen et al. (2011) has been used. The rupture displacement data ob- tained from the principal fault are scattered but are generally the most scattered near the fault rupture center and decrease rapidly toward the end of the rupture. In some earthquakes, including the Borgo Mountain earthquake in 1968, the most Assuming the mechanism of strike-slip and trace of the Tabriz fault as a simple trace (due to the lack of surface rup- tures in instrumental data), as well as considering two sce- narios (Mw ∼7.7, 645 years, and Mw ∼7.3, 300 years) and a fault section with a length of 50–60 km (as a probabilis- tic surface rupture in future), the probabilistic displacement and the annual exceedance rate are estimated by considering https://doi.org/10.5194/nhess-22-3571-2022 Nat. Hazards Earth Syst. Sci., 22, 3571–3583, 2022 3577 M. Hosseini and H. Rahimi: PFDHA for the north Tabriz fault M. Hosseini and H. Rahimi: PFDHA for the north Tabriz fault 3 Table 3. Different models used in principal fault attenuation relationships (Petersen et al., 2011). Analysis type Model Weight Bilinear ln(D) = 1.7969Mw + 8.5206(l/L) −10.2855, σin = 1.2906, l/L < 0.3 0.34 ln(D) = 1.7658Mw −7.8962, σin = 0.9624, l/L ≥0.3 Multivariate Quadratic 0.33 ln(D) = 1.7895Mw + 14.4696(l/L) −20.1723(l/L2) −10.54512, σin = 1.1346 Elliptical 0.33 ln(D) = 3.3041 q 1 − 1 0.52 (l/L) −0.5 2 + 1.7927Mw −11.2192, σin = 1.1348 Figure 3. Comparison of probability displacement: 5 % exceedance rate in 50, 475, and 2475 years for (a) D = 4.5 m and (b) D = 7.1 Figure 3. Comparison of probability displacement: 5 % exceedance rate in 50, 475, and 2475 years for (a) D = 4.5 m and (b) D = 7.1 m. on of probability displacement: 5 % exceedance rate in 50, 475, and 2475 years for (a) D = 4.5 m and (b) D = Figure 3. Comparison of probability displacement: 5 % exceedance rate in 50, 475, and 2475 years for (a one of the sites located on the Tabriz fault trace related to the entire segment as shown in Fig. 1. Also, for each scenario, two values of displacement (0 to 4.5 m and 0 to 7.1 m) were considered according to Hessami et al. (2003) and Ghassemi et al. (2016), respectively. 3.7 Attenuation relationship of strike-slip faults In the displacement of 7.1 m, the maximum displacements for the ﬁrst scenario of Mw ∼7.7 and 645 years for 5 % in 50, 475, and 2475 years were estimated at 186, 655, and 655 cm. For the second sce- nario (Mw ∼7.3, 300 years), the maximum displacements were calculated at 230, 655, and 655 cm, respectively, as shown in Fig. 3b. According to the results shown in Fig. 3a M. Hosseini and H. Rahimi: PFDHA for the north Tabriz fault M. Hosseini and H. Rahimi: PFDHA for the north Tabriz fault Figure 4. Comparison of probability displacement, different ﬁtting models for (a) 645-year return period and D = 4.5 m, (b) 645-year return period and D = 7.1 m, (c) 300-year return period and D = 4.5 m, and (d) 300-year return period and D = 7.1 m. Figure 4. Comparison of probability displacement, different ﬁtting models for (a) 645-year return period and D = 4.5 m, (b) 645-year return period and D = 7.1 m, (c) 300-year return period and D = 4.5 m, and (d) 300-year return period and D = 7.1 m. 4.4 Probabilistic displacement of the north Tabriz fault probability displacement has been compared using different ﬁtting models. According to passing a part of the north Tabriz fault through the ﬁfth district of Tabriz city, estimating the probabilistic displacement in this area is of great importance, and predict- ing the areas with a higher level of surface rupture hazard is an important matter. 4.2 Comparison of different ﬁtting models The ﬁtting models (bilinear, elliptical, and quadratic) have similar uncertainties. In this study, the bilinear model is used to obtain probabilistic displacements. In Fig. 4, estimated https://doi.org/10.5194/nhess-22-3571-2022 Nat. Hazards Earth Syst. Sci., 22, 3571–3583, 2022 3578 4.3 Annual exceedance rate of 5 % in 50 years In the next step, for both 4.5 and 7.1 m displacements, the annual exceedance rate of 5 % in 50 years, at distances of 64 and 120 m from the assumed site, has been examined and shown in Fig. 5. For both scenarios (Mw ∼7.7, 645 years, and Mw ∼7.7, 645 years), the results are shown in Fig. 5a and b. In the case of displacement of 4.5 m, the annual ex- ceedance rates of displacement of D = 4 m at distances of 64 and 120 m for the ﬁrst and second scenarios are 1.8 × 10−4, 7.5×10−6, 2.16×10−4, and 1.32×10−5, respectively. In the case of displacement of 7.1 m, the annual exceedance rates of displacement of D = 4 m at distances of 64 and 120 m for the ﬁrst and second scenarios are estimated as 1.88 × 10−4, 7.98 × 10−6, 2.22 × 10−4, and 1.39 × 10−5, respectively. Considering a section with a length of 2 km from the north Tabriz fault according to Figs. 6, 7, and 8, the possible 2D displacements for the north Tabriz fault have been estimated. Figure 6 shows the probabilistic displacement of the two sce- narios for 5 % in 50 years (by the blue color spectrum). The probabilistic displacements for the 4.5 and 7.1 m displace- ments for the ﬁrst scenario are shown in Fig. 6a and b, and for the second scenario, results are shown in Fig. 6c and d, respectively. For the second scenario, the probabilistic dis- placement values have a higher level of hazard that can be seen at greater distances from the assumed sites. The prob- abilistic displacements of two scenarios for 5 % in 475 and 2475 years are shown in Figs. 7 and 8, respectively (using the blue to red color spectrum). https://doi.org/10.5194/nhess-22-3571-2022 Nat. Hazards Earth Syst. Sci., 22, 3571–3583, 2022 3579 M. Hosseini and H. Rahimi: PFDHA for the north Tabriz fault M. Hosseini and H. Rahimi: PFDHA for the north Tabriz fault Figure 5. Comparison of the annual exceedance rate of displacement for (a) D = 4.5 m displacement and (b) D = 7.1 m displacement. Figure 5. Comparison of the annual exceedance rate of displacement for (a) D = 4.5 m displacement and (b) D = 7.1 m displacement. f the annual exceedance rate of displacement for (a) D = 4.5 m displacement and (b) D = 7.1 m displacement Figure 6. Fault displacement hazard map for a 5 % probability of exceedance in 50 years for (a) Mw ∼7.7 and return period of 645 years for D = 4.5 m, (b) Mw ∼7.7 and return period of 645 years for D = 7.1 m, (c) Mw ∼7.3 and return period of 300 years for D = 4.5 m, and (d) Mw ∼7.3 and return period of 300 years for D = 7.1 m. Figure 6. Fault displacement hazard map for a 5 % probability of exceedance in 50 years for (a) Mw ∼7.7 and return period of 645 years for D = 4.5 m, (b) Mw ∼7.7 and return period of 645 years for D = 7.1 m, (c) Mw ∼7.3 and return period of 300 years for D = 4.5 m, and (d) Mw ∼7.3 and return period of 300 years for D = 7.1 m. https://doi.org/10.5194/nhess-22-3571-2022 https://doi.org/10.5194/nhess-22-3571-2022 https://doi.org/10.5194/nhess-22-3571-2022 Nat. Hazards Earth Syst. Sci., 22, 3571–3583, 2022 Nat. Hazards Earth Syst. Sci., 22, 3571–3583, 2022 M. Hosseini and H. Rahimi: PFDHA for the north Tabriz fault 3580 3580 M. Hosseini and H. Rahimi: PFDHA for the north Tabriz fault Figure 7. Fault displacement hazard map for a 5 % probability of exceedance in 475 years for (a) Mw ∼7.7 and return period of 645 years for D = 4.5 m, (b) Mw ∼7.7 and return period of 645 years for D = 7.1 m, (c) Mw ∼7.3 and return period of 300 years for D = 4.5 m, and (d) Mw ∼7.3 and return period of 300 years for D = 7.1 m. Figure 7. Fault displacement hazard map for a 5 % probability of exceedance in 475 years for (a) Mw ∼7.7 and return period of 645 years for D = 4.5 m, (b) Mw ∼7.7 and return period of 645 years for D = 7.1 m, (c) Mw ∼7.3 and return period of 300 years for D = 4.5 m, and (d) Mw ∼7.3 and return period of 300 years for D = 7.1 m. of hazard and is more critical and can be considered as the worst-case scenario. The values of displacement perpendicular to the assumed site and the amount of probability hazard in the area were investigated and are illustrated in Fig. 9. The two scenarios (Mw ∼7.7, 645 years, and Mw ∼7.3, 300 years) were com- pared. According to Fig. 9a, for 5 % in 50 years, the scenario Mw ∼7.3 and 300 years has a higher hazard level and can be considered the worst-case scenario. The numerical value of the displacement is obtained equally in the two displacement cases (4.5 and 7.1 m). The ﬁrst scenario, given that it has a larger magnitude than the second scenario (1m = 0.4) but due to the higher return period, has a lower hazard level than the second scenario. At about 5 % in 50 years as shown in Fig. 9a, the second scenario has a higher level of hazard than the ﬁrst scenario due to the shorter return period. In the case of 5 % in 475 and 2475 years, according to Fig. 9b and c, un- like the case of 50 years, the ﬁrst scenario has a higher level 5 Conclusion The lack of large instrumental earthquakes in northwestern Iran leads to more signiﬁcant epistemic uncertainty in the obtained values. Due to the passing of the north Tabriz fault through the city of Tabriz and destructive historical earth- quakes, studying methods such as PFDHA is essential to pre- vent disasters and economic and human losses in this region. Despite following the Petersen et al. (2011) study and code, which have been associated with favorable properties such as fault mapping accuracy, and a survey of various ﬁt- ting models, we mention some weaknesses here. We hope that the hazard analysis community will be able to incorpo- rate these points in future studies. Nat. Hazards Earth Syst. Sci., 22, 3571–3583, 2022 https://doi.org/10.5194/nhess-22-3571-2022 M. Hosseini and H. Rahimi: PFDHA for the north Tabriz fault 3581 Figure 8. Fault displacement hazard map for a 5 % probability of exceedance in 2475 years for (a) Mw ∼7.7 and return period of 645 years for D = 4.5 m, (b) Mw ∼7.7 and return period of 645 years for D = 7.1 m, (c) Mw ∼7.3 and return period of 300 years for D = 4.5 m, and (d) Mw ∼7.3 and return period of 300 years for D = 7.1 m. M. Hosseini and H. Rahimi: PFDHA for the north Tabriz fault 3581 Figure 8. Fault displacement hazard map for a 5 % probability of exceedance in 2475 years for (a) Mw ∼7.7 and return period of 645 years for D = 4.5 m, (b) Mw ∼7.7 and return period of 645 years for D = 7.1 m, (c) Mw ∼7.3 and return period of 300 years for D = 4.5 m, and (d) Mw ∼7.3 and return period of 300 years for D = 7.1 m. 1. In the mentioned method, the geometry of the causal fault is not considered, so the characteristics of the source, such as dip, depth, and rake, are not used, which will increase the uncertainty in calculating numerical values. The north Tabriz fault has a slope to the north, so the displacement values on the north plate should be more than the displacement values on the south plate. ues in some cases. We can guess that one of the draw- backs of this method is the estimation of probabilis- tic displacement values for higher exceedance rates, in- cluding 2475 years. 4. 5 Conclusion The values obtained clearly show that the ﬁrst scenario (Mw ∼7.7, 645 years) has a higher hazard level such as in 475 and 2475 years because it has a higher mag- nitude. It is the worst case, and in 50 years, the second scenario has a higher level of risk due to the shorter re- turn period. We conclude that the return period is the most inﬂuential parameter in the lower years, and in the higher years, the magnitude is. 2. The attenuation relationships used for this hazard analy- sis are also taken from a minimal database that has used only 22 historical and instrumental earthquakes in the world. Surface rupture data in Asia, the United States, and other parts of the world, which have different seis- motectonic characteristics, cause high uncertainties in the displacement and exceedance rate. Due to the lack of instrumental data for high-magnitude earthquakes and surface ruptures, the discussion about the future probabilistic failure level of this active fault is uncer- tain. As a result, one of the ways to reduce the level of dam- 3. It is clear that probabilistic displacement values for the exceedance rate in 475 and 2475 years have similar val- 3. It is clear that probabilistic displacement values for the exceedance rate in 475 and 2475 years have similar val- https://doi.org/10.5194/nhess-22-3571-2022 Nat. Hazards Earth Syst. Sci., 22, 3571–3583, 2022 82 M. Hosseini and H. Rahimi: PFDHA for the north Tabriz fault gure 9. Comparison of drop in displacement values in the scenarios for (a) 50 years, (b) 475 years, and (c) 2475 years. 3582 M. Hosseini and H. Rahimi: PFDHA for the north Tabriz fault M. Hosseini and H. Rahimi: PFDHA for the north Tabriz fault mparison of drop in displacement values in the scenarios for (a) 50 years (b) 475 years and (c) 2475 yea Figure 9. Comparison of drop in displacement values in the scenarios for (a) 50 years, (b) 475 years, and (c) 2475 years. age and ﬁnancial and human losses is to avoid construction around this fault trace due to several terrible historical earth- quakes. Competing interests. The contact author has declared that neither of the authors has any competing interests. Competing interests. The contact author has declared that neither of the authors has any competing interests. Disclaimer. Publisher’s note: Copernicus Publications remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. Code availability. In this study, we have used the code related to Petersen et al. (2011, https://doi.org/10.1785/0120100035), which we received by request from Rui Chen. Special issue statement. This article is part of the special issue “Earthquake-induced hazards: ground motion ampliﬁcation and ground failures”. It is not associated with a conference. Data availability. The data used in this study can be obtained from the studies of Youngs et al. (2003), Hessami et al. (2003) and Ghas- semi et al. (2016). Acknowledgements. A sincere thanks to Mark Petersen and Rui Chen for providing computer programs and the editor and reviewers for their help in improving the paper. Author contributions. Mohammadreza Hosseini was responsible for methodology, software, and writing the original draft. Habib Rahimi was responsible for supervision, methodology, writing, re- viewing, and editing. Review statement. This paper was edited by Hans-Balder Havenith and reviewed by Robb E. S. Moss and one anonymous referee. References Katona, T. J., Tóth, L., and Gy˝ori, E.: Fault displace- ment hazard analysis based on probabilistic seismic haz- ard analysis for speciﬁc nuclear sites, Appl. Sci., 11, 7162, https://doi.org/10.3390/app11157162, 2021. Baize, S., Nurminen, F., Sarmiento, A., Dawson, T., Takao, M., Scotti, O., Azuma, T., Boncio, P., Champenois, J., Cinti, F. R., and Civico, R.: A worldwide and uniﬁed database of surface rup- tures (SURE) for fault displacement hazard analyses, Seismol. Res. Lett., 91, 499–520, https://doi.org/10.1785/0220190144, 2020. Mousavi-Bafrouei, S. H., Mirzaei, N., and Shabani, E.: A declus- tered earthquake catalog for Iranian plateau, Ann. Geophys- Italy., 57, 6, https://doi.org/10.4401/ag-6395, 2014. Nurminen, F., Boncio, P., Visini, F., Pace, B., Valentini, A., Baize, S., and Scotti, O.: Probability of occurrence and displacement regression of distributed surface ruptur- ing for reverse earthquakes, Front. Earth Sci., 8, 456, https://doi.org/10.3389/feart.2020.58160, 2020. Berberian, M.: Seismic Sources of the Transcaucasian Histori- cal Earthquakes. in: Historical and Prehistorical Earthquakes in the Caucasus’, NATO ASI Series 2, Environment-28, edited by: Giardini, D. and Balassanian, S., Kluwer Academic Press, the Netherlands, 233–311, https://doi.org/10.1007/978-94-011- 5464-2_13, 1997. Paul C. Rizzo Associates: Probabilistic Fault Displacement Hazard Analysis Krško East and West Sites Proposed Krško 2 Nuclear Power, Technical Report, Inc., 1, 1–22, 2013. Berberian, M. and Arshadi, S.: On the evidence of the youngest activity of the North Tabriz Fault and the seismicity of Tabriz city, Geological Survey of Iran, 39, 397–418, 1976. Petersen, M. D. and Wesnousky, S. G.: Fault slip rates and earth- quake histories for active faults in southern California, B. Seis- mol. Soc. Am., 84, 1608–1649, 1994. Berberian, M. and Yeats, R. S.: Patterns of historical earthquake rupture in the Iranian Plateau, B. Seismol. Soc. Am., 89, 120– 139, https://doi.org/10.1785/BSSA0890010120, 1999. Petersen, M. D., Dawson, T. E., Chen, R., Cao, T., Wills, C. J., Schwartz, D. P., and Frankel, A. D.: Fault displacement hazard for strike-slip faults, Bull. Seismol. Soc. Am., 101, 805–825, https://doi.org/10.1785/0120100035, 2011. Cornell, C. A.: Engineering seismic risk analysis, B. Seismol. Soc. Am., 58, 1583–1606, https://doi.org/10.1785/BSSA0580051583, 1968. Stepp, J. C., Wong, I., Whitney, J., Quittmeyer, R., Abraham- son, N., Toro, G., and Sullivan, T.: Probabilistic seismic hazard analyses for ground motions and fault displacement at Yucca Mountain, Nevada, Earthq. Spectra, 17, 113–151, https://doi.org/10.1193/1.1586169, 2001. Djamour, Y., Vernant, P., Nankali, H. R., and Tavakoli, F.: NW Iran- eastern Turkey present-day kinematics: Results from the Iranian permanent GPS network, Earth. Planet. Sc. Lett., 307, 27–34, https://doi.org/10.1016/j.epsl.2011.04.029, 2011. Ghassemi, M. M. Hosseini and H. Rahimi: PFDHA for the north Tabriz fault 3583 https://doi.org/10.5194/nhess-22-3571-2022 https://doi.org/10.5194/nhess-22-3571-2022 Nat. Hazards Earth Syst. Sci., 22, 3571–3583, 2022 Nat. Hazards Earth Syst. Sci., 22, 3571–3583, 2022 Nat. Hazards Earth Syst. Sci., 22, 3571–3583, 2022 Jackson, J.: Partitioning of strike-slip and convergent mo- tion between Eurasia and Arabia in eastern Turkey and the Caucasus, J. Geophys. Res.-Sol. Ea., 97, 12471–12479, https://doi.org/10.1029/92JB00944, 1992. References R.: Surface ruptures of the Iranian earth- quakes 1900–2014: Insights for earthquake fault rupture haz- ards and empirical relationships, Earth-Sci. Rev., 156, 1–13, https://doi.org/10.1016/j.earscirev.2016.03.001, 2016. Wells, D. L. and Coppersmith, K. J.: New Empirical Relation- ships among Magnitude, Rupture Length, Rupture Width, Rup- ture Area, and Surface Displacement, B. Seismol. Soc. Am., 84, 974–1002, 1994. Goda, K.: Potential Fault Displacement Hazard Assessment Us- ing Stochastic Source Models: A Retrospective Evaluation for the 1999 Hector Mine Earthquake, GeoHazards, 2, 398–414, https://doi.org/10.3390/geohazards2040022, 2021. Wesnousky, S. G.: Displacement and Geometrical Character- istics of Earthquake Surface Ruptures: Issues and Impli- cations for Seismic-Hazard Analysis and the Process of Earthquake Rupture, B. Seismol. Soc. Am., 98, 1609–1632, https://doi.org/10.1785/0120070111, 2008. Hessami, K., Pantosti, D., Tabassi, H., Shabanian, E., Abbassi, M. R., Feghhi, K., and Solaymani, S.: Paleoearthquakes and slip rates of the North Tabriz Fault, NW Iran: Preliminary results, Ann. Geophys.-Italy., 46, 903–916, https://doi.org/10.4401/ag- 3461, 2003. Youngs, R. R., Arabasz, W. J., Anderson, R. E., Ramelli, A. R., Ake, J. P., Slemmons, D. B., and Toro, G. R.: A methodology for prob- abilistic fault displacement hazard analysis (PFDHA), Earthq. Spectra, 19, 191–219, https://doi.org/10.1193/1.1542891, 2003. Jackson, J.: Partitioning of strike-slip and convergent mo- tion between Eurasia and Arabia in eastern Turkey and the Caucasus, J. Geophys. Res.-Sol. Ea., 97, 12471–12479, https://doi.org/10.1029/92JB00944, 1992. https://doi.org/10.5194/nhess-22-3571-2022 Nat. Hazards Earth Syst. Sci., 22, 3571–3583, 2022
https://openalex.org/W4376130373	https://www.researchsquare.com/article/rs-2908950/latest.pdf	English	null	In silico bioprospecting of receptors associated with the mechanism of action of Rondonin, an antifungal peptide from spider Acanthoscurria rondoniae hemolymph	Research Square (Research Square)	2,023	cc-by	6,553	In silico bioprospecting of receptors associated with the mechanism of action of Rondonin, an antifungal peptide from spider Acanthoscurria rondoniae hemolymph Elias Jorge Muniz Seif BUTANTAN INSTITUTE https://orcid.org/0000-0001-9914-8897 Marcelo Yudi Icimoto FEDERAL UNIVERSITY OF SÃO PAULO https://orcid.org/0000-0002-0746-5279 Pedro Ismael da Silva Junior (  pisjr@butantan.gov.br ) BUTANTAN INSTITUTE https://orcid.org/0000-0001-6619-6489 Research Article Page 1/23 Page 1/23 Abstract Multiresistant fungal species are associated with the development of diseases. Thus, there is a need to develop more efficient drugs for the treatment of these etiological agents. Rondonin is a peptide isolated from the hemolymph of spiders Acanthoscurria rondoniae. Previous studies showed that this peptide presented antifungal activity against Candida sp. and Trichosporon sp. strains, acting in genetic material. However, the molecular targets involved in your biological activity have not yet been described. To determine possible targets involved in the biological activity of Rondonin using bioinformatics tools. The Multiresistant fungal species are associated with the development of diseases. Thus, there is a need to develop more efficient drugs for the treatment of these etiological agents. Rondonin is a peptide isolated from the hemolymph of spiders Acanthoscurria rondoniae. Previous studies showed that this peptide presented antifungal activity against Candida sp. and Trichosporon sp. strains, acting in genetic material. However, the molecular targets involved in your biological activity have not yet been described. To determine possible targets involved in the biological activity of Rondonin using bioinformatics tools. The PharmMapper server was used to search microorganism targets for Rondonin. The PatchDock server was used to perform molecular docking. UCSF Chimera software was used to evaluate these intermolecular interactions. In addition, the I-TASSER server was used to predict the target ligand sites. In the end, all predictions were contrasted with the sites previously described in the literature. Rondonin was consistent with the ligand sites of the following targets: outer membrane proteins F (id:1MPF) and A (id:1QJP), responsible for allowing the passage of small molecules through the plasmatic membrane, subunit of the flavoprotein fumarate reductase (id:1D4E), responsible for the metabolism of nitrogenous bases, and ATP-dependent Holliday DNA helicase junction (id:1IN4), associated with histone proteins that fold the genetic material. These interactions corroborate previous in vitro studies on Rondonin, which acts on fungal genetic material without plasmatic membrane rupture. Therefore, the bioprospecting methods used in this research were considered satisfactory since they showed agreement with previous results obtained in vitro. Introduction Filamentous fungi and yeast belong to the fungal kingdom, which can be either multicellular or unicellular creatures with a double lipid bilayer and a cellular wall made of chitin, β-glucan, and proteins (Galagan et al., 2005). Decomposers are responsible for transforming organic to inorganic material (Baron et al., 2019), but there are free-living (Wennig et al., 2020) and parasite fungi (Barnett and Binder, 1973). Marjory fungi are helpful to people and are produced for use in agriculture (Ortega et al., 2020), food production (Dufossé et al., 2014), cosmetics, and medicine (Dutta et al., 2022). However, some species have a connection to diseases known as mycoses, such as Trichophyton sp., Microsporum sp., Sporothrix sp., and Candida sp. These can affect the skin, mucosa, nails, hair, or body generally, and they are more common in those who have compromised immune systems (Hay, 2013). The prevalence of fungi causes widespread disease in humans, animals, and plants. This harms human health as well as the agricultural and food industries (Hymery et al., 2014; Bernardi et al., 2019; Hay, 2013). A significant representative organism is Candida sp. (Eggimann et al., 2003). The newly discovered yeast Candida auris, described in a coma patient, is antibiotic resistant and has a mortality rate between 33% and 66% depending on the age and clinical condition of the infected individual. This The prevalence of fungi causes widespread disease in humans, animals, and plants. This harms human health as well as the agricultural and food industries (Hymery et al., 2014; Bernardi et al., 2019; Hay, 2013). A significant representative organism is Candida sp. (Eggimann et al., 2003). The newly discovered yeast Candida auris, described in a coma patient, is antibiotic resistant and has a mortality rate between 33% and 66% depending on the age and clinical condition of the infected individual. This Page 2/23 species is typically seen in intensive care units in hospitals and has started an emergency situation for health authorities (Chakrabarti and Singh, 2020; Cortegiani et al., 2018). When conventional treatments are ineffective against these novel yeast strains, the pharmacy industry's research goal is to find new experimental drugs. Natural compounds are a good source of bioactive molecules (Fernandes et al., 2022; Heard et al., 2021) because they are already utilized by living things to survive and evolve in harsh environments (Rochlani et al., 2017). Introduction Peptides, which are amino acid polymers, have a variety of biological effects on organisms. These effects include antibacterial (Lee et al., 2019), antifungal (Garnier et al., 2020), antinociceptive (Wu et al., 2019), antitumoral (Karpiński and Adamczak, 2018), and structural effects, making them promising candidates for combating pathogenic species. Rondonin is a neutral and amphiphilic peptide first isolated from spider (Acanthoscurria rondoniae) hemolymph, with the amino acid sequence IIIQYEGHKH-NH2 and molecular mass 1236.776 Da. This peptide has antifungal activity against yeast strains such as Candida albicans (MDM8 and IOC 4558), Candida krusei (IOC 4559), Candida glabrata (IOC 4565), Candida parapsilosis (IOC 4564), Candida tropicalis (IOC 4560), and Candida guilliermondii (IOC 4557), presenting an MIC (microbial inhibitory concentration) of 40 µM, and is also effective against the filamentous fungus Trichosporon sp. (IOC 4569) (Riciluca et al., 2012). This peptide's mechanisms of action involve DNA binding as well as preventing cell proliferation, leading to fungal death. Importantly, the proteins that are part of your mechanism have not yet been identified (Riciluca et al., 2021). Biological and biomedical researchers increasingly use bioinformatics tools, applied as molecular dynamics, docking between receptor and ligand, molecular modeling, and simulations of biological activities, among other aspects (Ju and Zhang, 2015; Bao et al., 2014; Seif et al., 2023). These methods have advantages such as low costs, fast screening and environmental friendliness. When the investigation requires considerable processing power, it can be carried out on institutional or even web servers (Iyer et al., 2015; Quintero-Gil et al., 2017). Therefore, the aim of this study was to identify potential receptors associated with the antifungal activity of Rondonin using molecular docking, a bioinformatics tool (in silico). Peptide structure and physicochemical properties The physicochemical properties of the Rondonin amino acid sequence (IIIQYEGHKH-NH2) were predicted by the heliquest server (https://heliquest.ipmc.cnrs.fr/) (Gautier et al., 2008). Rondonin structure was built using the primary structure and the software UCSF Chimera (https://www.cgl.ucsf.edu/chimera/) (Pettersen et al., 2004). Then, to have more parsimony, this structure was minimized using default settings of the system except for the Steepest decendent step (1000). Moreover, hydrogens and charges Page 3/23 Page 3/23 were added for minimization calculation. Finally, the rondonin structure was saved in MOL2 and PDB for target prospecting and molecular docking analysis. Target prospecting by PharmMapper Fishing A search of protein targets of Rondonin was performed with PharmMapper (http://www.lilab- ecust.cn/pharmmapper/) (Wang et al., 2017), a reverse docking server for target identification. The rondonin structure was submitted to the server using default settings, except for Pharmacophore Mapping, which selected all target options. Then, the results were classified by the normalized fit score. Moreover, all variables provided by the server (PDB ID, target name, normalized fit score, protein classification, species origin and function) were collected for 300 major results. Only 4,8% (table S1) of all targets originated from the fungal kingdom, an organism that Rondonin presented as biologically active in this study. Only bacterial targets were selected due to evolutionary proximity, the top 50 in this list, totaling 14 targets. Molecular Docking by PatchDock Molecular docking was performed using the PatchDock server (https://bioinfo3d.cs.tau.ac.il/PatchDock/) (Schneidman-Duhovny et al., 2005). The server was set by Clustering RMSD (4.0) and Complex Type (Default). All 14 targets found by PharmMapper fishing were submitted as receptors, and Rondonin was used as the ligand. Better peptide and receptor binding was determined by the major score, and area (binding area), ACE (effective atomic contact energies) and ligand transformation were collected for each interaction. Ligand and receptor interactions Rondonin and receptor interactions were measured by UCSF Chimera (https://www.cgl.ucsf.edu/chimera/) software (Pettersen et al., 2004). The FindhBond tool set by relax constraints binds (2 Å and 20 degrees) only to hydrogen bonds and is less than 4 Å in length, as presented in this study. The donor and acceptor residues of each interaction are presented. Peptide structure The heliquest server physicochemical results indicated that Rondonin is a neutral peptide with a net charge of 0, molecular weight of 1236.77 g/mol and hydrophobic index/hydrophobic moment of 0.477/0.236. The helicoidal prediction did not indicate a hydrophobic side because nonpolar amino acids were present at all 3 sites of the peptide helix. The peptide minimized the free energy (-852.55 kJ/mol) (Fig. 1). Ligand Site Prediction It was used by the I-TASSER (protein function & function prediction) server (https://zhanggroup.org/I- TASSER/) to predict ligand binding sites for receptors selected by better results in molecular docking analysis. We used the fasta sequence of pyridoxine 5-phosphate synthase [1M5W], outer membrane protein F [1MPF], and fumarate reductase flavoprotein subunit [1D4E] for the three better results. In addition, the ligand binding site for the Holliday junction ATP-dependent DNA helicase ruvB [1IN4] and outer membrane protein A [1QJP] presented median dock scores. Page 4/23 Page 4/23 Target Fishing by PharmMapper l l d ki Table 1 Normalized Fit Score (value obtained by ratio of fit score and number of features), Origin (Species from the target was isolated), + (Gram-positive), - (Gram-negative). Normalized Fit Score (value obtained by ratio of fit score and number of features), Origin (Species from the target was isolated), + (Gram-positive), - (Gram-negative). Target Fishing by PharmMapper With bioprospecting of PharmMapper, 300 targets were found for Rondonin, ranked by normalized fit score, and this variable, origin, function, disease, indication and protein classification were collected (Table S2). Due to the low frequency of yeast and fungal origins of targets, organisms that Rondonin presented biologically active, in this study, only the microbial origin of the top 50 by normalized fit score (0.9954–0.9454) was used. Fourteen potential receptors for Rondonin activity were selected (Table 1). After a PharmMapper search, it was found that outer membrane protein F [1MPF] (0.9954), parathion hydrolase [1P6B] (0.9952), and malate dehydrogenase [1UXH] (0.9873) presented the top 3 normalized fit scores, presenting 2, 3 and 8 respective general ranks. Methyl-accepting chemotaxis protein II [1LIH] (0.9454), Holliday junction ATP-dependent DNA helicase ruvB [1FUP] (0.9516), and Glycine oxidase [1NG3] (0.9529) presented lower values, with values of 50, 48 and 41, respectively, in all search ranks (Table 1). Gram-negative (-) was the major prevalent organism origin, representing approximately 79% of all targets. Page 5/23 Page 5/23 Table 1 Target name found by PharmMapper search using Rondonin as ligand. Target was classified by major normalized fit score and microorganism origin. PM Rank PDB ID Target Name Normalized Fit Score Origin Gram 2 1MPF Outer membrane protein F 0.9954 Escherichia coli - 3 1P6B Parathion hydrolase 0.9952 Flavobacterium sp. - 8 1UXH Malate dehydrogenase 0.9873 Chloroflexus aurantiacus - 13 1QJP Outer membrane protein A 0.9778 Escherichia coli BL21(DE3) - 14 1CTT Cytidine deaminase 0.9778 Escherichia coli - 24 1M5W Pyridoxine 5-phosphate synthase 0.9625 Escherichia coli - 25 1D4E Fumarate reductase flavoprotein subunit 0.9625 Shewanella oneidensis - 30 1BLH Beta-lactamase 0.9596 Staphylococcus aureus + 37 2EEO Aspartate 1-decarboxylase 0.9538 Thermus thermophilus HB8 - 39 1UZR Ribonucleoside- diphosphate reductase subunit beta 0.9532 Mycobacterium tuberculosis + 40 1NG3 Glycine oxidase 0.9529 Bacillus subtilis + 41 1FUP Fumarate hydratase class II 0.9516 Escherichia coli - 48 1IN4 Holliday junction ATP- dependent DNA helicase ruvB 0.9465 Thermotoga maritima - 50 1LIH Methyl-accepting chemotaxis protein II 0.9454 Salmonella enterica subsp. enterica serovar Typhimurium - PM Rank (General PharmMapper rank search), PDB ID (Identification code in protein data bank), Normalized Fit Score (value obtained by ratio of fit score and number of features), Origin (Species from the target was isolated), + (Gram-positive), - (Gram-negative). Molecular docking Dock results showed score (9910 to 5536), area (1496.2 to 709.3) and ACE (367.78 to -298.47). The receptor rank obtained by the PatchDock server was distinct from that presented by the PharmMapper search, and all results are presented in Table S3. In molecular docking analysis, pyridoxine 5-phosphate Page 6/23 synthase [1M5W] (score = 9910), outer membrane protein F [1MPF] (score = 8958), and fumarate reductase flavoprotein subunit [1D4E] (score = 8796) had the highest score values, and these targets presented 24, 2 and 25 PM ranks, respectively (Table 2). synthase [1M5W] (score = 9910), outer membrane protein F [1MPF] (score = 8958), and fumarate reductase flavoprotein subunit [1D4E] (score = 8796) had the highest score values, and these targets presented 24, 2 and 25 PM ranks, respectively (Table 2). However, methyl-accepting chemotaxis protein II [1LIH] (Score = 5536), beta-lactamase [1BLH] (Score = 6180), and aspartate 1-decarboxylase [2EEO] (Aspartate 1-decarboxylase) showed lower score values, with PM ranks of 50, 30 and 37, respectively. Two targets presented intermediary results, Holliday junction ATP-dependent DNA helicase ruvB [1IN4] (Score = 7932) and Outer membrane protein A [1QJP] (Score = 7248), because their function is correlated with the activity of Rondonin against yeast and fungi cells in in vitro assays (Table 2). Table 2 Docking results by the Patchdock server. Rondonin was used as the ligand, and PharmMapper targets were used as the receptor. Receptor names are classified by dock score. PM Rank PDB ID Receptor Name Score Area ACE (kj/mol) 24 1M5W Pyridoxine 5-phosphate synthase 9910 1330.8 367.78 2 1MPF Outer membrane protein F 8958 1080.2 303.83 25 1D4E Fumarate reductase flavoprotein subunit 8796 1496.2 205.69 8 1UXH Malate dehydrogenase 8686 1334.1 -298.47 40 1NG3 Glycine oxidase 8218 1169.7 -19.01 48 1IN4 Holliday junction ATP-dependent DNA helicase ruvB 7932 1069.4 311.18 41 1FUP Fumarate hydratase class II 7918 964.4 125.84 39 1UZR Ribonucleoside-diphosphate reductase subunit beta 7722 892 402.64 13 1QJP Outer membrane protein A 7248 932.1 -56.23 3 1P6B Parathion hydrolase 6958 900.4 -99.68 14 1CTT Cytidine deaminase 6580 739 -8.03 37 2EEO Aspartate 1-decarboxylase 6216 721.1 -55.63 30 1BLH Beta-lactamase 6180 836.1 -114.07 50 1LIH Methyl-accepting chemotaxis protein II 5536 709.3 22.08 PM Rank (General PharmMapper rank search), PDB ID (Identification code in protein data bank), ACE (Atomic effective contact energy). Table 2 Receptor and peptide interactions Page 7/23 To facilitate the understanding of the docking results, only h-bound (≤ 4 Å) interactions were presented (Table 3); however, all interactions can be accessed in Table S4. In this way, for all interactions presenting Rondonin as an electron donor and targets as acceptors, targets with lower scores did not show h-bonds between the receptor and Rondonin interaction. However, major dock score values did not present a major number of interactions. 1M5W (1 bound) is between residues MET 224. Receptor and GLN 4. Ligand, while 1MPF (2 bound) was among residues GLY 78. Receptor, SER 125. R and HIS 8. Ligand, LYS 9. Ligand, respectively, with 1D4E (1 bound) between residues LYS 69. Receptor and ILE 1. Ligand. In the end, 1IN4 (1 bound) is between residues SER 83. Receptor and ILE 1. Ligand, 1QJP (0 bound) presents any h-bound interaction (Table 3). Page 8/23 Table 3 Binding results between Rondonin and its receptors. Only residue H bonds (≤ 4 Å) are presented. PM Rank PDB ID Receptor Name Donor Acceptor D-A distance (Å) 24 1M5W Pyridoxine 5-phosphate synthase GLN 4.L NE2 MET 224.RB O 3.438 2 1MPF Outer membrane protein F HIS 8.L NE2 LYS 9.L NZ GLY 78.R O SER 125.R OG 2.688 3.610 25 1D4E Fumarate reductase flavoprotein subunit ILE 1.L N LYS 69.R O 2.631 8 1UXH Malate dehydrogenase GLN 4.L NE2 SER 15.RA OG 3.720 40 1NG3 Glycine oxidase LYS 9.L NZ ARG 329.RA O 2.677 48 1IN4 Holliday junction ATP-dependent DNA helicase ruvB ILE 1.L N SER 83.R OG 3.061 41 1FUP Fumarate hydratase class II ILE 1.L N ILE 2.L N GLY 432.RB O GLY 432.RB O 2.778 3.309 39 1UZR Ribonucleoside-diphosphate reductase subunit beta GLN 4.L NE2 HIS 8.L NE2 LYS 9.L NZ ASN 135.RB D1 GLU 229.RA O GLU 151.RA E1 2.823 3.698 3.501 13 1QJP Outer membrane protein A N/B N/B N/B 3 1P6B Parathion hydrolase GLN 4.L NE2 PHE 132.RA O 2.544 14 1CTT Cytidine deaminase HIS 8.L NE2 HIS 10.L NE2 SER 224.R O GLN 193.R OE1 2.277 3.880 37 2EEO Aspartate 1-decarboxylase N/B N/B N/B Page 9/23 PM Rank PDB ID Receptor Name Donor Acceptor D-A distance (Å) 30 1BLH Beta-lactamase N/B N/B N/B 50 1LIH Methyl-accepting chemotaxis protein II N/B N/B N/B Donor (electron donor residue), Acceptor (electron acceptor residue), D-A distance (distance between heavy atoms of electron acceptor and donor), GLY 1. Receptor and peptide interactions L N (Residue name/ Number/ Chain ID/ Shared electron atom), L (Ligand ID), Rx (ReceptorSubunit ID). Ligand binding site analysis Residues shared between both binding site models are in bold. PM Rank PDB ID C- Score Cluster size Ligand bind site residues (Prediction) Ligand bind site residues (Found) 24 1M5W 0.92 135 9, 72, 94, 133, 153, 193, 194, 213, 214, 215, 216 29RD, 224RB, 225RG(2), the prediction, ranges [0–1]), Cluster size (total number of templates in a C-Score (confidence score of the prediction, ranges [0–1]), Cluster size (total number of templates in a cluster). Ligand binding site analysis All ligand site predictions are presented in Table S5. Bind site predictions did not show colocalization for prediction and found binding sites for 1M5W (C-Score = 0.92; Cluster Size = 135) and 1MPF (C-Score = 0.08; Cluster Size = 14). Although they presented major dock score values, Rondonin interacted closely with the site (Figs. 2 and 3). For 1D4E (C-Score = 0.9; Cluster Size = 129) and 1IN4 (C-Score = 0.97; Cluster Size = 307), colocalization of receptor residues predicted by the I-TASSER server was found two times in 276 for 1D4E and for 1IN4 at 65 and two times for residues 66 and 217 (Fig. 4–5) (Table 4). The receptor 1QJP (C-Score = 0.05; Cluster Size = 6) also did not present colocalization between both models (Fig. 6). For this research, only the best prediction model and all the predictions can be accessed in Table S4. For all channel proteins, the ligand site model was found to have low C-SCORE values, indicating the absence of this site. Page 10/23 Table 4 Study of the binding site prediction of the receptor using the I-TASSER server compared with docking analysis by PatchDock. Residues shared between both binding site models are in bold. PM Rank PDB ID C- Score Cluster size Ligand bind site residues (Prediction) Ligand bind site residues (Found) 24 1M5W 0.92 135 9, 72, 94, 133, 153, 193, 194, 213, 214, 215, 216 29RD, 224RB, 225RG(2), 228RG, 228RD(2), 228RE, 229RH, 229RB, 2 1MPF 0.08 14 19, 21, 334, 336, 338 16, 42, 64, 68, 78, 80, 102, 125, 132, 168 25 1D4E 0.9 129 132, 133, 134, 135, 136, 137, 156, 157, 158, 162, 163, 164, 165, 167, 168, 169, 170, 171, 276, 277, 278, 312, 313, 314, 336, 337, 338, 344, 375, 504, 505, 534, 535, 545, 548, 549, 550, 551, 554 78(2), 79(2), 102, 103, 105, 274, 276(2), 294, 341(2), 343 48 1IN4 0.97 307 26, 27, 28, 59, 60, 61, 62, 63, 64, 65, 66, 188, 192, 216, 217, 220 17, 65, 66(2), 83(2), 109, 217(4), 224 13 1QJP 0.05 6 41, 43, 51, 53, 55, 79 16, 75, 117, 120 C-Score (confidence score of the prediction, ranges [0–1]), Cluster size (total number of templates in a cluster) Table 4 Study of the binding site prediction of the receptor using the I-TASSER server compared with docking analysis by PatchDock. Discussion The biological activity of peptides is directly associated with their physicochemical properties, such as charge, hydrophilicity, chain size and amino acid sequence. Because of interactions compatible with this structure, these properties are related to the mechanism of action of this molecule (Hopkins et al., 2014). Peptides can interact with proteins, enzyme lipid membranes, and nucleic acids in this manner, inactivating or restructuring important biomolecules required for microorganism maintenance, ultimately leading to death (Pelay-Gimeno et al., 2015; Nevola and Giralt, 2015). The heliquest server was used to measure the physicochemical properties of Rondonin, a neutral and hydrophobic peptide (Fig. 1). Cationic amphiphilic peptide mechanisms of action are usually associated with membrane disruption. Because of the presence of peptidoglycans in bacterial and proteoglycans in fungal cellular walls, this Page 11/23 Page 11/23 mechanism occurs due to a potential difference between the peptide, which is positively charged, and the microorganism membrane, which is negatively charged (Jenssen et al., 2006; Brogden, 2005; Wenzel et al., 2014). As a result of their hydrophobicity, these peptides are attracted by plasmatic membranes and can interact with them, forming toroidal pore Ludtke et al., 1996), barriel-stave (Ehrenstein and Lecar, 1977), and carpet (Pouny et al., 1992), causing the disruption of membranes. Rondonin, however, has a neutral charge rather than the cited mechanism. Additionally, the described biological activity revealed that Rondonin had no hemolysis potential with human red blood cells and did not interact in the mimetic system membrane POPC (Riciluca et al., 2012, 2021). It is believed that for antifungal activity, peptide internalization is required with the help of a channel protein present in the plasmatic membrane before Rondonin interacts with DNA structures and associated proteins (Walrant et al., 2017). PharmMapper, an online reverse dock server, was used to identify the targets involved in these interactions. To avoid selection bias and evolutionary proximity with the fungus kingdom, all targets were grouped by main normalized score value and filtered by Gram-positive or Gram-negative origin. As the next step, we selected all targets from PharmMapper to perform molecular docking simulation using PatchDock. In order, the best ranked results from PatchDock resulted in targets observed in Table 2. pyridoxine 5-phosphate synthase (IDpdb:1M5W), Outer membrane protein F (IDpdb:1MPF) and Fumarate reductase flavoprotein subunit (IDpdb:1D4E) are good candidates. Moreover, a few other targets presented good values of dock score. Discussion Finally, the holliday junction ATP-dependent DNA helicase ruvB (IDpdb:1IN4), outer membrane protein A (IDpdb:1QJP), histone and channel protein (Table 2) were selected due to the described effect of Rondonin's interaction with histone proteins (Riciluca et al., 2021). For all selected targets, only 1M5W 1MPF did not show residue colocalization between the predicted ligand binding site by the I-TASSER server and the dock Rondonin interaction (Table 4). Pyridoxine 5-phosphate synthase is an enzyme responsible for producing precursors to the biosynthesis of vitamin B6 in Escherichia coli. This vitamin is an essential cofactor in amino acid and glycogenolysis metabolism pathways. The residues GLU72, ARG20, GLU72, and HIS193, (Yeh et al., 2002) are found in its active site, but none of them was present in the predicted site and interacted with Rondonin (Fig. 2). Outer membrane protein F (OmpF) is one of the major membrane porins, and this protein usually forms a channel allowing diffusion of small hydrophobic molecules, 𝛃-lactans and several antibiotics across the plasmatic membrane. Among the amino acids found as internal sites of this protein, ARG42, ARG132, and (Jeanteur et al., 1994) were both found in Rondonin as possible targets. However, this is due to low C- Score values, indicating low accuracy for prediction. The fumarate reductase flavoprotein subunit is an important enzyme in fumarate respiration in anaerobic microorganisms and presents a periplasmic tetrahymena related to energy production. This protein is expressed in Shewanella putrefaciens MR-1. Your active sites are formed by ARG544, ARG401, MET235, THR376, MET374, HIS364, (Leys et al., 1999) none of this residues was found in Rondonin interaction, however the prediction site presented colocalization with real binding site. Page 12/23 Holliday junction ATP-dependent DNA helicase ruvB is present in hexamer RuvB and is responsible for migration, recombination and rescue of DNA replication forks present in bacterial cells. The Rondonin and receptor interaction presented colocalization with motif ADP and sensor 2 presented by residues THR65 and ARG217, respectively (Putnam et al., 2001). Both residues were also found in the predicted binding sites. Holliday junction ATP-dependent DNA helicase ruvB is present in hexamer RuvB and is responsible for migration, recombination and rescue of DNA replication forks present in bacterial cells. The Rondonin and receptor interaction presented colocalization with motif ADP and sensor 2 presented by residues THR65 and ARG217, respectively (Putnam et al., 2001). Both residues were also found in the predicted binding sites. Conclusion In silico bioprospecting was an efficient method to discover targets possibly involved in the antifungal potential of Rondonin, and pyridoxine 5-phosphate synthase and outer membrane protein F were listed as two principal receptors involved in the act mechanism, corroborating prior in vitro research. In this way, Rondonin can cross the cellular membrane through OmpF interacting with histones in the genetic material, therefore imbibing DNA duplication and consequently disturbing fungal proliferation. This study opens the way for future research to find these targets in individual fungi or in vitro studies to measure the interaction between peptides and these receptors as well as to design analogs to improve biological activity. Discussion Outer membrane protein A (OmpA) is an important protein for bacterial conjugation and maintaining the structural stability of the outer membrane, and it is very abundant in Escherichia coli. The active or ligand site of this protein was not found, but Rondonin interacted with the extracellular site and did not colocalize with the predicted binding site (Pautsch and Schulz, 2000). This was expected due to low C- Score values, indicating low accuracy for prediction. The colocalization of Rondonin with the internal face of the channel protein OmpF and motif ADP and Sensor 2 of the Holliday junction ATP-dependent DNA helicase ruvB corroborates the in vitro results of a previous study (Riciluca et al., 2021). Therefore, one might suggest that Rondonin is capable of crossing the cellular membrane through OmpF, interacting with histones present in genetic material. This inhibits cellular division, and consequently, organism growth leads the individual to death. Conflicts 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. https://zhanggroup.org/I-TASSER/. All files used in this study are available in https://github.com/eliasseif/Rondonin_prospecting.git. https://zhanggroup.org/I-TASSER/. All files used in this study are available in https://github.com/eliasseif/Rondonin_prospecting.git. Acknowledgments We thank all the team of the Protein Chemistry Laboratory at the Laboratory for Applied Toxinology (LETA - Butantan Institute, Brazil) for the constant support and encouragement. Additionally, we thank the technicians Rosa Maria Carmo and Priscila do Nascimento Nanni. Author Contributions Conceptualization, E.J.M.S., M.Y.I. and P.I.S.J. ; methodology E.J.M.S. and P.I.S.J. ; software, E.J.M.S. and P.I.S.J. ; validation, E.J.M.S. and P.I.S.J. ; formal analysis, E.J.M.S., M.Y.I. and P.I.S.J. ; investigation, E.J.M.S. and P.I.S.J. ; resources, P.I.S.J. ; data curation, E.J.M.S., M.Y.I. and P.I.S.J. ; writing-original draft preparation, E.J.M.S. ; writing, review and editing, E.J.M.S., M.Y.I. and P.I.S.J. ; supervision, P.I.S.J. ; project administration, P.I.S.J. ; funding acquisition, P.I.S.J. All authors have read and agreed to the published version of the manuscript. Funding This research received financial support from the Research Support Foundation of the State of São Paulo (FAPESP/CeTICS), grant number 2013/07467-1, and from the Brazilian National Council for Scientific and Technological Development (CNPq), grant numbers 472744/2012-7 and 161722/2021-0. Data and software availability The physicochemical proprieties were determined via the Heliquest server https://heliquest.ipmc.cnrs.fr/. Potential receptors were searched using PharmMapper available at http://www.lilab- ecust.cn/pharmmapper/. A receptor sequence file was obtained from the website protein data bank https://www.rcsb.org/. Molecular docking was performed by PatchDock https://bioinfo3d.cs.tau.ac.il/PatchDock/. Ligand and receptor interactions and molecular presentation were built by the free software UCSF chimera (version 1.16) https://www.cgl.ucsf.edu/chimera/. For the prediction of ligand sites, the I-TASSER (protein function & function prediction) server was used https://bioinfo3d.cs.tau.ac.il/PatchDock/. Ligand and receptor interactions and molecular presentation were built by the free software UCSF chimera (version 1.16) https://www.cgl.ucsf.edu/chimera/. For the prediction of ligand sites, the I-TASSER (protein function & function prediction) server was used Page 13/23 References 1. Bao,R. et al. (2014) Review of current methods, applications, and data management for the bioinformatics analysis of whole exome sequencing. journals.sagepub.com, 13, 67–82. 2. Barnett,H.L. and Binder,F.L. (1973) The Fungal Host-Parasite Relationship. Annu. Rev. Phytopathol., 11, 273–292. 3. Baron,N.C. et al. (2019) Filamentous fungi in biological control: Current status and future perspectives. Chil. J. Agric. Res., 79, 307–315. 4. Bernardi,A.O. et al. (2019) Food industry spoilage fungi control through facility sanitization. Curr. Opin. Food Sci., 29, 28–34. 5. Brogden,K.A. (2005) Antimicrobial peptides: Pore formers or metabolic inhibitors in bacteria? Nat. Rev. Microbiol., 3, 238–250. Page 14/23 6. Chakrabarti,A. and Singh,S. (2020) Multidrug-resistant Candida auris: an epidemiological review. Expert Rev. Anti. Infect. Ther., 18, 551–562. 7. Cortegiani,A. et al. (2018) Epidemiology, clinical characteristics, resistance, and treatment of infections by Candida auris. J. Intensive Care, 6. 8. Dufossé,L. et al. (2014) Filamentous fungi are large-scale producers of pigments and colorants for the food industry. Curr. Opin. Biotechnol., 26, 56–61. 9. Dutta,B. et al. (2022) Fungi in Pharmaceuticals and Production of Antibiotics. Springer, Cham, pp. 233–257. 10. Eggimann,P. et al. (2003) Epidemiology of Candida species infections in critically ill non- immunosuppressed patients. Lancet Infect. Dis., 3, 685–702. 11. Ehrenstein,G. and Lecar,H. (1977) Electrically gated ionic channels in lipid bilayers. Q. Rev. Biophys., 10, 1–34. 12. Fernandes,L. et al. (2022) Essential Oils as a Good Weapon against Drug-Resistant Candida auris. Antibiotics, 11, 977. 13. Galagan,J.E. et al. (2005) Genomics of the fungal kingdom: Insights into eukaryotic biology. Genome Res., 15, 1620–1631. 14. Garnier,L. et al. (2020) Antifungal activity of fermented dairy ingredients: Identification of antifungal compounds. Int. J. Food Microbiol., 322, 108574. 15. Gautier,R. et al. (2008) HELIQUEST: A web server to screen sequences with specific α-helical properties. Bioinformatics, 24, 2101–2102. 16. Hay,R. (2013) Superficial fungal infections. Med. (United Kingdom), 41, 716–718. 013) Superficial fungal infections. Med. (United Kingdom), 41, 716–718. 16. Hay,R. (2013) Superficial fungal infections. Med. (United Kingdom), 41, 7 17. Heard,S.C. et al. (2021) Antifungal natural products. Curr. Opin. Biotechnol., 69, 232–241. 18. Hopkins,A.L. et al. (2014) The role of ligand efficiency metrics in drug discovery. Nat. Rev. Drug Discov., 13, 105–121. 19. Hymery,N. et al. (2014) Filamentous fungi and mycotoxins in Cheese: A review. Compr. Rev. Food Sci. Food Saf., 13, 437–456. 20. Iyer,P. et al. (2015) In silico identification of targets for a novel scaffold, 2-thiazolylimino-5-benzylidin- thiazolidin-4-one. Mol. Divers., 19, 855–870. 21. Jeanteur,D. et al. References (1994) Structural and functional alterations of a colicin-resistant mutant of OmpF porin from Escherichia coli. Proc. Natl. Acad. Sci. U. S. A., 91, 10675–10679. H. et al. (2006) Peptide antimicrobial agents. Clin. Microbiol. Rev., 19, 491–5 22. Jenssen,H. et al. (2006) Peptide antimicrobial agents. Clin. Microbiol. Re 23. Ju,F. and Zhang,T. (2015) Experimental Design and Bioinformatics Analysis for the Application of Metagenomics in Environmental Sciences and Biotechnology. Environ. Sci. Technol., 49, 12628– 12640. 24. Karpiński,T.M. and Adamczak,A. (2018) Anticancer Activity of Bacterial Proteins and Peptides. Pharm. 2018, Vol. 10, Page 54, 10, 54. Page 15/23 25. Lee,H. et al. (2019) Conjugation of Cell-Penetrating Peptides to Antimicrobial Peptides Enhances Antibacterial Activity. ACS Omega, 4, 15694–15701. 26. Leys,D. et al. (1999) Structure and mechanism of the flavocytochrome c fumarate reductase of Shewanella putrefaciens MR-1. Nat. Struct. Biol., 6, 1113–1117. 27. Nevola,L. and Giralt,E. (2015) Modulating protein-protein interactions: The potential of peptides. Chem. Commun., 51, 3302–3315. 28. Ortega,H.E. et al. (2020) Patents on endophytic fungi for agriculture and bio-and phytoremediation applications. Microorganisms, 8, 1–26. 29. Pautsch,A. and Schulz,G.E. (2000) High-resolution structure of the OmpA membrane domain. J. Mol. Biol., 298, 273–282. 30. Pelay-Gimeno,M. et al. (2015) Structure-Based Design of Inhibitors of Protein-Protein Interactions: Mimicking Peptide Binding Epitopes. Angew. Chemie - Int. Ed., 54, 8896–8927. 31. Pettersen,E.F. et al. (2004) UCSF Chimera - A visualization system for exploratory research and analysis. J. Comput. Chem., 25, 1605–1612. 32. Pouny,Y. et al. (1992) Interaction of Antimicrobial Dermaseptin and its Fluorescently Labeled Analogs with Phospholipid Membranes. Biochemistry, 31, 12416–12423. 33. Putnam,C.D. et al. (2001) Structure and mechanism of the RuvB holliday junction branch migration motor. J. Mol. Biol., 311, 297–310. 34. Quintero-Gil,C. et al. (2017) In-silico design and molecular docking evaluation of peptides derivatives from bacteriocins and porcine beta defensin-2 as inhibitors of Hepatitis E virus capsid protein. VirusDisease, 28, 281–288. 35. Riciluca,K.C.T. et al. (2021) Rondonin: antimicrobial properties and mechanism of action. FEBS Open Bio, 11, 2541–2559. 36. Riciluca,K.C.T. et al. (2012) Rondonin an antifungal peptide from spider (Acanthoscurria rondoniae) haemolymph. Results Immunol., 2, 66–71. 37. Rochlani,Y. et al. (2017) Metabolic syndrome: Pathophysiology, management, and modulation by natural compounds. Ther. Adv. Cardiovasc. Dis., 11, 215–225. 38. Schneidman-Duhovny,D. et al. (2005) PatchDock and SymmDock: Servers for rigid and symmetric docking. Nucleic Acids Res., 33. 39. Seif,E.J.M. et al. (2023) In silico bioprospecting of receptors for Doderlin: an antimicrobial peptide isolated from Lactobacillus acidophilus. Silico Pharmacol., 11, 1–11. References 40. Walrant,A. et al. (2017) Membrane Crossing and Membranotropic Activity of Cell-Penetrating Peptides: Dangerous Liaisons? Acc. Chem. Res., 50, 2968–2975. 41. Wang,X. et al. (2017) PharmMapper 2017 update: A web server for potential drug target identification with a comprehensive target pharmacophore database. Nucleic Acids Res., 45, W356–W360. 42. Wennig,R. et al. (2020) Mushroom Poisoning. Dtsch. Arztebl. Int., 117, 701. Wennig,R. et al. (2020) Mushroom Poisoning. Dtsch. Arztebl. Int., 117, 701. Page 16/23 Page 16/23 43. Wenzel,M. et al. (2014) Small cationic antimicrobial peptides delocalize peripheral membrane proteins. Proc. Natl. Acad. Sci. U. S. A., 111. 44. Wu,T. et al. (2019) Spider venom peptides as potential drug candidates due to their anticancer and antinociceptive activities. J. Venom. Anim. Toxins Incl. Trop. Dis., 25. 45. Yeh,J.I. et al. (2002) Multistate binding in pyridoxine 5′-phosphate synthase: 1.96 Å crystal structure in complex with 1-deoxy-D-xylulose phosphate. Biochemistry, 41, 11649–11657. 45. Yeh,J.I. et al. (2002) Multistate binding in pyridoxine 5′-phosphate synthase: 1.96 Å crystal structure in complex with 1-deoxy-D-xylulose phosphate. Biochemistry, 41, 11649–11657. Figures g Figure 1 Characterization and physicochemical proprieties of Rondonin. (A) Three-dimensional structure designed by UCSF software relative to the position of each amino acid residue. In (B) peptide helicoidally projection, yellow represents the nonpolar residues, blue represents the basic residues, pink represents asparagine and glutamine, gray represents alanine and glycine, red represents glutamic acid, lilac represents serine and tyrosine, cyan represents histidine and green represents proline. The arrow indicates the hydrophobic portion of this projection. The table at the end shows the amino acid sequence, molecular weight, net charge (NC) and hydrophobicity index/hydrophobic moment (H/µH). 43. Wenzel,M. et al. (2014) Small cationic antimicrobial peptides delocalize peripheral membrane proteins. Proc. Natl. Acad. Sci. U. S. A., 111. Figure 1 Characterization and physicochemical proprieties of Rondonin. (A) Three-dimensional structure designed by UCSF software relative to the position of each amino acid residue. In (B) peptide helicoidally projection, yellow represents the nonpolar residues, blue represents the basic residues, pink represents asparagine and glutamine, gray represents alanine and glycine, red represents glutamic acid, lilac represents serine and tyrosine, cyan represents histidine and green represents proline. The arrow indicates the hydrophobic portion of this projection. The table at the end shows the amino acid sequence, molecular weight, net charge (NC) and hydrophobicity index/hydrophobic moment (H/µH). Page 17/23 Page 17/23 Figure 2 Molecular docking between Rondonin and pyridoxine 5-phosphate synthase. (A) Overview of Rond and receptor docking. (B) focuses on the Rondonin and receptor dock interaction. Red brick (Rond medium blue (Receptor), forest green (predicted ligand binding site residues), yellow line (intermol forces) and white label (residue name and sequence). Figure 2 Molecular docking between Rondonin and pyridoxine 5-phosphate synthase. (A) Overview of Rondonin and receptor docking. (B) focuses on the Rondonin and receptor dock interaction. Red brick (Rondonin), medium blue (Receptor), forest green (predicted ligand binding site residues), yellow line (intermolecular forces) and white label (residue name and sequence). Page 18/23 Page 18/23 Figure 3 Molecular docking between Rondonin and outer membrane protein F. (A) Overview of Rondonin and receptor docking. (B) focuses on the Rondonin and receptor dock interaction. Red brick (Rondonin), medium blue (Receptor), forest green (predicted ligand binding site residues), yellow line (intermolecu forces) and white label (residue name and sequence). Figure 3 Molecular docking between Rondonin and outer membrane protein F. (A) Overview of Rondonin and receptor docking. (B) focuses on the Rondonin and receptor dock interaction. Red brick (Rondonin), medium blue (Receptor), forest green (predicted ligand binding site residues), yellow line (intermolecular forces) and white label (residue name and sequence). Page 19/23 Page 19/23 Figure 4 Molecular docking between Rondonin and the fumarate reductase flavoprotein subunit. (A) Overview o Figure 4 Molecular docking between Rondonin and the fumarate reductase flavoprotein subunit. (A) Overview o Rondonin and receptor docking. (B) focuses on the Rondonin and receptor dock interaction. Red brick (Rondonin), medium blue (Receptor), forest green (predicted ligand binding site residues), yellow line (intermolecular forces) and white label (residue name and sequence). Figure 4 Molecular docking between Rondonin and the fumarate reductase flavoprotein subunit. (A) Overview Rondonin and receptor docking. (B) focuses on the Rondonin and receptor dock interaction. Figure 1 Red bric (Rondonin), medium blue (Receptor), forest green (predicted ligand binding site residues), yellow line (intermolecular forces) and white label (residue name and sequence). Figure 4 Molecular docking between Rondonin and the fumarate reductase flavoprotein subunit. (A) Overview of Rondonin and receptor docking. (B) focuses on the Rondonin and receptor dock interaction. Red brick (Rondonin), medium blue (Receptor), forest green (predicted ligand binding site residues), yellow line (intermolecular forces) and white label (residue name and sequence). Page 20/23 Page 20/23 Figure 5 Molecular docking between Rondonin and the holliday junction ATP-dependent DNA helicase ruvB. (A Overview of Rondonin and receptor docking (B) focuses on the Rondonin and receptor dock interact Figure 5 Molecular docking between Rondonin and the holliday junction ATP-dependent DNA helicase ruvB. (A) Overview of Rondonin and receptor docking. (B) focuses on the Rondonin and receptor dock interaction Red brick (Rondonin), medium blue (Receptor), forest green (predicted ligand binding site residues), yellow line (intermolecular forces) and white label (residue name and sequence). Figure 5 Molecular docking between Rondonin and the holliday junction ATP-dependent DNA helicase ruvB. ( Overview of Rondonin and receptor docking. (B) focuses on the Rondonin and receptor dock interact Red brick (Rondonin), medium blue (Receptor), forest green (predicted ligand binding site residues), yellow line (intermolecular forces) and white label (residue name and sequence). Figure 5 Molecular docking between Rondonin and the holliday junction ATP-dependent DNA helicase ruvB. (A) Overview of Rondonin and receptor docking. (B) focuses on the Rondonin and receptor dock interaction. Red brick (Rondonin), medium blue (Receptor), forest green (predicted ligand binding site residues), yellow line (intermolecular forces) and white label (residue name and sequence). Page 21/23 Page 21/23 Figure 6 Figure 6 Molecular docking between Rondonin and outer membrane protein A. (A) Overview of Rondonin and receptor docking. (B) focuses on the Rondonin and receptor dock interaction. Red brick (Rondonin), medium blue (Receptor), forest green (predicted ligand binding site residues), yellow line (intermolecula forces) and white label (residue name and sequence). Molecular docking between Rondonin and outer membrane protein A. (A) Overview of Rondonin and receptor docking. (B) focuses on the Rondonin and receptor dock interaction. Red brick (Rondonin), medium blue (Receptor), forest green (predicted ligand binding site residues), yellow line (intermolecular forces) and white label (residue name and sequence). Page 22/23 Page 22/23 Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. S1table.xlsx S2table.xlsx S3table.xlsx S4table.xlsx S5table.xlsx S1table.xlsx S2table.xlsx S3table.xlsx S4table.xlsx S5table.xlsx S1table.xlsx S2table.xlsx Page 23/23
https://openalex.org/W2887395451	https://www.matec-conferences.org/articles/matecconf/pdf/2018/51/matecconf_iceast2018_02001.pdf	Chinese	null	Design and simulate of LQR-Fuzzy controller for unicycle robot with double flywheels	MATEC web of conferences	2,018	cc-by	5,004	Design and simulate of LQR-Fuzzy controller for unicycle robot with double flywheels Surachat Chantarachit* Department of Mechatronics Engineering, Rajamangala University of Technology Thanyaburi, Thailand Department of Mechatronics Engineering, Rajamangala University of Technology Thanyaburi, Thailand Abstract. This research is focus on design and simulate unicycle robot with double flywheels model with LQR-Fuzzy controller. Roll balancing torque is generated by gyroscopic effect. Pitch balancing torque is applied by inverted pendulum concept. To control the heading of the robot, the angular momentum from both flywheel is applied to control this. The robot model is based on Euler-Lagrange equations. The non-linear model is linearization by Taylor series expansion. The simulation results conducted by MATLAB/Simulink. LQR-Fuzzy is combination algorithm between LQR and Fuzzy controller. The main structure control is the LQR controller and use the Fuzzy controller to adjust the close loop controller gain. The simulation results is simulated and compared with conventional LQR. Fig. 1 Angular momentum of flywheel Fig. 2 Unicycle robot model Fig. 1 Angular momentum of flywheel 1. Introduction Unicycle robot is a mobile robot which has only one contact point on ground. The unicycle robot dynamic model is very challenging and interesting in research topic in dynamic control. Since, model is non-linear, unstable and difficult to control. A. Schoowinkel [1] is the first one who derives unicycle dynamic model by using the turntable mechanism concept. Z. Sheng and K. Yamafuji [2], who are successful to balance the unicycle robot. The next generation of unicycle balancing is gyroscope force (flywheel). S. Majima et al [3] proposed a single-flywheel technique for balancing a unicycle robot. The produced torque from this technique is higher than the turntable technique. To generate greater torque, the double flywheel is introduced in this paper [4]. There are many control algorithms which applied to control unicycle robot. LQR is well-known and widely used controller in robot control. This paper presents the combination of LQR and Fuzzy controller. The performance of LQR- Fuzzy [5] is evaluated and compared in this paper. Fig. 1 Angular momentum of flywheel Fig. 1 Angular momentum of flywheel Fig. 2 Unicycle robot model Fig. 2 Unicycle robot model © 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: surachat_c@rmutt.ac.th https://doi.org/10.1051/matecconf/201819202001 https://doi.org/10.1051/matecconf/201819202001 MATEC Web of Conferences 192, 02001 (2018) ICEAST 2018 2. Concept Design https://doi.org/10.1051/matecconf/201819202001 MATEC Web of Conferences 192, 02001 (2018) ICEAST 2018 MATEC Web of Conferences 192, 02001 (2018)       2 2 F1 F3 w1 2 F1 F3 2 2 2 2 F1 F2 F3 11 Cos Sin Cos Cos Cos 12 Cos Cos Sin Cos Cos Sin Cos Sin Cos 13, 14 0 , 21 12 22 Cos Sin Sin Cos Cos F F F F F w F F F M i i i rm r l m l r l r m M i i l m r l M M M M M i i i                                      2 2 2 2 w2 w3 2 2 2 Sin Cos 0.5 0.5 Cos2 Cos 0.5 Cos[ 2 ] 0.5 Cos[ 2 ] 0.75 0.25Cos2 0.125Cos[2( )] 0.25Cos2 F F F F F F F F F F F F F F i i r m m r r l m r l m r l m l m l m l m                         2 2 2 2 2 F2 2 2 2 2 w2 2 2 2 2 0.125Cos[2( )] Sin 23 Sin Cos Sin Sin 24 Sin Cos Sin Sin Sin Cos Cos Sin Cos Sin Sin Cos Sin F F F F w F F F F F F F F F F w l m l m r m M i r l m l m M i r m r m r l m r l m r m                                  2 2 2 F2 2 2 w2 Sin Sin 31 13 , 32 23 33 34 Cos 41 14 , 42 4 24 , 3 44 34 w F F F F F w r m M M M M M i l m M rl m M M M M M i r m r m M M                3. Robot Dynamic Model 2 2 2 2 F1 F2 F3 22 Cos Sin Sin Cos Cos M i i i          2 2 2 2 w2 w3 2 2 2 Sin Cos 0.5 0.5 Cos2 Cos 0.5 Cos[ 2 ] 0.5 Cos[ 2 ] 0.75 0.25Cos2 0.125Cos[2( )] 0.25Cos2 F F F F F F F F F F F F F F i i r m m r r l m r l m r l m l m l m l m                         2 2 2 2 0.125Cos[2( )] Sin F F F F w l m l m r m        2 2 2 2 F1 F2 F3 22 Cos Sin Sin Cos Cos M i i i          2 2 2 2 w2 w3 2 2 2 Sin Cos 0.5 0.5 Cos2 Cos 0.5 Cos[ 2 ] 0.5 Cos[ 2 ] 0.75 0.25Cos2 0.125Cos[2( )] 0.25Cos2 F F F F F F F F F F F F F F i i r m m r r l m r l m r l m l m l m l m                         2 2 2 2 0.125Cos[2( )] Sin F F F F w l m l m r m        2 2 2 2 F1 F2 F3 22 Cos Sin Sin Cos Cos M i i i          2 2 2 2 w2 w3 2 2 2 Sin Cos 0.5 0.5 Cos2 Cos 0.5 Cos[ 2 ] 0.5 Cos[ 2 ] 0.75 0.25Cos2 0.125Cos[2( )] 0.25Cos2 F F F F F F F F F F F F F F i i r m m r r l m r l m r l m l m l m l m                         2 2 2 2 0.125Cos[2( )] Sin F F F F w l m l m r m        Fig. 3. Robot Dynamic Model 3 Wheel and Body model The Dynamic model is derived by using Euler-Lagrange Fig. 3 Wheel and Body model F1 F3 w2 2 2 F1 F1 F2 2 2 2 2 F3 F3 F1 2 2 F2 1 2Cos Sin 2Cos Sin Cos Cos Cos Cos Sin Cos Cos Cos Cos Sin Cos Sin Sin Cos Sin Cos Cos Si C i i i i i i i i i i                                                       2 2 F3 w2 2 2 w3 2 2 n Cos Sin Cos Sin Cos Cos Sin Cos Cos Sin Cos Cos Cos Sin 2 Sin Cos Cos w F F F i i i rm r r r r r l m l                                                                Fig. 3 Wheel and Body model The Dynamic model is derived by using Euler-Lagrange (Eq.1). Friction in the system is small and negligible. The Dynamic model is derived by using Euler-Lagrange (Eq.1). Friction in the system is small and negligible. The Dynamic model is derived by using Euler-Lagrange (Eq.1). Friction in the system is small and negligible. d L L f dt q q             (1) (1) L is the difference between total kinetic energy and potential energy. From (Eq.1), the nonlinear equation will be presented in 4 terms and rearranged in to non-linear dynamic equation. The equation of the unicycle robot is shown in (Eq. 2). M is inertia matrix. C is Coriolis matrix. G is gravity matrix, and D is disturbance matrix. Since the robot is operated at low speed, viscous friction is small and negligible (D=0). 2. Concept Design Fig. 2 Unicycle robot model Fig. 2 Unicycle robot model Unicycle robot can be falling in lateral and longitudinal direction. In lateral direction, the gyro scope is the concept to generate torque. There are two flywheels with produce the rolling torque. Both of them are rotated in difference direction with high speed in order to generate angular momentum. By opposite rotating of both flywheels, there are some component of angular momentum effect from rolling torque, it can be applied to control heading of the robot. The angular momentum vector presents in Fig.1. The green vector is the rolling torque component and purple vector is the heading torque component. Last balancing axis is the longitudinal direction, the invert pendulum concept is applied to generate torque. The unicycle robot model presents in Fig.2. 3. Robot Dynamic Model The dynamic model of the unicycle robot is derived into two main parts: wheel and body are presented in Fig.3. The model is referred to Chantarachit, S. [4] model. Unicycle robot model is coupling model between each axis (x y and z). 5.2 LQR-Fuzzy Controller The input of Fuzzy control block in Fig.4 is position state error of the unicycle robot: roll yaw pitch and wheel position. There are four inputs and one output. The output of the fuzzy controller is torque ratio. By This torque ratio, the LQR controller gains are adjusted depend on environment of each state input. This technique can produce good dynamic and static performance. LQR-Fuzzy algorithm is designed for decreasing overshoot and increasing performance of the system. The input of Fuzzy control block in Fig.4 is position state error of the unicycle robot: roll yaw pitch and wheel position. There are four inputs and one output. The output of the fuzzy controller is torque ratio. By This torque ratio, the LQR controller gains are adjusted depend on environment of each state input. This technique can produce good dynamic and static performance. * M is the symmetrical positive definite matrix of the robot inertia and evaluated at the upright position. There are four inputs and one output. The output of the fuzzy controller is torque ratio. By This torque ratio, the LQR controller gains are adjusted depend on environment of each state input. This technique can produce good dynamic and static performance. 3. Robot Dynamic Model     , M q q C q q G q D F     (2) Where, (2) 11 12 13 14 1 21 22 23 24 2 , , , 31 32 33 34 3 41 4 1 2 3 2 43 4 4 4 4 M M M M C M M M M C M C G F M M M M C M M M M C G G G G                                                                     The details of M C and G matrices which are derived from unicycle robot model are shown below The details of M C and G matrices which are derived from unicycle robot model are shown below 2 MATEC Web of Conferences 192, 02001 (2018) ICEAST 2018 https://doi.org/10.1051/matecconf/201819202001   2 2 F3 F2 F3 ratio 2 2 2 F3 F3 2 2 2 F1 2 2 2 3 Cos Sin Cos Cos Cos Cos Sin Cos Cos Sin Cos Sin Cos Cos Sin Cos Cos Sin Sin Sin e t F F K K C i i i RG i i i r r l m                                                       2 2 2 2 2 2 2 2 Sin 2 Cos Cos 0.5 Sin 0.5 Cos Sin 0.5 Sin Sin 0.5Sin2 (0.5Cos[2 ] Cos 0.5Cos[2 ]) ( 0.25Sin2 0.125Sin[2( )] 0.125Sin[2( )]) F F r r r r l m                                                             2 2 w2 ratio 2 2 2 2 2 4 Cos 2 Cos 2 Cos Sin 2Sin Sin 2Cos Cos Cos Sin Sin Sin e t F w F F K K C i r m r m RG rl m                                                 1 Sin Cos Sin 2 0 , 4 0 3 Cos Sin F F w F F G m g r l gm r G G G g l m            4.1 LQR Controller x Ax Bu   (3) where: 1 7 3 4 2 3 7 2 6 2 2 3 7 4 2 7 1 5 5 2 6 2 6 - 0 0 0 0 1 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 1 0 0 0 2 0 2 0 0 0 * 0 0 0 0 0 0 0 2 0 2 2 0 0 F w F F F F F F F w w A i i i l m M gl m rl m i i                                                          1 1 1 * * * 0 0 0 0 0 0 0 0 0 0 0 0 - , - 1 0 0 0 1 0 * * * 0 0 1 0 0 1 B u M M M                                                                                                                       x Ax Bu   (3) where: 1 7 3 4 2 3 7 2 6 2 2 3 7 4 2 7 1 5 5 2 6 2 6 - 0 0 0 0 1 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 1 0 0 0 2 0 2 0 0 0 * 0 0 0 0 0 0 0 2 0 2 2 0 0 F w F F F F F F F w w A i i i l m M gl m rl m i i                                                          1 1 1 * * * 0 0 0 0 0 0 0 0 0 0 0 0 - , - 1 0 0 0 1 0 * * * 0 0 1 0 0 1 B u M M M                                                                                                                       4.1 LQR Controller Taylor series expansion method is applied to linearize robot model and rearranged to state space form as shows in (Eq.3) ( q ) x Ax Bu   (3) where: 1 7 3 4 2 3 7 2 6 2 2 3 7 4 2 7 1 5 5 2 6 2 6 - 0 0 0 0 1 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 1 0 0 0 2 0 2 0 0 0 * 0 0 0 0 0 0 0 2 0 2 2 0 0 F w F F F F F F F w w A i i i l m M gl m rl m i i                                                          1 1 1 * * * 0 0 0 0 0 0 0 0 0 0 0 0 - , - 1 0 0 0 1 0 * * * 0 0 1 0 0 1 B u M M M                                                                                                                       where:       2 1 2 4 2 3 4 1 2 3 2 2 5 1 2 3 2 2 6 7 1 3 - - 2 - - - 2 2 - - F F w F F F F F F F F F F F w F F F F F F F F F F w F F g r l m rm rl m l m rl m l m i i i i i i i rl m l m r m rl m r m i i                       * M is the symmetrical positive definite matrix of the robot inertia and evaluated at the upright position. 4.1 LQR Controller The conventional LQR controller is the optimal controller that optimally determines the gains by compromising the state and control-input cost. The LQR cost function is expressed by   0 T T J x Qx u Ru dt     (4) (4) The Q and R matrices are the state and control weighting matrices. The control signal follows (Eq. 5) u Kx  (5) (5) 2 2 w2 ratio 2 2 2 2 2 4 Cos 2 Cos 2 Cos Sin 2Sin Sin 2Cos Cos Cos Sin Sin Sin e t F w F F K K C i r m r m RG rl m                                            The optimal gain matrix is solved by the algebraic Riccati equation (Eqs. 6-7). The optimal gain matrix is solved by the algebraic Riccati equation (Eqs. 6-7). 1 0 T T A P PA Q PBR B P      (6) 1 T K R B P   (7) (6) (7) ( ) (7)   1 Sin Cos Sin 2 0 , 4 0 3 Cos Sin F F w F F G m g r l gm r G G G g l m            (7) 2 0 , 4 0 G G   The metrics Q and R are the weighting metrics which define depend on significant state. The Q matrix is combined of many states of the unicycle robot. The balancing states (roll and pitch) of the unicycle robot are set to highest priority than other states. The unicycle robot equation of motion is in non-linear form. Taylor series expansion method is applied to linearize robot model and rearranged to state space form as shows in (Eq.3) The unicycle robot equation of motion is in non-linear form. 5.2 LQR-Fuzzy Controller (3) The LQR-Fuzzy applies both advantages from LQR and Fuzzy controller. LQR controller gain is optimal gain which designed by the cost function (4). It can easily satisfy the response of the system by set the priority of the Q matrix. And Fuzzy controller can handle the non-linear term of unicycle robot system. Therefore, the combination between LQR and Fuzzy controller can improve the performance of the robot. The block diagrams shows in Fig.4. Fig. 4 LQR-Fuzzy Block diagrams Fig. 4 LQR-Fuzzy Block diagrams where: where:       2 1 2 4 2 3 4 1 2 3 2 2 5 1 2 3 2 2 6 7 1 3 - - 2 - - - 2 2 - - F F w F F F F F F F F F F F w F F F F F F F F F F w F F g r l m rm rl m l m rl m l m i i i i i i i rl m l m r m rl m r m i i                       where:       2 1 2 4 2 3 4 1 2 3 2 2 5 1 2 3 2 2 6 7 1 3 - - 2 - - - 2 2 - - F F w F F F F F F F F F F F w F F F F F F F F F F w F F g r l m rm rl m l m rl m l m i i i i i i i rl m l m r m rl m r m i i                       Fig. 4 LQR-Fuzzy Block diagrams LQR-Fuzzy algorithm is designed for decreasing overshoot and increasing performance of the system. The input of Fuzzy control block in Fig.4 is position state error of the unicycle robot: roll yaw pitch and wheel position. LQR-Fuzzy algorithm is designed for decreasing overshoot and increasing performance of the system. References Fig. 7 Rule base of Fuzzy controller 1. Schoonwinkel, A. (1988). Design and test of a computer-stabilized unicycle. 4. Control Algorithm This section is presented the implemented control algorithm. By combination LQR with Fuzzy controller, the details presents below. 3 MATEC Web of Conferences 192, 02001 (2018) ICEAST 2018 https://doi.org/10.1051/matecconf/201819202001 Fig. 8 Simulation results of Roll Yaw Pitch and Wheel Position The input membership function is Gaussian type. There are two numbers of membership function of each input. For example, the membership function of the roll angle is combined with two Gaussian sets. The first set is normal period angle, and another one is aggressive angle as show in Fig.5. All member ship functions are set similar to roll angle. The input membership function is Gaussian type. There are two numbers of membership function of each input. p p For example, the membership function of the roll angle is combined with two Gaussian sets. The first set is normal period angle, and another one is aggressive angle as show in Fig.5. All member ship functions are set similar to roll angle. Fig. 5 Input of membership function Fig. 5 Input of membership function Fig. 5 Input of membership function The membership function of the output is combined with three Gaussian sets: low normal and aggressive. The defuzzification output is solved by centroid concept. p y p Fig. 6 Output of membership function Fig. 7 Rule base of Fuzzy controller Fig. 6 Output of membership function Fig. 8 Simulation results of Roll Yaw Pitch and Wheel Position From the simulation results, the LQR-Fuzzy is designed which meets requirements by decreasing the overshoot and increasing the performance of the unicycle robot. The LQR-Fuzzy can decease the overshoot about fifty percent compares with conventional LQR. From the simulation results, the LQR-Fuzzy is designed which meets requirements by decreasing the overshoot and increasing the performance of the unicycle robot. The LQR-Fuzzy can decease the overshoot about fifty percent compares with conventional LQR. Fig. 6 Output of membership function Fig. 6 Output of membership function 6. Conclusion In this paper, the unicycle robot model is simulated by LQR-Fuzzy controller. LQR controller is optimal control for multivariable feedback and known the good performance for linear model. Fuzzy controller ability is to deal with non-linearity of the system. The combination of both controller can increase the performance of robot. 5. Simulation results 2. Sheng, Z., & Yamafuji, K. (1995, May). Realization of a human riding a unicycle by a robot. In Robotics and Automation, 1995. Proceedings., 1995 IEEE International Conference on (Vol. 2, pp. 1319-1326). IEEE. In this section, the balancing performances of the unicycle robot are simulated and compared with convention LQR. The robot simulation is simulated by MATLAB/Simulink. In the simulation parts, there are initial conditions in each states of the robot. The results show that the robot can reject disturbances and try to convert each states to set point (zero). The comparison of LQR-Fuzzy and LQR are simulate, presents in Fig.8. 3. Majima, S., Kasai, T., & Kadohara, T. (2006, November). A design of a control method for changing yaw direction of an underacuatted unicycle robot. In TENCON 2006. 2006 IEEE Region 10 Conference (pp. 1-4). IEEE. 4. Chantarachit, S., & Parnichkun, M. (2016). Development and control of a unicycle robot with double flywheels. Mechatronics, 40, 28-40. 5. Yazdanpanah, R., Mahjoob, M. J., & Abbasi, E. (2013). Fuzzy LQR controller for heading control of an unmanned surface vessel. In International Conference in Electrical and Electronics Engineering (pp. 73-78). 4
https://openalex.org/W2138403851	https://storage.googleapis.com/jnl-up-j-jime-files/journals/1/articles/32/submission/proof/32-1-430-1-10-20100909.pdf	English	null	Scholarly Rhetoric in Digital Media (or: Now that we have the technology, what do we do with it?)	Journal of interactive media in education	2,000	cc-by	9,963	Submission to Jnl. Interactive Media in Education: PrePrint under review Submission to Jnl. Interactive Media in Education: PrePrint under review 1. Introduction It is clear that the impact of contemporary information and communications technologies (ICTs) on Higher Education is and will be almost unimaginably profound. However, there is one key area of impact that has, as yet, I believe received too little consideration. That is the impact of this technology on the ways in which we construct scholarly argument1. For most of the last 400 years scholarly argument has largely been published to the world via the medium of print. Scholarly argument is, of course, also published via other media, e.g. lectures, seminars and tutorials. However, since we largely valorise those arguments that are eventually published in print, I propose to restrict my remarks to a consideration of the impact of this technology on the construction of print(able) arguments. Bruce.Ingraham@tees.ac.uk Abstract: This article addresses the hypothesis that scholarly argument as it is presently pursued is mediated through print; but the advent of modern ICT offers alternative media to support scholarly publication. However, few academics have much expertise with these media. Accordingly, if this technology is to be fully exploited the academic community will need to acquire such expertise and this may have significant implications or the way in which scholarly argument is constructed. This hypothesis is addressed from a rhetorical perspective and consideration is given to what the impact of alternative publication media may be on the structure of scholarly argument. Keywords: Rhetoric, hypertext, multimedia, scholarly discourse 1.1 The Hypothesis The hypothesis that I want to offer up for consideration is that: Teaching Fellow, Centre for Lifelong Learning, University of Teesside, Middlesbrough, UK Teaching Fellow, Centre for Lifelong Learning, University of Teesside, Middlesbrough, UK Bruce.Ingraham@tees.ac.uk Scholarly Rhetoric in Digital Media (or: Now that we have the technology, what do we do with it?) Bruce Douglas Ingraham eaching Fellow, Centre for Lifelong Learning, University of Teesside, Middlesbrough, UK 2 Finally, since the challenge of multimedia rather than that of hypertext, lies at the heart of this article there should arguably be a fourth, multimedia, version; and perhaps one day there will be. However, for the moment, band-width limitations persuaded me to proceed with only three versions. The hypothesis that I want to offer up for consideration is that: Scholarly argument as it is presently pursued is mediated through print. The academic community has developed considerable expertise in presenting argument in this medium. However, the advent of modern ICT offers alternative modes of 1 Throughout this paper I have used the word ‘argument’ to denote a specific subset of some area of scholarship such as might be contained in a book or journal article. Similarly, I have used the word ‘discourse’ to denote the wider area of scholarship out of which a specific ‘argument’ emerges. This is in keeping with contemporary discourse analysis. This is in keeping with contemporary discourse theory. [A brief, but useful introduction to discourse theory can be found in the entry for discourse in A Dictionary of Cultural and Literary Theory (Payne, 1997).] 1 publication in which, as yet, few academics have much expertise. If we are to fully exploit this technology we will need to acquire this expertise and this may compel us to re-think the ways in which scholarly argument is constructed. publication in which, as yet, few academics have much expertise. If we are to fully exploit this technology we will need to acquire this expertise and this may compel us to re-think the ways in which scholarly argument is constructed. Underlying this hypothesis is, as will doubtless become apparent, a very real concern, of the post-modernist kind, about the legitimacy of current modes of scholarly argument. However, I do not really wish to enter into that debate here. Rather I want to address the problem from a somewhat more practical point of view by asking, first, what contemporary ICT has to offer to scholarly argument; and, then, by asking what, in practical terms, are the implications of that for scholarly argument as it presently pursued. 1.2 The Presentation of This Hypothesis Since the manner in which scholarly argument is presented is the principal concern of this article, it may be wise to reflect briefly on how and why it is presented in the ways that it is. Originally this ‘paper’ was conceived of as a conventional print publication. Its primary purpose was, and is, to place its argument on the agenda of contemporary scholarly authors. However, various pressures and opportunities have led to this article being re-cast in three2 forms: the initial print version; a simple hypertext suitable for internet access; and, I hope most interestingly, in the form of a Navihedron. The Print Version is organised in a conventional manner with footnotes, references, and bibliography; and is divided into a series of numbered sections [and sub-sections]. These are: 1. Introduction 2. Contemporary ICT 3. The Information Revolution 4. The Rhetoric of Scholarly Argument 5. Electronically Mediated Scholarly Argument 5.1. The Rhetoric of Electronic Text 5.2. The Rhetoric of Hypertext 5.3. The Rhetorics of Multimedia 6. Rhetorical Models for non-print(able) Argument 7. How do you write a multimedia essay? From a rhetorical perspective [see The Rhetoric of Scholarly Argument below] these 1. Introduction 2. Contemporary ICT 3. The Information Revolution 6. Rhetorical Models for non-print(able) Argument 7. How do you write a multimedia essay? From a rhetorical perspective [see The Rhetoric of Scholarly Argument below] these divisions serve two purposes. First, they serve to articulate the line of the argument. Second, they provide a further means of navigating one’s way around the article and the argument. 2 Finally, since the challenge of multimedia rather than that of hypertext, lies at the heart of this article there should arguably be a fourth, multimedia, version; and perhaps one day there will be. However, for the moment, band-width limitations persuaded me to proceed with only three versions. 2 That is, they serve as a table of contents or index might in a larger work to allow the reader to move more easily from one section of the argument to another. Although the ‘line of one’s argument’ is of great importance, it is equally important to remember that scholarly reading is rarely linear. Normally, academics at least begin by examining such things as subject heads and indices to get a sense of the overall argument [but see The Rhetoric of Hypertext below]. 1.2 The Presentation of This Hypothesis Similarly, the use of expressions like “see below” serve to offer alternative routes through the argument. Finally, the use of notes, references, bibliographies etc. serve to place this line of argument in the context of a wider discourse [again see The Rhetoric of Hypertext below]. The Hypertext Version of this article is conceived of essentially as an electronic publication of the printed article; and, as such, makes only limited use of the full potential of hypertext [see The Rhetoric of Hypertext below]. It differs from the printed version only in the following ways. The section headings appear as a hyper-linked table of contents to facilitate navigation. Although they are perhaps superfluous in a hypertext, I have retained the section numbering since this may be said to emphasize the ‘line of reasoning’ being pursued [see The Rhetoric of Scholarly Argument below]. Expressions such as ‘see above’ and ‘see below’ have been replaced with hyperlinks (indicated by blue text) to the relevant sections. Similarly notes and references are hyperlinked for ease of navigation. [Readers using browsers 4.0 or above will also find that merely pointing at the reference date will display the full reference, while clicking will take them to the bibliography.] There is, however, one significant change between the print version and the hypertext. The hypertext includes a section on post-modernism that is not included in the printed text. Arguably, this section could have been included in the print version as an appendix. Indeed, from a post-modern perspective such an appendix might sensibly have been viewed as ‘the tale that wags the dog’. However, such a strategy didn’t seem to be entirely appropriate. On the one hand, it would be a rather unusual appendix to an otherwise conventional argument; and, on the other, these reflections perhaps more appropriately sit beside rather than follow on from the central argument. The Navihedron version [http://www.navihedron.com/nav/bruce/now]is a much more radical departure from conventional scholarly presentation. Navihedrons™ are tools for composing hyper-information structures developed by Roy Stringer and his colleagues at Amaze Ltd. [http://www.amaze.co.uk]. More information, examples and the opportunity to experiment with Navihedra™ can be found at http://www.navihedron.com/. In brief, however, Navihedra™ are 3D models based on Platonic solids and relationships between pieces of information are articulated in terms of the spatial relationships represented by the vertices of the polyhedron. 1.2 The Presentation of This Hypothesis That is, units of information (of any kind, media, size or complexity) are attached to a specific vertex and bi-directionally hyperlinked to all the immediately adjacent vertices. The overall structure being determined by some perceived relevance reflected in proximity. Proximate vertices are understood to locate units of information/argument that are more closely related to one another than units of information that are not directly hyperlinked Furthermore this 3 dimensional arrangement can be rotated in space so that hyperlinked. Furthermore, this 3 dimensional arrangement can be rotated in space so that differing patterns of inter-relatedness can be viewed. Creating such an arrangement is much more difficult than it might appear and requires an author to consider the structure/presentation of even a simple argument like the one contained in this article with at g pp q structure/presentation of even a simple argument like the one contained in this article with at least as much care as a more conventional presentation. In the present Navihedron™ version the Navihedron™ appears at the left and takes on the navigation functions of the table of contents in the hypertext version and the section headings in the print version; but, significantly, presents the argument as a polyhedron of potential relationships rather than as a ‘line’. However, I do not wish to claim to have been wholly successful in re-presenting the sections of this argument in an optimum pattern. In 3 order to facilitate comparison, I made a conscious decision to keep the Navihedron™ version as close to the other versions as possible. To this end I intended to retain the same headings as in the other versions but without the numbering in order to encourage alternative views of the argument. However, current Navihedra™ technology limits the number of characters attached to each node and it was necessary to slightly revise the section labels. order to facilitate comparison, I made a conscious decision to keep the Navihedron™ version as close to the other versions as possible. To this end I intended to retain the same headings as in the other versions but without the numbering in order to encourage alternative views of the argument. However, current Navihedra™ technology limits the number of characters attached to each node and it was necessary to slightly revise the section labels. 1.2 The Presentation of This Hypothesis Similarly, as current Navihedra™ won’t rotate in response to internal hyperlinks, readers must use Navihedron to navigate to internal references like ‘See Hypertexts’. Similarly while pointing at reference dates displays the whole reference, readers must use the Navihedron to navigate to the bibliography. Note however that such references are no longer ‘above’ or ‘below’ since they may lie anywhere in the line of reasoning being pursued by the reader. However, apart from these two changes and some necessary revisions to make sub-sections less dependent on those immediately preceding or following, the text of the Navihedron™ version is identical to that of the simple hypertext version. In some respects this is probably a mistake since it tended to limit the reconsideration of the underlying complexity of the argument. Nonetheless, the Navihedron™ version of the argument is, I hope, at least suggestive of a genuinely alternative approach to structuring scholarly argument. data/information dissemination/communication. With respect to data capture it may perhaps be understood to begin with Daguerre in 18383 and includes: the development of photography, sound recording (1877), moving pictures (1888), videotape recording (1956), and, more recently, computer imaging (including scientific visualisation) and VR. With respect to communications, it includes: telegraphy(1837), telephony (1876), wireless telegraphy (1896), radio (1920), TV (1926), digital computing (1946), and, since the 1980’s, PCs, email, MPCs, LANs, WANs, MANs, Intranets and the Internet. These last are of particular importance because they provide the key to easy access to any information that can be recorded in a digital format; and thus easy access to human discourse that is not conducted in print. It is this easy access that provides the novelty in the current phase of the information revolution. At the time of writing, these key elements are only 5 to 10 years old. The first MPC (Multimedia PC) standard was articulated in the early 1990’s and, although the roots of the ‘Web’ go back nearly thirty years, easy access and wide-spread use of it only became available with the advent of Mosaic, Netscape etc. in the mid-1990’s. Perhaps still more crucially, the sort of high bandwidth networks needed to complete the link between the scholar’s desktop and the full range of available digital resources are only now beginning to become readily available.4 Nonetheless it is clear that within the immediately foreseeable future the technology needed to support easy, real-time access to any sort of digital information will be available to scholars5. 3 As a semiotician I am conscious that the inclusion of photography, or indeed, any still images in this argument is perhaps a little contentious. However, it seems reasonable to view the development of photography alongside telegraphy as the first in a series of technologies that lead to and are encompassed by what we now understand by the information revolution. 4 In the UK a number of separate metropolitan area networks serving various academic communities have been developed. These include ScotMan, NorMAN and others and it is reasonable to suppose that these will be upgraded and integrated via SuperJANET to form a very high bandwidth network in the service of British academia. 2. Contemporary Information and Communications Technology Developments in Information and Communication Technologies would seem to offer the scholar three sorts of things that are likely to have an impact on the way scholarly argument is conducted. These are: • alternative ways of presenting print-based arguments; • easy access to information in formats other than print; • ways of presenting information for scholarly consideration in formats other than print. Arguably the second of these is the most obviously pressing for scholarship. As scholars we have an obligation to consider any information pertinent to our particular area of study irrespective of the medium in which it is recorded. However, until comparatively recently unless information was recorded in a printable format, it was not readily available on a scholars desk. For example, the mechanisms for accessing information stored in time-based media (audio, video, film recordings etc.) were, until the advent of audio and video cassette technology very limited, and even that technology has only been widely available for about 30 years. Still further some kinds of information, e.g. the 3D and other computer models currently used in scientific visualisation, were effectively unrecordable until very recently. However, with the development of networked multimedia personal computing easy access to such information has become much more possible. I do not wish to appear to be naïve about this access. Access to a film on a PC or a video screen is not the same thing as viewing it in the cinema anymore than a print of a painting is the same thing as the painting itself, but in all these cases these alternative representations serve at worst as acceptable aide memoire within scholarly argument. They are, in short, very much better than nothing. At first glance this may not seem to be all that important until we remind ourselves that the information era/society/revolution, call it what you will, is not actually all that new. 4 The ‘information revolution’ is more than 160 years old and takes two forms: The ‘information revolution’ is more than 160 years old and takes two forms: data/information dissemination/communication. 5 A useful example of this can be seen in the progress made in a joint BUFVC/JISC project on Networking Moving Images for University Teaching and Research, the interim results of which were published at a workshop held at Robinson College Cambridge, 17-18 December 1998. “In January 1997, following a further meeting at the British Film Institute with members of JISC’s Committee on Electronic Information, funding was agreed to support a joint pilot project which began work in January 1998. Since then the project has been working to deliver moving picture content to two pilot sites: the South Wales Metropolitan Area Network led by the University of Glamorgan and the University of Glasgow with the Performing Arts Data Service. More than thirty hours of moving pictures, relevant to study in Medicine, Social History and Film Studies, have been selected predominantly from the British Film Institute’s National Film and Television Archive. These items have been encoded at the University of Manchester and then delivered to the pilot sites ready for integration with associated metadata. The sites have now developed their own file access ystems and these are now ready for demonstrations online and for use in teaching trials.” [ http://www.bufvc.ac.uk/networkconf.htm ]. 5 With this access comes the opportunity to include within scholarly discourse both information in non-print(able) formats and non-print(able) discourse. However, this opportunity also problematises scholarly discourse, because scholarly argument is, I submit, fundamentally rooted in print. In saying that scholarly argument is fundamentally rooted in print I simply mean that academics know how to prepare scholarly arguments for print. We have been conducting scholarly discourse through print for more than 400 years. We are trained at each stage of our studies, undergraduate and post-graduate in increasingly refined methods of using the medium and, I suggest, this effects how we do it. We may not wish to go to far down the route indicated by Marshal McLuhan’s “the medium is the message”[1968], but there is clearly good reason to suppose that to some degree, at least, we think in ways that are suitable for printing. However, the significant issue for this paper is that whatever the impact of academics’ print- oriented skills on their argument, few academics possess such skills with respect to ‘electronic’ media. There is, of course, a distinguished history of educationalists who have exploited the full range of media -- radio, film, television, computing, etc. data/information dissemination/communication. Of these perhaps the UK’s Open University has been the most successful and harbours the greatest concentration of expertise; but the OU is only 30 years old and its experts represent only a tiny fraction of the total academic population. Similarly, there is that body of academics whose field of study lies within one or another of these alternative media and who, it may be assumed, have some expertise in their use. Again, film, television and media studies are comparative newcomers to the academic scene and employ only a small number of academics. In short, while there is a growing body of academic expertise in the use of these media it is not, as yet, widespread and it needs to become more so if we are to exploit the opportunity offered by contemporary technology. Another way of looking at this is to ask the question ‘How do you write a multimedia essay?’ or, perhaps more importantly, ‘How do you mark one?’ What are the conditions that need to be met that would allow the scholarly community to valorise arguments that include non- print(able) data and discourse and/or are themselves conducted in non-printable discourse? We mark other student work in terms of the degree to which it approximates to the work of scholars. We teach strategies for analysing data, constructing logical arguments that are clearly located in the wider discourse of the discipline and of representing these in print (or spoken) discourse. However, at this early stage of multimedia scholarship there are as yet no well-developed scholarly models. There is, as yet, little that can pass for good practice, because, frankly, there has been too little practice. Nonetheless, there are perhaps some avenues that may be explored which may at least suggest places in which to look for appropriate models against which to measure the quality of electronically mediated scholarship (see below). 4. The Rhetoric of Scholarly Argument From a semiotic perspective the problem can be understood to be one of rhetoric. By rhetoric I mean “The art of using language so as to persuade or influence others....” The Oxford English Dictionary “The art of using language so as to persuade or influence others....” The Oxford English Dictionary In Rethinking University Teaching Diana Laurillard argued that “Teaching is essentially a rhetorical activity, seeking to persuade students to change the way they experience the world. It 6 has to create the environment that will enable students to learn the descriptions of the world devised by others.” [Laurillard, 1993, p. 28] I would wish to extend this to include all academic discourse or certainly all academic argument. The purpose of any academic argument is to persuade others to accept the legitimacy of some viewpoint. Developing a persuasive argument is, of course, partly a matter of logic (that is some accepted code of reasonableness6) and partly of evidence; but it is also partly a matter of presenting one’s reasonable analysis of appropriate evidence through established print or other representational conventions. A quick glance at the immediately preceding paragraph will give some indication of what I mean. In that paragraph I introduce the issue of rhetoric into the current argument by defining it and defending the definition by referring the reader to a highly respected reference source. By so doing I suggest to the reader that the definition is uncontentious (at least for present purposes) and widely accepted throughout the academic community; and, as such, a reasonable basis upon which to proceed. Most importantly for the present argument, however, that rhetorical move, that step in the argument, was done not by reason, but through the accepted conventions (in English) of placing a string of text in quotations marks and following it with another string in italic. That combination serves the purpose of notifying the reader without further explanation that the quoted words are not mine and that the source of the words is The Oxford English Dictionary. The fact that the source is widely regarded as reputable then completes the rhetorical move of persuading the reader that they don’t need to worry about proceeding with an argument that rests on a proposition that has been valorised by the wider academic community. A similar move is made in the next sentence of that paragraph with the quotation from Diana Laurillard that extends the definition of rhetoric and applies it directly to the present context. Again simple print conventions are used to signal the idea that other reputable scholars share the views being expressed. In short, what is done is to place the current argument within a wider discourse. “The art of using language so as to persuade or influence others....” The Oxford English Dictionary We all know that elements of that wider discourse may be contentious and to some the rhetorical force of the reference may be less than convincing. Indeed, in this case, the careful reader may wonder what sins of omission are signed by the three ellipses (...) that indicate that something has been left out of the original definition.7 However, it is not my objective to deconstruct my own argument. It is merely to reflect upon how print conventions allow us to make rhetorical moves without at every turn needing to defend in detail every element of our argument. Most scholarly argument is supported by a cloud of referentiality that embeds one argument in the wider discourse of the discipline(s) from which it emerges and upon which it impinges; and this cloud is often signaled by simple print-based conventions. It is this sort of thing that is meant by suggesting that scholars know how to construct an argument in print. 7 In fact the words omitted were“... the body of rules to be observed by a speaker or writer in order that he may express himself with eloquence.” However, I leave the reader to muse over why I felt the rhetorical force of those words might not contribute advantageously to my overall argument. 6 According to the OED logic is “The branch of philosophy that treats of the forms of thinking in general, and more especially of inference and the scientific method.” and also “...the art of reasoning....” 5.1 The rhetoric of electronic text I do not propose to say much about this issue here, because the other issues are still more problematic. However, it is important to recognize that printed text is not the same as electronic text. Although very similar to print, it is another of the ‘new media’ subsumed under the heading multimedia and scholars will need to become adept at understanding and exploiting the differences between electronic and printed text. Among the most obvious of these are things like font choice and page layout. For example, there has been considerable discussion about how much text should be displayed on a computer screen at any one time; and the tendency for electronic texts to be laid out in ‘landscape’ can be a significant problem for scholars wishing to create electronic access to archives of print documents that are, as general rule, laid out in ‘portrait’. More generally, the problem is simply that electronic text has only been around for about 40 years and only really controllable for 15 or 20, while print has been with us for 500 years. There simply hasn’t yet been time enough to develop the same level of expertise or accepted conventions for electronic text. As in many electronic texts, the electronic versions of this article use colour to indicate various things; but, as yet, these do not constitute a generally accepted strategy like the use of quotation marks, italic or the Harvard referencing system. our activities8; and , I submit, electronically mediated scholarly argument is problematised by three considerations: ur activities8; and , I submit, electronically mediated scholarly argument is problematised b hree considerations: our activities8; and , I submit, electronically mediated scholarly argument is problematised by three considerations: • the rhetoric of electronic text • the rhetoric of electronic text • the rhetoric of hypertext • the rhetoric(s) of multimedia 5. Electronically Mediated Scholarly Argument However, when it comes to the preparation of arguments for electronic publication our lack of comparable levels of experience and shared conventions problematises or should problematise 7 In fact the words omitted were“... the body of rules to be observed by a speaker or writer in order that he may express himself with eloquence.” However, I leave the reader to muse over why I felt the rhetorical force of those words might not contribute advantageously to my overall argument. 7 7 5.2 The rhetoric of hypertext Similarly, a variety of menus, maps, and labeling systems has arisen to preserve one’s orientation within a particular hyper-information environment. Significantly for the present argument, these emerging navigational strategies and tools are analogous to the use of italics, brackets, quotations marks and so forth referred to above. They are ‘extra’, or perhaps ‘supra’, textual rhetorical markers that can serve to advance an argument or at least place it within the wider discourse. Within the preceding description of hypertext it is possible to see images of two different aspects of conventional scholarly activity. The well-structured hypertext can easily be a conventional scholarly article whose reading is enhanced by easy access to the sources referred to. It is analogous to the sort of situation that arises when scholars check references and follow- up leads that are suggested by the reading of a particular article -- just potentially quicker and easier. The less well structured hypertext, depending on its size, is perhaps more like a database or library of information available for scholarly exploration -- again facilitated by search algorithms or other electronic enhancements. For the moment the scale of such electronic resources means that they are perhaps more like that subset of information that one frequently finds on the scholar’s desktop -- a group of texts, articles, books, manuscripts, what have you, through which the scholar moves, checking references and reflecting upon the relationships between the information under study. However, advances in networking technology mean that the scale of material available will rapidly exceed even that of our best libraries. In any event, from both these perspectives, hypertext can simply be viewed as an electronically enhanced version of normal scholarly practice. It simply provides a means of locating the reader within that cloud of referentiality that represents the ongoing discourse of any discipline.9 However, in so doing hypertext also tends to make more visible the ‘rhetorical’ character of the moves made to provide the reader with a particular perspective on the discourse. 5.2 The rhetoric of hypertext Hypertext, however, is clearly a radical departure from printed text. The ability to move seamlessly from one point in a document to another or to a point in another document clearly has enormous potential for scholarly study. At the very least it can provide ‘fancy footnoting’ by allowing direct access to the complete bibliographical citation and thus obviating the need for the reader to turn to the end of the article or the bottom of the page. Still more usefully, of course, hypertext can, in principle, not only provide the citation but also direct access to the document cited; and, if that document were itself a similarly annotated hypertext, to other documents included within a chain or network of related information. Such a body of documents can be more or less structured, but freedom of movement within such a corpus is clearly one of the virtues of hypertext. Hypertext can be understood as “…a system of accessing textual data in which the data is understood to be stored in no particular sequence. The data must, of course, be stored in an orderly manner, but this order is not intended to influence the order in which it is accessed. Ultimately, of course, the data is accessed sequentially, but the sequence is determined by the 8 Locke Carter ( http://english.ttu.edu/carter/), who describes himself as a “techno-rhetorician”, is another writer who has addressed some of these issues from perspective of rhetoric. His Ph.D. dissertation ‘Arguments in Hypertext’ (1997) and another related study (Rickly 1995) are available from his website or at http://labyrinth.daedalus.com/dissertations/. 8 end user rather than by the original author. As such, hypertexts are usually understood to be very open data structures which the user is 'free' to explore at will.” [Ingraham et al, 1994, pp107-8] end user rather than by the original author. As such, hypertexts are usually understood to be very open data structures which the user is 'free' to explore at will.” [Ingraham et al, 1994, pp107-8] Of course, it was recognized early that this ‘freedom’ was itself problematic [e.g. Emery, 1993, p. 73] and a whole series of navigational strategies has evolved to overcome the problem of becoming ‘lost in hyperspace’. These include variations in font colour, size or face to signal possible hypertextual links or various search algorithms to provide still more open linking. 5.2 The rhetoric of hypertext Furthermore, to the degree that hypertext (by, for example, incorporating full-text citations) facilitates the reader’s independent exploration of the discourse, it also tends to make more visible the many inter-dependent strands and possibilities of which the discourse is composed and the particular argument is thus revealed as precisely that, a particular argument -- one of a number of possible, competing and not necessarily mutually exclusive perspectives upon the problem in hand. As such, hypertext may tend to reveal levels of complexity in the discourse that traditional modes of argument may tend or are specifically designed to obscure; and, 9 Indeed, David Kolb in a paper presented at Hypertext 97 [Kolb, 1997] goes still further and argues that hypertext allows us to more accurately represent that ongoing debate between scholars that typifies all scholarly activity by allowing the reader to move smoothly and easily between the various interlocutors. Interestingly, although Kolb was unaware of it the time, the Journal of Interactive Multimedia in Education (JIME -- http://www-jime.open.ac.uk ) had already begun to publish in a way that clearly instantiated the sort of discourse for which Kolb was implicitly calling. The issue of collaborative discourse has attracted still further discussion. A good introduction to the literature on this subject is available from Simon Shum’s website at the Open University’s Knowledge Media Institute ( http://kmi.open.ac.uk/sbs/ ). 9 perhaps invites us to consider whether there may be alternative approaches to the structure of scholarly argument that may better reveal the full complexity of the discourse.10 perhaps invites us to consider whether there may be alternative approaches to the structure of scholarly argument that may better reveal the full complexity of the discourse.10 In Socrates in the Labyrinth (1995) David Kolb tackled this issue by setting out to create a hypertext consideration of the potential role of hypertext in philosophical discourse. Socrates in the Labyrinth was created using a tool called StorySpace11 which provides a range of tools for ‘mapping’, displaying and navigating one’s way through the complex of inter-relationships that makes up Kolb’s arguments. Regrettably the tools available in Storyspace all tend to represent the argument as a two dimensional network and this tends to make the structure of the argument needlessly obscure.12 Be that as it may, in Socrates in the Labyrinth and more recently in Hypertext as Subversive? 5.2 The rhetoric of hypertext Kolb (2000) has nonetheless demonstrated that it is certainly possible to approach the development of a genuinely scholarly argument in a radically different way. Furthermore, with the advent of inexpensive software to support 3D modeling and low-end VR, tools are emerging, the Navihedron™’ used in one version of this article, for example, that make modeling hypertexts in three (or more?) dimensions much easier. However, whether or not one chooses to explore the question of whether hypertext offers a genuinely alternative structure for scholarly argument, it is clear that the electronic presentation of textual material (hypertext or otherwise) does impact on the print-oriented traditions that largely dominate scholarly discourse. On the one hand, we will need to reflect on issues of text design (font, layout etc.) for electronic media. On the other, hypertext may both ease (movement via indices, tables of contents, citations and notes becoming transparently easy) and disrupt the flow of the argument. Minimally, the route followed by the argument needs to be clearly sign- posted in order to avoid the risk of becoming lost in a text, especially as the capacity for full text citation increases. Of course, one may wish to argue that the appropriate response to this challenge to traditional academic discourse is to disregard it. Our current systems are clearly very effective and, as such, we might be ill advised to change them. Perhaps the scholarly community will only exploit the electronic dissemination of texts in the most minimal ways, but the potential advantages of easy access to information would seem to make that unlikely.13 10 In Hypertext 2.0, George Landow (1997) explores these and related issues at length. 11 Storyspace is available from Eastgate Systems (http://www.eastgate.com/). See also Bolter, J.D. et al, 1996. 12 Recent comments about Storyspace in Computers and Texts (Grigar & Corwin, 1998) are more positive, which perhaps suggests that my concerns with Socrates in the Labyrinth have other origins. 13 Another perspective from which to view this is that suggested by Maddux et al (1997) who argue that computer use in education can be divided into two types of applications. Type I are those which simply make what we already do easier, while Type II allow us to do new things. Hypertext can be seen as an example of either a Type I or Type II application, but I suspect that its convenience as a Type I application will gradually lead to its wider uptake as Type II. (See also Harlow and LaMont Johnson, 1998). 5.3 The rhetoric(s) of multimedia Although each of these disciplines would be likely to reflect differently on evidence drawn from a news broadcast, there is no doubt that such broadcasts provide evidence that is as valid as many print-based sources, newspapers for example. However, that evidence must, in part, be understood through the rhetoric of the medium if an appropriately critical perspective is to be maintained by scholars using such evidence. For example, understanding the position of a story in the running order can contribute significantly to our understanding of how the story’s importance was perceived at the time. In short, just as 400 years of scholars have evolved critical strategies for dealing with print-based evidence, we must evolve similar strategies for non-printable sources. Nor is it realistic to suppose that scholarly discourse can simply ignore such evidence. In some cases such evidence may be crucial to the resolution of a particular line of inquiry. For example, it would seem unthinkable that anyone should seek to write a history the Gulf war of the early 90s without reference to the archives of CNN or to the to role played by CNN in shaping opinion concerning that conflict. Certainly if the latter issue is of significance to scholarly inquiry, then access to the archival sources would be essential. Still further one would require some strategy for analysing and interpreting those sources; and any such strategy could only evolve against a background of both historical and media studies. Still more problematically the rhetoric of each medium in any mix of multimedia will also interact with and be modified by the rhetorics of the others. As such, multimedia will not only need to respect the rhetorics of the media it exploits -- whether as evidence or as the primary channel of the argument (see below) -- it will also have to evolve its own particular rhetoric(s) out of the various mixes of media that are exploited. In the simplest terms, this means asking questions about how the various media are deployed. Is video or graphics shown in a window? Is that window fixed in size and/or place? Does video sometimes/always take over the full screen? Is text displayed above, below, beside, on top of video or graphics? How are ‘hyper- moves’ signaled in text, graphics, video or to interactors who may have some sensory impairment, etc., etc, etc.? 5.3 The rhetoric(s) of multimedia In any case the challenges thrown up by the advent of electronic text are still further problematised by the advent of multimedia. As mentioned above the challenge of multimedia is twofold. First it provides access to data that exists in formats that are not print(able) and second it makes possible scholarly discourse that is not print(able). That is, it both makes it possible to include within an electronic text material, like film or scientific models, that it would otherwise be impossible to include; and, at least in principle, it makes possible the creation of scholarly discourse in some medium other than print. 13 Another perspective from which to view this is that suggested by Maddux et al (1997) who argue that computer use in education can be divided into two types of applications. Type I are those which simply make what we already do easier, while Type II allow us to do new things. Hypertext can be seen as an example of either a Type I or Type II application, but I suspect that its convenience as a Type I application will gradually lead to its wider uptake as Type II. (See also Harlow and LaMont Johnson, 1998). 10 I will return to this second challenge shortly, but with respect to the first, it is important to remember as contemporary ICT gives access to information contained in the full range of available media, that each of them, like print, has its own rhetoric. Media are not transparent conveyors of information. Each of them has developed or, as in the case of VR14 is developing their own particular ways of creating meaning and of persuading those who interact with them. These ‘rhetorics’ are usually well understood by practitioners within these disciplines and in some cases, e.g. film and television studies, also by scholars who have directed their study towards them. However, these ‘rhetorics’ are not necessarily well understood by other scholars who may wish to use the data represented in such media in the course of constructing arguments in some other discipline.15 An obvious case in point would be the use of television news in the context of some historical, political, or sociological discourse. 14 A useful introduction to thinking in VR circles about these issues can be found in a paper presented by Clive Fencott at the 1999 Virtual Systems and Multimedia Conference in Dundee [ http://www-scm.tees.ac.uk/users/p.c.fencott/vsmm99 ]. 5.3 The rhetoric(s) of multimedia HCI literature deals at length with these and other related issues, but rarely if at all has it addressed these questions from the point of view of how such questions impact on the structure of scholarly argument. 14 A useful introduction to thinking in VR circles about these issues can be found in a paper presented by Clive Fencott at the 1999 Virtual Systems and Multimedia Conference in Dundee [ http://www-scm.tees.ac.uk/users/p.c.fencott/vsmm99 ]. 15 Pursuing a rather different line of argument, Collins et al argue that in the preparation of multimedia learning resources one needs to select the most appropriate media for any task and make a reasonably detailed analysis of the strengths and weakness, what they call “affordances and constraints” of a range of ‘new media’. [ http://www.apc.src.ncu.edu.tw/apc/allanmedia.htm ] 11 In those terms access to non-printable data first challenges us to understand that data on its own terms and to adopt an appropriately critical stance with respect to its rhetorical force. However, as indicated above, it also invites us to consider not only including such data in our arguments, but also adopting one or more of them as our primary channel of communication. It invites us to consider the creation of scholarly arguments within non-printable discourse. This invitation presents itself in two ways. The first of these is simply because we can. Print became the medium of choice for scholars because it was the most efficient means of disseminating their work; and, in an electronic incarnation, it may, via the internet, remain so. However, other media, notably television, and perhaps especially television in an interactive digital incarnation, clearly is in competition. The other media and mixes of media that are now available to us may offer more efficient and effective channels for scholarly communication and just as it behooves scholars to consider any data available to us, it behooves us to explore these alternatives. Second, there is a question to be addressed about whether text is the best medium in which to consider all other, and perhaps especially time-based, media. Clearly, we can and do discuss other media through the medium of text, but should we? It may be possible to represent these in text. 16 At the moment, much of what passes for electronically mediated scholarly argument is of this type. Even in the more ambitious electronic journals such as JiME, The Journal of Interactive Media in Education [http://www-jime.open.ac.uk] or JoDI, The Journal of Digital Information [http://jodi.ecs.soton.ac.uk/] most of the articles are simple hypertexts or illustrated hypertexts. One very interesting exception to this is Hardy & 5.3 The rhetoric(s) of multimedia We can include a still image drawn from a film or computer model or the script of a broadcast, but scripts and stills are not the same thing as the time-based media they represent and certainly lack the ‘rhetorical force’ of the original. Even in the case of scientific modeling a still image of a molecular model may well be ‘less convincing’, lack the rhetorical force, of an on-line model with which an investigator can interact. The inclusion of fully functioning time-based media within an electronic text is certainly likely to enhance such discourse, but it also invites the question of why use printed text as the primary channel. After all, we give lectures and these are frequently enhanced with ‘audio-visual aides’ (multimedia). Such performances are recordable and therefore a television presentation of the material without necessarily any loss of scholarly rigour is at least imaginable. I don’t mean to suggest that we can or should simply record lectures. Doing so largely fails to understand the rhetoric of both media (TV & lectures). I am merely pointing out that there are alternative models within contemporary scholarly practice in which printed text is not the primary channel of communication. Significantly, there are televisual models for conducting discourse about non-printable data objects -- e.g. film review programmes for film studies -- that may provide scholars with useful models (see below). 6. Rhetorical Models for non-printable discourse It is these alternative models of discourse drawn from non-printable media that returns us to the question of “How to write a multimedia essay”. Clearly one way is simply to interpolate multimedia data within an electronic text. This generates what might be described as argument by quotation or illustration and is much the same as including a photograph, graph or other still illustration in a printed text. That is, subject to treating that data and its rhetoric with appropriate rigour, one could then proceed with what would in essence remain a conventional print-based argument. In short, such scholarly arguments simply proceed as traditional texts with some enhancements from other media or advances in text manipulation.16 16 At the moment, much of what passes for electronically mediated scholarly argument is of this type. Even in the more ambitious electronic journals such as JiME, The Journal of Interactive Media in Education [http://www-jime.open.ac.uk] or JoDI, The Journal of Digital Information [http://jodi.ecs.soton.ac.uk/] most of the articles are simple hypertexts or illustrated hypertexts. One very interesting exception to this is Hardy & 12 However, there are models other than traditional scholarly discourse -- lectures, articles, monographs -- to which we might turn to better exploit the potential of contemporary ICT in pursuit of the best medium in and through which to conduct scholarly discourse. We could, for example, look at the ways in which meaning is created in visual media. At the radical end of the spectrum, this could offer what might be described as ‘argument by montage’17. In such argument spatial and/or temporal juxtaposition between any images/texts/sounds drawn from any media might indicate reasonable relationships (associations) that would be difficult or even impossible to articulate in text (even illustrated text) alone. Montage can through the juxtaposition of images both simultaneously and sequentially engender meaning that can not be easily paraphrased. In Rethinking University Teaching Diana Laurillard of the Open University argues precisely such a case for the value of educational television. “In educational broadcasting, given my position that academic knowledge is essentially rhetoric anyway, the medium can legitimately fulfill its potential.... 6. Rhetorical Models for non-printable discourse It hardly matters if a student fails to remember some constituent item within a sequence or programme; if the medium is being used as I have argued it should be, to persuade the viewer of a line of argument, or a way of seeing the world, then the important question is whether they understood the point being made.” [Laurillard, 1993, p115] Later in the book she extends this to include hypertext/multimedia and does so in a way which suggests that she similarly views the rhetorical force of multimedia arguments as, at least in part, emerging from the montage of images. “The combination of a hypertext system with audio-visual media to give ‘multimedia’ brings together the best of both. It is easier to create image-argument synergy in a system that can handle both text and visuals, and since that feature of video referred explicitly to an associative link between image and argument -- the identification of one with the other -- it follows that this is precisely where the associative links offered by hypertext can be valuable.” [Laurillard, 1993, p223] Although filmic montage can and does include the simultaneous juxtaposition of images (e.g. sound and vision), it is essentially time-based and to that degree exercises some control over how the ‘argument’ unfolds. However, 3D objects like Roy Stringer’s ‘Navihedra™’ [see above] might provide a more architectural, and perhaps still more radical, means of displaying the relationships between the constituent elements of an argument. In the end, of course, an argument will still unfold in time, but such a ‘solid’ overview of the subject can perhaps support a more open exploration of the possibilities contained within the discourse; and such exploration may result in a more perspicacious view of the subject. However, as elsewhere in this paper, recognizing the force of the alternative rhetorics available within the new media doesn’t necessarily force us to adopt a radical de[re]construction of conventional academic practice. As already indicated, there are other more conventional models to which we may turn that may provide alternative routes to the ‘writing’ of multimedia essays. Portelli’s (1999) "aural essay" entitled “I Can Almost See the Lights of Home ~ A Field Trip to Harlan County, Kentucky” in the Journal of Multimedia History [http://www.albany.edu/jmmh/] in which sound is the primary medium of communication. 17 The writings of the Russian film director Sergei Eisenstein (1994 ) provide the locus classicus for the study of montage. Portelli’s (1999) "aural essay" entitled “I Can Almost See the Lights of Home ~ A Field Trip to Harlan County, Kentucky” in the Journal of Multimedia History [http://www.albany.edu/jmmh/] in which sound is the primary medium of communication. 6. Rhetorical Models for non-printable discourse 17 The writings of the Russian film director Sergei Eisenstein (1994 ) provide the locus classicus for the study of montage. 13 To develop but one example18, documentary television could provide a reasonably straight- forward alternative to the creation of articles and monographs that could fully exploit the rhetoric of that medium without abandoning the rigours of traditional academic discourse. Certainly there are examples of academically sound documentaries and yet few would ordinarily be regarded as such by the academic community. Few, if any, would, for example, count towards the RAE19. Why? In the case of some of the more popular -- ‘prime time’ -- documentaries, the BBC’s Horizon or much of what is best on the Discovery channel, for example, the level of argument is perhaps too superficial. Even though the underlying scholarship may be sound, too much is compressed in too small a space. However, that is by no means always the case. Recently Twin Cities Public Television produced a documentary history of the American War of Independence entitled Liberty which certainly had the hallmarks of sound scholarship, offered a novel perspective on its subject and proceeded at a level of detail seemingly commensurate with scholarly rigour. A specialist American historian might disagree with that assertion, but, at the very minimum, this series conducts its discourse at a level at least appropriate to under-graduates. From this we may at the very least conclude that the level of discourse in the medium of broadcast television doesn’t necessarily have to be superficial. However, even such an admirable series does have a serious drawback from the perspective of scholarly argument. Broadcast television isn’t good at locating the argument being presented clearly within the wider discourse. In short, there are no footnotes. However sound the argument, the one thing contemporary television documentaries never are is ‘well-documented’.20 To be sure Professor X or Y will be called upon to comment. Their presence lends rhetorical force to the argument in much the same way as a footnote, but one would normally be hard pressed to follow up such reference, access the relevant materials, or otherwise behave in a conventionally critical manner towards the argument. This, however, is a problem intrinsic to the current state of non-interactive broadcast television21. It need not be an issue for a multimedia documentary. 6. Rhetorical Models for non-printable discourse It is clearly possible to embed a conventional television documentary within a multimedia presentation that provides easy access to the full panoply of scholarly reference. In short, to use contemporary ICT to clearly situate a genuinely televisual presentation of a scholarly argument within that cloud of referentiality that constitutes the wider discourse and in so doing place fellow academics in a position to judge the value of that argument. However, in doing so they must learn to judge not only the force of the argument, but also the rhetorical force given to it by the medium or mix of media they encounter. Before leaving this section, it would be naive not to call attention to the cost implications of the above argument. Whatever the scholarly or pedagogical importance of exploiting the new technologies, the cost of doing so will be enormous, possibly even prohibitive. However, the 18 Other examples might include talk shows or interviews, computer games or other ‘virtual’ worlds of discourse. 19 The RAE (Research Assessment Exercise) is one of the funding mechanisms for UK Higher Education. Through the RAE HEI’s receive additional funding according to the quantity and quality of their research activities. 19 The RAE (Research Assessment Exercise) is one of the funding mechanisms for UK Higher Education. Through the RAE HEI’s receive additional funding according to the quantity and quality of their research activities. 20 Indeed, in the case of Liberty even the date of production wasn’t documented. 20 Indeed, in the case of Liberty even the date of production wasn’t documented. 21 It is interesting to note the UK’s Open University has largely abandoned broadcasting lecture material in favour of distributing a combination of video tapes and text that permits a greater degree of interaction. 21 It is interesting to note the UK’s Open University has largely abandoned broadcasting lecture material in favour of distributing a combination of video tapes and text that permits a greater degree of interaction. 14 question of whether we can afford to allow the new technologies to impinge on scholarly argument is really separate from what their impact can or should be; and, as such, lies outside the scope of this paper. 7. How, then, do you write a multimedia essay? As I said at the outset, there are no easy answers. Approaching the question from the perspective of semiotics suggests that the issue is at least in part a matter of rhetoric. As such, we must, first, recognize that none of the media that we study is a transparent vehicle for its content. Each medium constructs its content in its own way and we must take that into consideration if we are to understand the evidence that we find therein. Second, we must recognize that mixing evidence from various media together will impact on the rhetorics of each and of our argument as a whole. Finally, we must ask what is the most suitable medium or mix of media in and through which to articulate our arguments. What are the "affordances and constraints" [Collins et al] of each varying medium or combination of media. In short, we must examine, modify and develop the models of discourse that are available to us with a view to devising the most convincing strategies for pursuing our arguments. Finally, the question should perhaps be asked whether this is really important. The answer must be ‘yes’. As scholars we can not ignore data or discourse that doesn’t ‘fit to print’. Once that is accepted I do not think we can, in good conscience, ignore the challenge to ask whether or not print is actually the best or only medium in which to conduct scholarly argument; and, still further, whether the reasoning strategies that have evolved in our print oriented scholarship are really as sound as they might be. However, as Diana Laurillard points out the academic community is intrinsically, and probably rightly, intellectually conservative; but “The implementation of new technology methods cannot take place without the system around it adjusting to the intrusion of this new organism.... If academe is to preserve what is good in its traditions and also preserve its mission to develop knowledge and educate others, then the higher education system needs a more robustly adaptive mechanism than it has had to develop hitherto.” [Laurillard, 1993, p223] Furthermore, if academia doesn’t take up the challenge of this technology in the interest of education, someone else might. Not all the pressures motivating the desire to widen access to education and promote lifelong learning have their roots in an unselfish desire to improve the lot of humankind. 8. References Bolter, J.D. et al (1996) Getting Started with Storyspace for Windows, Eastgate Systems, Watertown, Ma., http://www.eastgate.com. Bolter, J.D. et al (1996) Getting Started with Storyspace for Windows, Eastgate Systems, Watertown, Ma., http://www.eastgate.com. BUFVC/JISC, Networking Moving Images for University Teaching and Research, Robinson College Cambridge, 17-18 December 1998. http://www.bufvc.ac.uk BUFVC/JISC, Networking Moving Images for University Teaching and Research, Robinson College Cambridge, 17-18 December 1998. http://www.bufvc.ac.uk Carter, L. (1997), Arguments in Hypertext, Ph.D. dissertation University of Texas. Available at http://labyrinth.daedalus.com/dissertations/. Carter, L. (1997), Arguments in Hypertext, Ph.D. dissertation University of Texas. Available at http://labyrinth.daedalus.com/dissertations/. Carter, L. (1997), Arguments in Hypertext, Ph.D. dissertation University of Texas. Available at http://labyrinth.daedalus.com/dissertations/. Collins, A., Neville, P. and Bielaczyc, K., “The Role of Different Media in Designing Learning Environments”, http://www.apc.src.ncu.edu.tw/apc/allanmedia.htm. Collins, A., Neville, P. and Bielaczyc, K., “The Role of Different Media in Designing Learning Environments”, http://www.apc.src.ncu.edu.tw/apc/allanmedia.htm. Collins, A., Neville, P. and Bielaczyc, K., “The Role of Different Media in Designing Learning Environments”, http://www.apc.src.ncu.edu.tw/apc/allanmedia.htm. Eisenstein, S.M. (1994), Selected Works, Volume II, Towards a Theory of Montage (Glenny, M. & Taylor, R., eds) British Film Institute, London. Eisenstein, S.M. (1994), Selected Works, Volume II, Towards a Theory of Montage (Glenny, M. & Taylor, R., eds) British Film Institute, London. 15 Emery, C. (1993), “Integrating New Technology: From Multimedia to Hypermedia”, in Coleman & Rouxville, Integrating New Approaches, CiLT, London. Fencott, C. (1999), Content and Creativity in Virtual Environment Design http://www- scm.tees.ac.uk/users/p.c.fencott/vsmm99 Grigar, D. & Corwin, M. (1998), “The Loom and the Weaver”, Computers & Texts, Winter, 1998, No. 16-17. Hardy, C. & Portelli, A (1999) “I Can Almost See the Lights of Home ~ A Field Trip to Harlan County, Kentucky”, Journal of Multimedia History, Vol. 2, http://www.albany.edu/jmmh/. Harlow, S.D. & LaMont Johnson, D. (1998), “An Epistemology of Technology”, Educational Technology Review, Spring/Summer 1998, No. 9. Ingraham.B, Chanier, T., Emery, C. (1994), “Language Training for Various Purposes in Several Languages on a Common Hypermedia Framework”, Computers and Education, Vol. 23, No. 1/2, pp 107-115. Kolb, D. (1995), Socrates in the Labyrinth, Eastgate Systems, Watertown, Ma., USA. Storyspace Storyspace Kolb, D. (1997) “Scholarly Hypertext: Self-Represented Complexity”, Hypertext 97, ACM, New York. Kolb, D. (2000), “Hypertext as Subversive?”, Culture Machine, 2, http://culturemachine.tees.ac.uk Landow, G. (1997), Hypertext 2.0, The Johns Hopkins University Press, London. Laurillard, D. (1993), Rethinking University Teaching, Routledge, London. Maddux, C.D., Johnson, D.L. & Willis, J.W. (1997), Educational computing: Learning with tomorrow’s technologies, Allyn & Bacon, London. McLuhan, M. (1968), Understanding Media, Sphere Books Ltd., London. Payne, M. (1997), A Dictionary of Cultural and Literary Theory, Basil Blackwell, Oxford. Rickly, R. (1995), Exploring the Dimensions of Discourse, Ph.D. Dissertation, Ball State University. Available at http://labyrinth.daedalus.com/dissertations/. Shum, S., Computer Supported Collaborative Work, http://kmi.open.ac.uk/sbs/. Stringer, R., http://www.amaze.co.uk and http://www.navihedron.com/. Twin Cities Public Television, Liberty, The American War of Independence 16
https://openalex.org/W3200149922	https://www.cadernos.prodisa.fiocruz.br/index.php/cadernos/article/download/822/847	Portuguese	null	O papel do Poder Local no combate à pandemia de COVID-19	Cadernos Ibero-Americanos de Direito Sanitário	2,021	cc-by	3,243	1 Doutor em Direito Civil; diretor, Centro de Direito Biomédico, Faculdade de Direito, Universidade de Coimbra, Coimbra, Portugal. https://orcid.org/0000-0003-4793-3855. E-mail: andreper@fd.uc.pt Resumo Resumo A obra Poder Local em tempos de COVID-19 analisa o impacto das restrições provocadas pela COVID-19 no funcionamento da democracia local e da democracia participativa, abordam-se os efeitos da legislação COVID-19 no emprego público, nas finanças locais, na contratação pública e na digitalização da administração pública; analisa a cooperação territorial e institucional que se verificou entre os municípios e o poder estadual, o Serviço Nacional de Saúde e as Freguesias. No final, considera os riscos e potencialidades que podem estar associados à fase de recuperação, deixando-se um alerta para os perigos de descontrolo orçamental nos municípios e no sector empresarial local, para a importância de se combaterem os riscos de corrupção associados a esta fase com medidas sólidas aptas a assegurar uma governação sustentável e transparente, e para a necessidade de se fazer da recuperação uma oportunidade para se assegurar a efetiva implementação nos municípios dos Objetivos de Desenvolvimento Sustentável da ONU. Palavras-chave Poder Local. COVID-19. Políticas Públicas. Abstract This work analyzes the impact of the restrictions caused by COVID-19 on the functioning of local democracy and participatory democracy, addressing the effects of the COVID-19 legislation on public employment, local finance, public procurement and the digitization of public administration; analyzes the territorial and institutional cooperation that took place between the municipalities and the State power, the National Health Service and the local Authorities. In the end, it considers the risks and potentialities that may be associated with the recovery phase, leaving an alert to the dangers of lack of budgetary control in municipalities and in the municipal enterprises sector, to the importance of combating the risks of corruption associated with this phase with solid measures capable of ensuring sustainable and transparent governance, and the need to turn recovery into an opportunity to ensure the effective implementation of the UN Sustainable Development Goals in municipalities. Resenha O papel do Poder Local no combate à pandemia de COVID-19 Review The role of local government in combating the COVID-19 pandemic Resenha O papel do Poder Local no combate à pandemia de COVID-19 Review The role of local government in combating the COVID-19 pandemic El papel del gobierno local en la lucha contra la pandemia de COVID-19 André Dias Pereira1 Resumen Resumen Este trabajo analiza el impacto de las restricciones provocadas por COVID-19 sobre el funcionamiento de la democracia local y la democracia participativa, abordando los efectos de la legislación COVID-19 sobre el empleo público, las finanzas locales, la contratación pública y la digitalización de la administración pública; analiza la cooperación territorial e institucional que se dio entre los municipios y el poder estatal, el Serviço Nacional de Sanidad y los ayuntamientos. Al final, considera los riesgos y potencialidades que se pueden asociar a la fase de recuperación, dejando una alerta a los peligros de la falta de control presupuestario en los municipios y en el sector empresarial local, a la importancia de combatir los riesgos de corrupción asociados a esta fase, con medidas sólidas capaces de asegurar una gobernanza sostenible y transparente, y la necesidad de convertir la recuperación en una oportunidad para asegurar la implementación efectiva de los Objetivos de Desarrollo Sostenible de la ONU en los municipios. Palabras clave Poder Local COVID-19 Políticas Públicas A obra Poder Local em tempos de COVID-19 – em dois volumes (1, 2) – reúne textos de autores de proveniência científica diversa: Ciência Política, Direito, Sociologia, Comunicação, Economia, Auditoria, Políticas públicas, Engenharia informática, entre outras e de profissões diversas: autarcas, professores, magistrados, advogados, investigadores, técnica superior de município. Encontramos autores portugueses de origens geográficas distintas: Aveiro, Bragança, Coimbra, Minho, Lisboa. Assim, para além da alta representatividade geográfica, esta obra reúne um total de 20 capítulos, nos quais participam 32 autores com um amplo conhecimento e experiência nas lides autárquicas e é uma obra com equilíbrio de género. Poderá ser injusto destacar alguém deste naipe, mas todos compreendem que refira o nome de José Tavares, Juiz Conselheiro, Presidente do Tribunal de Contas e do Conselho de Prevenção da Corrupção, dos meus colegas – professores da Faculdade de Direito da Universidade de Coimbra, Pedro Gonçalves e Fernanda Paula Oliveira – e do Professor Pedro Pitta Barros, ilustre economista da saúde e que foi colega do signatário deste texto no Conselho Nacional de Ética para as Ciências da Vida (CNECV), de 2015 a 2021. Autores que garantem a alta qualidade desta obra! Parabéns aos coordenadores Luís Filipe Mota Almeida, do Direito Público; Luís de Sousa, da Ciência Política; e Manuel Ferreira Ramos, jurista e consultor da Associação Nacional de Assembleias Municipais. Trata-se de uma publicação temporalmente acertada e de conteúdo de excelência! Keywords y Municipal Authorities. COVID-19. Public Policies. Municipal Authorities. COVID-19. Public Policies. 235 Cad. Ibero-amer. Dir. Sanit., Brasília, 10(3): jul./set., 2021 https://doi.org/10.17566/ciads.v10i3.822 Resumen Este trabajo analiza el impacto de las restricciones provocadas por COVID-19 sobre el funcionamiento de la democracia local y la democracia participativa, abordando los efectos de la legislación COVID-19 sobre el empleo público, las finanzas locales, la contratación pública y la digitalización de la administración pública; analiza la cooperación territorial e institucional que se dio entre los municipios y el poder estatal, el Serviço Nacional de Sanidad y los ayuntamientos. Al final, considera los riesgos y potencialidades que se pueden asociar a la fase de recuperación, dejando una alerta a los peligros de la falta de control presupuestario en los municipios y en el sector empresarial local, a la importancia de combatir los riesgos de corrupción asociados a esta fase, con medidas sólidas capaces de asegurar una gobernanza sostenible y transparente, y la necesidad de convertir la recuperación en una oportunidad para asegurar la implementación efectiva de los Objetivos de Desarrollo Sostenible de la ONU en los municipios. Palabras clave P d L l COVID 19 P líti Públi Cad. Ibero-amer. Dir. Sanit., Brasília, 10(3): jul./set., 2021 https://doi.org/10.17566/ciads.v10i3.822 Resumen Um marco fundamental nesta área do saber e com importância para o Direito, as Políticas Públicas e a ação concreta dos decisores políticos. Esta é a primeira obra que, relativamente às autarquias locais, analisa o impacto da crise sanitária, económica e social provocada pela COVID-19. Está dividida em 2 volumes e Cad. Ibero-amer. Dir. Sanit., Brasília, 10(3): jul./set., 2021 https://doi.org/10.17566/ciads.v10i3.822 236 contém a Parte I sobre A Qualidade da Democracia Local em Tempos de COVID-19. Aqui se revelam as limitações das plataformas eletrónicas para o verdadeiro debate político; demonstram-se as dificuldades (Luís Mota de Almeida) do exercício do direito de oposição durante as pandemias e a presidencialização (Marco Caldeira) do Executivo – também ele elevado a responsável pela proteção civil pelo artigo 35.º da Lei n.º 27/2006 – Lei da Proteção Civil –, na sua reforma de 2015. contém a Parte I sobre A Qualidade da Democracia Local em Tempos de COVID-19. Aqui se revelam as limitações das plataformas eletrónicas para o verdadeiro debate político; demonstram-se as dificuldades (Luís Mota de Almeida) do exercício do direito de oposição durante as pandemias e a presidencialização (Marco Caldeira) do Executivo – também ele elevado a responsável pela proteção civil pelo artigo 35.º da Lei n.º 27/2006 – Lei da Proteção Civil –, na sua reforma de 2015. contém a Parte I sobre A Qualidade da Democracia Local em Tempos de COVID-19. Aqui se revelam as limitações das plataformas eletrónicas para o verdadeiro debate político; demonstram-se as dificuldades (Luís Mota de Almeida) do exercício do direito de oposição durante as pandemias e a presidencialização (Marco Caldeira) do Executivo – também ele elevado a responsável pela proteção civil pelo artigo 35.º da Lei n.º 27/2006 – Lei da Proteção Civil –, na sua reforma de 2015. Por seu turno, a Democracia participativa ficou limitada nestes mais de 18 meses (Roberto Falanga e Fiovani Allegretti), numa pandemia que é para além de uma patologia do corpo, uma patologia da sociedade. Ainda assim, os autores deste capítulo revelam casos de sucesso e inovação, na área da democracia participativa, em Lisboa, Cascais e Valongo... que merecem leitura atenta. Resumen Na Parte II - A Governação Local em Tempos de COVID-19 – encontramos textos sobre O papel dos municípios na resposta à crise sanitária, social e económica, de Carlos José Batalhão, que destaca o papel excecional dos municípios portugueses no combate à crise pandémica, designadamente no plano da atuação sanitária, da atuação informativa, da atuação financeira e da contratação pública, oferecendo refeições e outros apoios sociais e socioeconómicos, isenções e benefícios fiscais e apoios diretos às empresas. Por coincidência, o pacote legislativo de descentralização entrou em vigor neste ano e meio da pandemia – trata-se da transferência do Estado para os Municípios várias competências em matéria de saúde e de educação, entre outras, que resulta da Lei n.º 50/2018 de 16 de agosto (Lei-quadro da transferência de competências para as autarquias locais e para as entidades intermunicipais) – e, no específico domínio da saúde, o Decreto-Lei n.º 23/2019 de 30 de janeiro. Como afirma o autor “fique para memória futura (...) a relevância crucial das autarquias locais, nomeadamente em épocas de emergência...” e remata: “O Estado de Direito democrático não é nada sem autarquias locais.” O livro avança com perspectivas jurídico-financeiras (Hugo Flores da Silva e Joaquim Freitas da Rocha), que nos dão conta da flexibilização em vários domínios (contratação, prestação de contas, simplificação de procedimentos, etc...). Os autores terminam com algumas palavras de cautela e exigência de maior rigor e racionalidade na despesa pública. Segue-se uma explanação sobre a contratação pública local no contexto da pandemia COVID-19 de Pedro Gonçalves, de que se destaca a explicação sobre o regime excecional do ajuste direto. A terminar esta parte e o primeiro volume, Susana Ferreira dos Santos e Melanie Neiva Santos escrevem sobre o Emprego público municipal: teletrabalho e proteção Cad. Ibero-amer. Dir. Sanit., Brasília, 10(3): jul./set., 2021 https://doi.org/10.17566/ciads.v10i3.822 237 de dados em tempos de COVID-19. Aqui se anota que, por exemplo na Câmara Municipal de Lisboa, havia no dia 1 de março de 2021 – 41% dos trabalhadores em teletrabalho. de dados em tempos de COVID-19. Aqui se anota que, por exemplo na Câmara Municipal de Lisboa, havia no dia 1 de março de 2021 – 41% dos trabalhadores em teletrabalho. Passamos ao Vol. II e entramos na Parte III – Cooperação Territorial e Institucional em resposta à COVID-19. Resumen Sanit., Brasília, 10(3): jul./set., 2021 https://doi.org/10.17566/ciads.v10i3.822 238 de contingência, mapeia riscos no emprego de dinheiros públicos e recomenda medidas específicas de controlo.” E afirmam: “quanto maior é a tormenta, maior é a exigência colocada a todos os tripulantes da nau!” Com efeito, recordam: “O montante das múltiplas medidas COVID reportado pelo Ministério das Finanças cifra-se na escala dos milhares de milhões de euros”. de contingência, mapeia riscos no emprego de dinheiros públicos e recomenda medidas específicas de controlo.” E afirmam: “quanto maior é a tormenta, maior é a exigência colocada a todos os tripulantes da nau!” Com efeito, recordam: “O montante das múltiplas medidas COVID reportado pelo Ministério das Finanças cifra-se na escala dos milhares de milhões de euros”. de contingência, mapeia riscos no emprego de dinheiros públicos e recomenda medidas específicas de controlo.” E afirmam: “quanto maior é a tormenta, maior é a exigência colocada a todos os tripulantes da nau!” Com efeito, recordam: “O montante das múltiplas medidas COVID reportado pelo Ministério das Finanças cifra-se na escala dos milhares de milhões de euros”. Tiago Serrão discorre sobre o funcionamento dos órgãos colegiais autárquicos, sobretudo anotando a passagem da obrigatoriedade de reuniões, deliberações e votações presenciais, para a admissibilidade de meios telemáticos. O autor propõe que esta medida excecional seja adotada para futuro com carácter permanente. À semelhança do CPA, os meios telemáticos vieram para ficar nos órgãos colegiais autárquicos. É uma proposta que merece ponderação. O autor vai mais longe e defende a utilização de meios eletrónicos para a realização de convocatórias e a regulação da votação por escrutínio secreto. Tenho dúvidas de que esse seja o caminho, pois o contacto humano é importante para a vivência da polis e as decisões dos órgãos políticos beneficiam dessa dimensão corpórea e presencial. Constitui um tema que merece atenta reflexão. Pedro Camões aborda também o tema do risco do descontrolo orçamental em tempos de COVID-19. Deste texto, importa logo realçar que Portugal está na parte de baixo da tabela no que respeita ao peso comparativo das despesas da Administração Regional e Local no total da despesa pública. Temos que descentralizar mais e transferir mais verbas para as autarquias – é condição de desenvolvimento do território e das populações! Um contributo modesto para a mudança pode vir do Fundo de Solidariedade da União Europeia, no qual os municípios são as únicas entidades beneficiárias desses fundos. Cad. Ibero-amer. Dir. Sanit., Brasília, 10(3): jul./set., 2021 https://doi.org/10.17566/ciads.v10i3.822 Resumen Desde a cooperação entre o poder local e o poder estadual (Ana Fernanda Neves) – cujo quadro legal de importa reforçar, até ao papel das Freguesias (unidade administrativa mais próxima das populações, ao nível de bairro nas cidades ou de um conjunto próximo de populações, nas zonas rurais) (Cláudia Costa e Tânia Maia) que ofereceram respostas que importa não esquecer: aquisição e distribuição de equipamentos de proteção individual, atividades de desinfeção, higienização e limpeza, disponibilização ou financiamento de testes à COVID-19, distribuição de refeições e bens alimentares a pessoas carenciadas e alunos, atribuição ou empréstimo de computadores e equipamentos informáticos, apoios logísticos à população, linhas de apoio, campanhas de sensibilização, apoios ao comércio local, apoios ao plano de vacinação contra a COVID-19.... Um conjunto vasto de atividades que este poder autárquico de grande proximidade e com recursos financeiros muito limitados, mas que fez toda a diferença na vida das populações durante a crise pandémica. Enfim, uma miríade de atividades que revela a imaginação e dedicação das Juntas de Freguesia ao bem comum! Um pilar do poder democrático que importa qualificar, robustecer e promover! Pedro Pitta Barros apresenta-nos a hospitalização domiciliária como solução de futuro, na qual os municípios também se devem envolver. Lança o desafio aos municípios de criarem a sua unidade de Hospitalização Domiciliária. Apresenta ainda uma interessante análise na qual conclui: i) os concelhos com uma proporção maior na população do grupo etário com mais de 65 anos tiveram um maior choque associado à pandemia; ii) a densidade populacional do concelho – concelhos mais densamente povoados foram sendo mais duramente atingidos pela pandemia; iii) concelhos com maior proporção de desempregados apresentam maior peso de COVID-19. Finalmente, Manuel Ferreira Ramos explica o papel da ANAM e das Assembleias Municipais em tempos de pandemia. A Parte IV – Risco de Má Governação e outros Desafios em tempos de COVID-19 e de Recuperação – é escrita a pensar já na recuperação e na fase pós-pandémica. Aqui se coloca em evidência, pela mão de José Tavares, Presidente do Tribunal de Contas, e de Ana Paula Gil Garcês, o imperativo de Boa governação, gestão de riscos e transparência em contexto pandémico, realçando o papel decisivo do Tribunal de Contas: “aciona o plano Cad. Ibero-amer. Dir. Resumen O caminho seria, pois, que “cada governo local deve financiar, a partir de recursos ou receitas próprias as despesas sobre as quais decide.” Cuidado, porém, pois o princípio da solidariedade nacional implica transferências financeiras entre regiões. Júlia Gonçalves coloca-nos em alerta ao escrever Pré-insolvência municipal em tempos de COVID-19, mas apresenta propostas de solução, defendendo a responsabilidade subsidiária do município, na exata medida em que exerce o controlo em cadeia. Georgina Morais apresenta um estudo sobre A necessidade de implementar mecanismos de auditoria interna para uma recuperação sustentável nas autarquias. Um conjunto de autores – José Fidalgo, João António Joana Abreu, Tânia Correia, Jorge Cerol e Ricardo Reis – colocam em destaque Os Objetivos de desenvolvimento sustentável enquanto bússola da recuperação pós- COVID-19. É de aplaudir a convocação Cad. Ibero-amer. Dir. Sanit., Brasília, 10(3): jul./set., 2021 https://doi.org/10.17566/ciads.v10i3.822 239 deste instrumento-bússola internacional, para um mundo mais sustentável e com máximo respeito pelos direitos humanos. Finalmente, Fernanda Paula Oliveira escreve sobre Perspetivas e desafios urbanísticos num mundo pós-COVID: breves reflexões, texto no qual questiona se o eventual regresso ao campo ou a fuga às cidades terá consequências no direito urbanismo. A autora alerta para que não se deve voltar ao urbanismo em “mancha de óleo”... antes densificar as médias e pequenas cidades. Este é um desafio para os municípios e para as regiões! Por fim, destaco algumas ideias que muito me marcaram na leitura destes dois volumes: Por fim, destaco algumas ideias que muito me marcaram na leitura destes dois volumes: 1) A ligação entre o Poder Local e a Academia. A promoção de uma relação dialógica de enriquecimento comum e a produção de saber para conduzir, com mais qualidade, os destinos coletivos. 2) A colocação da saúde em todas as políticas em lugar cimeiro. Quer na política da União Europeia, do Estado, das Regiões, dos Municípios... em todas as políticas: de educação, de urbanismo, de turismo, de economia e indústria, de ambiente, etc. Devemos introduzir os indicadores de saúde e ambientais nas métricas económicas e não apenas o PIB! E ter uma visão holística da saúde, fugindo do sistema hospitalocêntrico e não cair noutro centralismo dos cuidados de saúde primários. Com efeito, como a pandemia está a demonstrar, a saúde está para além dos edifícios físicos e seus profissionais. 3) Daí, a compreensão da importância da saúde pública e da saúde mental no sistema de saúde. Resumen Temos que reivindicar esta centralidade – mesmo para além dos períodos pandémicos. 4) O não afunilamento das discussões aos estereótipos do século XX, designadamente na frágil dicotomia público-privado, mas antes a compreensão da necessidade do envolvimento de todos os setores e a devida dignificação do setor social. 5) A preocupação com a corrupção, o desvio de fundos e a má gestão, quer durante a pandemia, quer após a pandemia. É um imperativo ético de todos nós. 5) A preocupação com a corrupção, o desvio de fundos e a má gestão, quer durante a pandemia, quer após a pandemia. É um imperativo ético de todos nós. 1. Almeida LFM de, Sousa L de, Ramos MF (coord.). Poder Local em tempos de COVID- 19. Vol. 1, A Qualidade da Democracia Local e Governação em Tempos de COVID-19. Coimbra: Almedina; 2021. ISBN: 978-972-40-97512. Cad. Ibero-amer. Dir. Sanit., Brasília, 10(3): jul./set., 2021 https://doi.org/10.17566/ciads.v10i3.822 Dias Pereira A. O papel do Poder Local no combate à pandemia de COVID-19. Cadernos Ibero-Americanos de Direito Sanitário. 2021 jul./set.;10(3):235-241. Referências 1. Almeida LFM de, Sousa L de, Ramos MF (coord.). Poder Local em tempos de COVID- 19. Vol. 1, A Qualidade da Democracia Local e Governação em Tempos de COVID-19. Coimbra: Almedina; 2021. ISBN: 978-972-40-97512. 240 Cad. Ibero-amer. Dir. Sanit., Brasília, 10(3): jul./set., 2021 https://doi.org/10.17566/ciads.v10i3.822 2. Almeida LFM de, Sousa L de, Ramos MF (coord.). Poder Local em tempos de COVID- 19. Vol. 2, A Cooperação Territorial e Institucional no Combate à Pandemia e os Desafios da Recuperação, Coimbra: Almedina; 2021. ISBN: 978-972-40-97169. 2. Almeida LFM de, Sousa L de, Ramos MF (coord.). Poder Local em tempos de COVID- 19. Vol. 2, A Cooperação Territorial e Institucional no Combate à Pandemia e os Desafios da Recuperação, Coimbra: Almedina; 2021. ISBN: 978-972-40-97169. Submetido em: 24/08/21 Aprovado em: 01/09/21 Como citar este artigo Dias Pereira A. O papel do Poder Local no combate à pandemia de COVID-19. Cadernos Ibero-Americanos de Direito Sanitário. 2021 jul./set.;10(3):235-241. https://doi.org/10.17566/ciads.v10i3.822 Submetido em: 24/08/21 Aprovado em: 01/09/21 https://doi.org/10.17566/ciads.v10i3.822 Submetido em: 24/08/21 Aprovado em: 01/09/21 Como citar este artigo Como citar este artigo Como citar este artigo 241
https://openalex.org/W2396330268	https://europepmc.org/articles/pmc4877366?pdf=render	English	null	Within-Leaf Nitrogen Allocation in Adaptation to Low Nitrogen Supply in Maize during Grain-Filling Stage	Frontiers in plant science	2,016	cc-by	10,717	Keywords: bioenergetics, light harvesting, phosphoenolpyruvate carboxylase, photosynthetic rate, photosynthetic nitrogen use efﬁciency, pyruvate orthophosphate dikinase, ribulose-1,5-bisphosphate carboxylase, thylakoid nitrogen Within-Leaf Nitrogen Allocation in Adaptation to Low Nitrogen Supply in Maize during Grain-Filling Stage Xiaohuan Mu Qinwu Chen Fanjun Chen Lixing Yuan and Guohua Mi* Xiaohuan Mu, Qinwu Chen, Fanjun Chen, Lixing Yuan and Guohua Mi* Center for Resources, Environment and Food Security, College of Resources and Environmental Science, China Agricultural University, Beijing, China Nitrogen (N) plays a vital role in photosynthesis and crop productivity. Maize plants may be able to increase physiological N utilization efﬁciency (NUtE) under low-N stress by increasing photosynthetic rate (Pn) per unit leaf N, that is, photosynthetic N-use efﬁciency (PNUE). In this study, we analyzed the relationship between PNUE and N allocation in maize ear-leaves during the grain-ﬁlling stage under low N (no N application) and high N (180 kg N ha−1) in a 2-year ﬁeld experiment. Under low N, grain yield decreased while NUtE increased. Low-N treatment reduced the speciﬁc N content of ear leaves by 38% without signiﬁcant inﬂuencing Pn, thereby increasing PNUE by 54%. Under low-N stress, maize plants tended to invest relatively more N into bioenergetics to sustain electron transport. In contrast, N allocated to chlorophyll and light-harvesting proteins was reduced to control excess electron production. Soluble proteins were reduced to shrink the N storage reservoir. We conclude that optimization of N allocation within leaves is a key adaptive mechanism to maximize Pn and crop productivity when N is limited during the grain-ﬁlling stage in maize under low-N conditions. Edited by: Soumitra Paul, University of Calcutta, India Reviewed by: Dimah Habash, SECUREWHEAT Consultancy, UK Ümit BarI¸s Kutman, Konya Food and Agriculture University, Turkey Correspondence: Guohua Mi miguohua@cau.edu.cn Edited by: Soumitra Paul, University of Calcutta, India Reviewed by: Dimah Habash, SECUREWHEAT Consultancy, UK Ümit BarI¸s Kutman, Konya Food and Agriculture University, Turkey Correspondence: Guohua Mi miguohua@cau.edu.cn ORIGINAL RESEARCH published: 24 May 2016 doi: 10.3389/fpls.2016.00699 INTRODUCTION In modern crop production systems, nitrogen (N) plays a vital role in yield formation; it is the mineral element required in the greatest amounts by plants, and is often the growth-limiting nutrient. N is a fundamental constituent of many cell components. In leaves, forms of N include soluble components such as nitrates, amino acids and proteins, and insoluble components in cell walls, membranes and other structures. N used in the photosynthetic apparatus can be divided into two categories, namely, that associated with photosynthetic enzymes and thylakoid N. The main photosynthetic enzymes, ribulose-1,5-bisphosphate carboxylase (Rubisco), phosphoenolpyruvate carboxylase (PEPC) and pyruvate orthophosphate dikinase (PPDK), are involved in carbon reduction reactions and are the most abundant enzymes in photosynthesis (Tazoe et al., 2005). Thylakoid N is distributed between two types of proteins: (1) proteins related to bioenergetics, including Cyt b/f and CF1/CF0 involved in electron transport and photophosphorylation, and (2) light-harvesting proteins, such as photosystem I (PSI), photosystem II (PSII), and light-harvesting complex II (LHCII) proteins, that are associated with the light reactions of photosynthesis (Makino et al., 1997; Takashima et al., 2004; Tazoe et al., 2005; Uribelarrea et al., 2009). When grown under high light, C3 leaves typically invested 58% of leaf N to soluble protein (about 40% of which is Specialty section: This article was submitted to Plant Nutrition, a section of the journal Frontiers in Plant Science Received: 25 February 2016 Accepted: 06 May 2016 Published: 24 May 2016 Citation: Mu X, Chen Q, Chen F, Yuan L and Mi G (2016) Within-Leaf Nitrogen Allocation in Adaptation to Low Nitrogen Supply in Maize during Grain-Filling Stage. Front. Plant Sci. 7:699. doi: 10 3389/fpls 2016 00699 Specialty section: This article was submitted to Plant Nutrition, a section of the journal Frontiers in Plant Science Received: 25 February 2016 Accepted: 06 May 2016 Published: 24 May 2016 Plant Materials and Growth Conditions In spite of these ﬁndings, the underlying physiological mechanism of photosynthetic N use eﬃciency remains unclear. The experiment consisted of a randomized block design with four replicates, with each plot 15 m long and 9 m wide. Zhengdan 958, the most popular commercial hybrid across North and Northeast China, was sown on April 28, 2013, and April 29, 2014, and was harvested on September 7 and September 3 of 2013 and 2014, respectively. The plots were over-seeded using a hand planter, and then thinned at the seeding stage to 60,000 plants ha−1. Distances between rows and plants were 60 and 28 cm, respectively. Plots were kept free of weeds, insects and diseases during the growth season. During 2013, rainfall was adequate during the entire growth period and no irrigation was applied. Because a severe drought occurred during the silking stage in 2014, irrigation was applied during that period to ensure normal plant growth. In general, C4 plants exhibit a greater PNUE than C3 plants (Brown, 1978; Schmitt and Edwards, 1981; Sage and Pearcy, 1987, 2000; Makino et al., 2003). This higher PNUE is mainly because C4 plants can eliminate photorespiration by increasing CO2 levels in the vicinity of Rubisco. In maize, a C4 plant, the N cost for C4-cycle enzymes (PPDK and PEPC) is not large; in addition, the lower amount of Rubisco in maize allows a greater N investment to be made in the thylakoid components compared with rice, a C3 plant (Makino et al., 2003). And NADP-malic enzyme (ME) C4 grasses have a higher PNUE and NUE than NAD-ME C4 grasses (Ghannoum et al., 2005). The main reason is that they have less leaf N and soluble protein but a faster kcat of Rubisco in NADP-malic enzyme (ME) C4 grasses (Ghannoum et al., 2005). Gas Exchange and Chlorophyll Fluorescence Parameters Measurements At the silking stage, six plants with the same silking date were tagged. At 20 and 23 days after silking in 2013 and 2014, respectively, the ear-leaf net photosynthetic rate (Pn) of six tagged plants per plot was determined. The average of the Pn values of the six plants in each plot was taken as a replicate. Pn was measured with a portable photosynthesis system (Li6400; LI-COR, Lincoln, NE, USA) coupled to a standard red/blue LED broadleaf cuvette (6400-02B; LI-COR) and a CO2 mixer (6400-01; LI-COR) at a light intensity of 1,600 µmol m−2 s−1. Plant Materials and Growth Conditions Plant Materials and Growth Conditions Field experiments were conducted in 2013 and 2014 at the Shangzhuang Experimental Station, China Agricultural University, Beijing, China (116◦11′ N, 40◦8′ E). The ﬁeld soil was a typical Ustochrept soil with the following physical and chemical characteristics (0–20 cm) at the start of the experiment: 92.4 kg N ha−1 CaCl2-extracted mineral N (Nmin), 16.2 mg kg−1 available phosphorus (Olsen-P), 122.6 mg kg−1ammonium acetate extractable potassium (K), 11.6 g kg−1 organic matter, and a pH (H2O) of 8.0. Before plowing, the ﬁeld was irrigated, plowed (immediately before plowing) and sprinkled with a base fertilizer consisting of 135 kg P2O5 ha−1 [as superphosphate (Ca (H2PO4)2·H2O)] and 75 kg K2O ha−1 (as K2SO4). Additional N was applied at two diﬀerent treatment levels: 180 kg N ha−1 (HN) and no N application (LN). For the HN treatments, 30% of the N fertilizer was applied before sowing and the remaining at the V6 stage (six expanded leaves). In the previous research conducted in the same ﬁeld, N application at 180 kg ha−1 was found suﬃcient to achieve the maximum yield (data not shown). At harvest, the residual soil Nmin (0–20 cm) was 52 and 38 kg N ha−1 in 2013 and 2014, respectively. y Under N-limited conditions, maize plants increased N use eﬃciency by increasing N uptake eﬃciency (NUpE) and/or N utilization eﬃciency (NUtE; Moll et al., 1982; Hirel et al., 2007). Eﬃcient N acquisition is believed to rely on a large and deep root system (Lawlor, 2002; Wang et al., 2005; Mi et al., 2010; Lynch, 2013). NUtE, which is deﬁned as grain yield produced per unit of plant N (Moll et al., 1982; Hirel et al., 2007), is an indicator of how plant N is used for photosynthetic production. On a whole- plant level, an optimal N distribution in the plant canopy may improve photosynthesis without additional N input (Dwyer and Stewart, 1986; Drouet and Bonhomme, 1999; Hikosaka, 2014). How N can be eﬃciently used to produce as much photosynthate in single leaves, especially under N-limited conditions, is less clear. Photosynthetic N use eﬃciency (PNUE), the rate of photosynthesis per unit leaf N, is increased under low-N stress (Sage and Pearcy, 1987; Ghannoum et al., 2005; Uribelarrea et al., 2009; Hawkesford, 2012). In a recent ﬁeld study, an N-eﬃcient maize genotype was found to have a higher PNUE than an ineﬃcient one (Chen et al., 2014). Citation: Mu X, Chen Q, Chen F, Yuan L and Mi G (2016) Within-Leaf Nitrogen Allocation in Adaptation to Low Nitrogen Supply in Maize during Grain-Filling Stage. Front. Plant Sci. 7:699. doi: 10.3389/fpls.2016.00699 May 2016 \| Volume 7 \| Article 699 1 Frontiers in Plant Science \| www.frontiersin.org Within-Leaf Nitrogen Allocation Mu et al. Rubisco) and 22% to thylakoids (Evans, 1989a; Poorter and Evans, 1998; Makino et al., 2003). While in C4 plant, about 45% leaf N partitioned into soluble protein (about 20% of which is Rubisco) and 28% into thylakoids (Makino et al., 2003; Ghannoum et al., 2005; Tazoe et al., 2005). And about 80% of thylakoids N in C3 plants and 75% of thylakoids N in C4 plants allocated in light-harvesting proteins, the other allocated in bioenergetics (Evans, 1989b; Makino et al., 2003). A strongly positive correlation has been widely reported between photosynthetic capacity and N content per unit leaf area (Makino et al., 2003; Takashima et al., 2004; Tazoe et al., 2005). N supply thus has substantial eﬀects on plant growth and development and yield formation. Rubisco) and 22% to thylakoids (Evans, 1989a; Poorter and Evans, 1998; Makino et al., 2003). While in C4 plant, about 45% leaf N partitioned into soluble protein (about 20% of which is Rubisco) and 28% into thylakoids (Makino et al., 2003; Ghannoum et al., 2005; Tazoe et al., 2005). And about 80% of thylakoids N in C3 plants and 75% of thylakoids N in C4 plants allocated in light-harvesting proteins, the other allocated in bioenergetics (Evans, 1989b; Makino et al., 2003). A strongly positive correlation has been widely reported between photosynthetic capacity and N content per unit leaf area (Makino et al., 2003; Takashima et al., 2004; Tazoe et al., 2005). N supply thus has substantial eﬀects on plant growth and development and yield formation. (Sugiharto et al., 1990; McCullough et al., 1994; Makino et al., 2003), less is known about the physiological determinants of PNUE during the grain-ﬁlling stage under ﬁeld conditions. The aim of this study was consequently to uncover the relationship between within-leaf N allocation and PNUE. Frontiers in Plant Science \| www.frontiersin.org Plant Materials and Growth Conditions Measurements were obtained at a leaf temperature of 30 ± 0.5◦C Yield, N Uptake and Physiological N Utilization Efﬁciency (NUtE) p Rubisco, PEPC, and PPDK contents of frozen leaves were determined using the method of Makino et al. (2003) with minor modiﬁcations. Using a chilled mortar and pestle, the frozen leaves were homogenized in extraction buﬀer containing 0.2 mM EDTA-Na2, 10 mM dithiothreitol, 2 mM iodoacetic acid, 0.1% Triton X-100 and 100 mM Tris-HCl at pH 6.8. The homogenate was centrifuged at 12,000 g for 20 min at 4◦C. A portion of the supernatant was used for determination of total soluble protein content using a protein assay kit based on the Bradford method (Bio-Rad Protein Assay; Bio-Rad, CA, USA). The supernatant was treated with double-strength loading buﬀer [5% (w/v) lithium dodecyl sulfate, 5% β-mercaptoethanol, 0.1% bromophenol blue, 5% glycerol and 25 mM Tris-HCl at pH 6.8], heated at 100◦C for 5 min, and analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Rubisco, PEPC, and PPDK were determined spectrophotometrically by formamide extraction of their Coomassie-Brilliant-Blue-R-250- stained bands, which corresponded to 99 kDa for PEPC (Uedan and Sugiyama, 1976), 94 kDa for PPDK (Sugiyama, 1973) and 52 and 15 kDa for Rubisco (Makino et al., 2003). Protein concentration was calculated using bovine serum albumin as a standard (Makino et al., 1986; Fukayama et al., 2001). Cell wall biomass and N content were analyzed according to Onoda et al. (2004) with modiﬁcations. The pellet was resuspended with Tris-HCl buﬀer containing 3% (w/v) SDS, incubated at 90◦C for 5 min, and then centrifuged at 2,500 g for 5 min. This procedure was repeated eight times. To remove small amounts of cytoplasmic protein contamination, the pellet was washed six times with 0.2 M KOH followed by centrifugation at 2,500 g for 5 min. These procedures removed proteins weakly bound to cell walls, leaving behind only tightly bound (structural) proteins. After six washes with distilled water and six washes with ethanol, the tube containing the pellet was dried in an oven at 75◦C to a constant weight. After drying, the cell wall materials were At silking and physiological maturity, ﬁve consecutive plants per plot were cut at the soil surface and separated into leaves, stalks (including leaf sheaths and tassels), husks/cobs and grains. All samples were dried at 65◦C to a constant weight. Gas Exchange and Chlorophyll In maize, around 80–100% of dry matter for grain yield formation is contributed by post-silking photosynthesis (Tollenaar and Lee, 2006; Chen et al., 2013, 2014; Kosgey et al., 2013; Antonietta et al., 2014). During the post-silking period, the maintenance of a high rate of whole-plant photosynthesis is diﬃcult, as N is continuously remobilized from the leaves, especially under N-limited conditions. An increase in PNUE would potentially resolve this problem (Chen et al., 2014). Most of the research on PNUE has been conducted at seedling stage May 2016 \| Volume 7 \| Article 699 Frontiers in Plant Science \| www.frontiersin.org 2 Mu et al. Within-Leaf Nitrogen Allocation weighed and their total N concentrations were determined using the NC analyzer. and a CO2 concentration inside the chamber of 400 ± 1 µmol CO2 (mol air)−1 (Ding et al., 2005a; Uribelarrea et al., 2009). The following day, the same plants that had been used for the photosynthetic rate measurements in each plot were used to obtain chlorophyll ﬂuorescence using Li6400. The plants were continuously illuminated at least 1 h. CO2 concentration inside the chamber was 400 µmol mol−1 and holding incident irradiance at 1600 µmol m−2 s−1. The actual quantum yield of PSII photochemistry (8PSII) and electron transport rate (ETR, µmol e−s−1 m−2) were calculated as deﬁned by Ding et al. (2005b). Leaf area was calculated according to Sanderson et al. (1981) as leaf length × maximum width × k, where k is a shape factor equal to 0.75 (Birch et al., 2003). Chlorophyll (Chl) was extracted from leaf disks with acetone and ethanol. The absorbance of the extracts was spectrophotometrically measured at 645 and 663 nm (Porra et al., 1989). Nitrate was extracted from fresh leaves with distilled water and quantiﬁed following Cataldo et al. (1975). Free amino acids were determined according to Moore (1968). g Thylakoid membranes were prepared according to standard methods (Zhang et al., 1999; Peng et al., 2006). The leaves were homogenized with a chilled mortar and pestle in an ice-cold isolation buﬀer containing 400 mM sucrose, 50 mM HEPES- KOH (pH 7.8), 10 mM NaCl, and 2 mM MgCl2 and ﬁltrated through four layers of cheesecloth. The ﬁltrate was centrifuged at 5000 g for 10 min. The thylakoid pellets were washed with isolation buﬀer, recentrifuged, and suspended in isolation buﬀer. The thylakoid membrane pellets were dried in an oven at 75◦C to a constant weight. Biochemical Measurements After measurement of chlorophyll ﬂuorescence parameters, two leaves per plot were removed, immediately frozen in liquid N2 and stored at −80◦C for subsequent analysis. Two additional leaves were dried at 65◦C to a constant weight. After removal of midribs, the dried leaves were thoroughly ground and then analyzed for total N with an NC analyzer (Vario EL III; Elementar, Hanau, Germany). Speciﬁc leaf N (SLN) was calculated as N content per unit leaf area. Gas Exchange and Chlorophyll After drying, the thylakoid membrane was weighed and their total N concentrations were determined using the NC analyzer. Calculations Thylakoid N can be divided into two categories according to its allocation: (1) bioenergetics (associated with the electron transport chain and photophosphorylation) and (2) light- harvesting (involved in PSI, PSII, and LHC). N associated with light harvesting (Nh) was calculated assuming 37 mol mol−1Chl (Ghannoum et al., 2005), while N in bioenergetics (Nb) was calculated as NTH minus Nh. N allocated to Rubisco, PEPC, PPDK, and soluble protein was calculated assuming 16% N in proteins (Ghannoum et al., 2005). Yield, N Uptake and Physiological N Utilization Efﬁciency (NUtE) The silking stage was considered to be when 50% of the ears in a plot attained silking, whereas physiological maturity corresponded to the stage when a black layer was visible at the grain base in 50% of the ears. Dry samples were weighed and ground to a powder; N concentrations were determined using the semi-micro-Kjeldahl method. NUtE values were calculated as grain dry weight divided by total N content per plant. At physiological maturity, two rows of plants in each plot were harvested for determination of grain yield. Grain yield was standardized to 14% moisture. Frontiers in Plant Science \| www.frontiersin.org Effect of N Supply on N Investment into Soluble-N Components Nitrate content in LN-treated plants was higher than in the HN treatment (Table 4). Leaf nitrate content was very low, accounting for about 1% of total leaf N (Figure 3). The amount of N in amino acids was 51% lower under the LN treatment compared with HN treatment (Table 4). Soluble protein content was almost twofold less in plants grown under the LN treatment conditions (Table 4). Low N reduced the contents of Rubisco (including large and small subunits), PEPC and PPDK per unit leaf area by 52.0, 54.2, and 51.0%, respectively (Table 4, Figure 4; Supplementary Figure S1). When calculated on the basis of leaf N, Rubisco, PEPC, and PPDK concentrations per leaf N were, respectively, 24, 28, and 23% lower under LN treatment than under HN treatment (data not shown). RESULTS TABLE 1 \| Abbreviations, symbols used and their units used in the text. Symbol Deﬁnition Units 3-PGA 3-phosphoglyceric acid – CA Carbonic anhydrase – Chl Chlorophyll µmol m−2 Chl a Chlorophyll a µmol m−2 Chl b Chlorophyll b µmol m−2 Fd Ferredoxin − gbs Bundle-sheath conductance − LHCII Light harvest complex − LUS Rubisco large subunits − Mal Malate − MDH Malate dehydrogenase − N Nitrogen − Na Nitrogen in amino acids mg m−2 Nb Nitrogen in bioenergetic protein mg m−2 Nb/NTH Fraction of thylakoid nitrogen allocated to bioenergetics % Ncw Nitrogen in cell wall mg m−2 Nh Nitrogen in light-harvesting protein mg m−2 Nh/NTH Fraction of thylakoid nitrogen allocated to light-harvesting protein % ns Not signiﬁcant − Ns Nitrogen in the soluble proteins other than PEPC, PPDK, and Rubisco mg m−2 NT Nitrogen in thylakoid mg m−2 NTH/N Fraction of nitrogen allocated to thylakoid % OAA Oxaloacetic acid − PC Plastocyanin − PEPC Phosphoenolpyruvate carboxylase − Pn Photosynthetic rate µmol CO2 m−2 s−1 Pmax Maximum photosynthetic rate µmol CO2 m−2 s−1 PNUE Photosynthetic nitrogen use efﬁciency µmol CO2 g−1N s−1 PPDK Pyruvate orthophosphate dikinase − PQ Plastoquinone − PSI Photosystem I − PSII Photosystem II − Pyr Pyruvate − Rubisco Ribulose 1,5-bisphosphate carboxylase/oxygenase − RuBP Ribulose 1,5-bisphosphate − SLN Speciﬁc leaf nitrogen g m−2 SSU Rubisco small subunits − TABLE 1 \| Abbreviations, symbols used and their units used in the text. Effect of N Supply on Grain Yield, N Accumulation, NUtE, Pn, and PNUE Under LN stress, grain yield and total N uptake decreased by 32 and 57%, respectively (Table 2). In contrast, physiological N utilization eﬃciency (NUtE) increased by 57% compared with HN treatment. Effect of N Supply on Grain Yield, N Accumulation, NUtE, Pn, and PNUE Under LN stress, grain yield and total N uptake decreased by 32 and 57%, respectively (Table 2). In contrast, physiological N utilization eﬃciency (NUtE) increased by 57% compared with HN treatment. For the ear-leaf, LN treatment signiﬁcantly aﬀected SLN, but had little inﬂuence on Pn (Table 3). As a calculated result, PNUE was increased in the LN treatment. Compared with the HN treatment, SLN was 38% lower and PNUE was 54% higher under the LN treatment. Statistical Analysis Data across N treatments and years were ﬁrst pooled and subjected to a two-factor analysis of variance using the ANOVA procedure implemented in SPSS Statistics 17.0 (SPSS, Inc., Chicago, IL, USA). Diﬀerences were compared using the least signiﬁcant diﬀerence test at a 0.05 level of probability. It is found that the N treatment × Year interaction eﬀect is not signiﬁcant for almost all the measured parameters (Supplemental Table S1). May 2016 \| Volume 7 \| Article 699 Frontiers in Plant Science \| www.frontiersin.org Frontiers in Plant Science \| www.frontiersin.org 3 Mu et al. Within-Leaf Nitrogen Allocation PEPC, and PPDK declined 24, 28, and 23%, respectively, under LN treatment. In contrast, N allocation to bioenergetics processes increased by 62.5% under low N (4.0% at HN and 6.5% at LN). Therefore, the 2 years’ data was averaged for each parameter. All ﬁgures were constructed using GraphPad Prism 5 (GraphPad Software Inc., 2007). Effect of N Supply on the N Allocation into Structure-N Components NTH and Nh expressed per unit leaf area were 44.6 and 47.4% lower, respectively, in the LN treatment compared with HN (Table 4; Figure 4). There was no signiﬁcant diﬀerence in Nb between N treatments. Compared with HN treatment, Nh/NTH was reduced and Nb/NTH was increased under LN treatment, while NTH/SLN was no signiﬁcant diﬀerence (Figures 1 and 4). Ear-leaf cell-wall N content (Ncw) was 35% lowers in LN compared with the HN treatment (Table 4). However, the fraction of cell wall biomass was higher in the LN treatment (Figure 2). Effect of N Supply on Chlorophyll (Chl) and Chlorophyll Fluorescence Numbers followed by different letters indicate signiﬁcant differences (P < 0.05). TABLE 3 \| Effect of nitrogen (N) supply on net photosynthetic rate (Pn), speciﬁc leaf N (SLN) and photosynthetic N use efﬁciency (PNUE) in maize ear-leaves during the grain-ﬁlling stage. Pn was measured using a light intensity of 1600 µmol m−2 s−1. Measurements were made on ear-leaves between 09:00 and 12:00. Data are the means for the 2 years. Numbers followed by different letters indicate signiﬁcant differences (P < 0.05). In accord with previous studies (Pons and Westbeek, 2004; Ding et al., 2005a), LN treatment led to reduced total Chl, Chl a and Chl b contents (Figure 5). A reduction in Chl, together with an increase in zeaxanthin formation and the capacity for excitation energy dissipation in PSII (Khamis et al., 1990; Lu et al., 2001), are believed to be strategies for protection of PSII function (Khamis et al., 1990). The lower capacity of Pn under low-N conditions means that the greater excess of excitation energy may potentially lead to increased susceptibility of PSII to photo-inhibition (Ramalho et al., 1997; Grassi et al., 2001; Lu et al., 2001). Although the degree of low-N stress in our study was insuﬃcient to reduce leaf Pn, the leaves responded by reducing Chl content to avoid excess production of excitation energy. The change in N distribution within thylakoids was consistent with this change in Chl. Although NTH in the LN treatment was less than that in the HN treatment (Table 4), the percentage of N allocated to thylakoids was similar between N rates (30–31%; Figure 1). These values are close to those uncovered by Makino et al. (2003), who reported that 34% of leaf N was allocated to TABLE 4 \| Effect of nitrogen (N) supply on the contents of N compounds in maize ear-leaves during the grain-ﬁlling stage. TABLE 4 \| Effect of nitrogen (N) supply on the contents of N compounds in maize ear-leaves during the grain-ﬁlling stage. N treatment Parameter HN LN Nitrate mg m−2 22b 26a Soluble protein mg m−2 885a 447b PEPC mg m−2 85a 39b PPDK mg m−2 68a 33b Rubisco mg m−2 355a 170b NTH mg m−2 775a 452b Nb mg m−2 107a 101a Nh mg m−2 668a 351b Ncw mg m−2 243a 158b Na mg m−2 112a 55b Data are the means for the 2 years. DISCUSSION As is well documented, low-N stress causes stunted plant growth by reducing leaf expansion and Pn (Radin and Boyer, 1982; Snir and Neumann, 1997; Gastal and Lemaire, 2002; Jovanovic et al., 2004; KavanovA et al., 2008). Reduction in leaf expansion (leaf area) helps sustain leaf N concentration, thereby contributing to the maintenance of Pn (Radin and Boyer, 1982; Gastal and Lemaire, 2002) this seems to be especially true in N-eﬃcient maize genotypes (McCullough et al., 1994). Under the experimental conditions of our study, N deﬁciency caused a relative reduction of 36% in ear-leaf leaf area index (data not shown) and 38% in SLN, from 2.48 g m−2 under HN treatment to 1.55 g m−2 under LN treatment. Pn, however, was not signiﬁcantly aﬀected. Thus, PNUE was greatly increased, by 54%, under the LN treatment. According to Sinclair and Horie (1989), for each N allocation to leaves, an optimum N content exists to maximize crop biomass production. The optimal SLN for maximum Pn has been reported to be approximately 1.5 in ﬁeld-grown maize (McCullough et al., 1994; Muchow and Sinclair, 1994; Paponov et al., 2005; Vos et al., 2005), which ﬁts well with the value obtained under LN treatment in the present study. A higher PNUE enables maize plants to use N eﬃciently for biomass production and to increase N use eﬃciency (Table 2). A genotypic diﬀerence in PNUE has in fact been observed in maize, with N-eﬃcient genotypes having higher PNUE than ineﬃcient ones (McCullough et al., 1994; Paponov and Engels, 2003; Echarte et al., 2008; Chen et al., 2014). This variation suggests that genetic improvement of PNUE is possible, and elucidation of the physiological mechanism underlying high PNUE is therefore essential. Because the high PNUE observed under LN treatment contributed to a decrease in SLN rather than an increase in Pn, within-leaf optimization of N allocation potentially explains high PNUE. Effect of N Supply on the Leaf N Budget Effect of N Supply on the Leaf N Budget The percentage of leaf N allocated to diﬀerent N components is summarized in Figure 3. Relative to HN treatment levels, the LN treatment signiﬁcantly reduced the percentage of N allocated to soluble proteins (including Rubisco, PEPC, and PPDK) and light- harvesting proteins. Among the three major enzymes—Rubisco, PEPC, and PPDK —the percentage of leaf N allocated to Rubisco, May 2016 \| Volume 7 \| Article 699 Frontiers in Plant Science \| www.frontiersin.org 4 Mu et al. Within-Leaf Nitrogen Allocation (Figure 5). There were no signiﬁcant diﬀerences in 8PSII and ETR between N treatment (Figure 6). (Figure 5). There were no signiﬁcant diﬀerences in 8PSII and ETR between N treatment (Figure 6). (Figure 5). There were no signiﬁcant diﬀerences in 8PSII and ETR between N treatment (Figure 6). Unexpectedly, the allocation of N to nitrate and the remaining N components were also higher under low N. The proportion of N in amino acid was lower 22% under LN treatment. The percentage of N allocated to cell walls was similar between N treatments. Unexpectedly, the allocation of N to nitrate and the remaining N components were also higher under low N. The proportion of N in amino acid was lower 22% under LN treatment. The percentage of N allocated to cell walls was similar between N treatments. Effect of N Supply on Chlorophyll (Chl) and Chlorophyll Fluorescence The contents of Chl, Chl a and Chl b per unit leaf area were reduced by LN treatment (Figure 5). Chl content decreased by 47% in LN leaves than in HN leaves; conversely, the Chl a/b ratio was signiﬁcantly higher in LN leaves than in HN leaves TABLE 2 \| Effect of nitrogen (N) supply on grain yield, N accumulation at maturity and N utilization efﬁciency (NUtE). N treatments Grain yield (kg ha−1) N accumulation in maturity (g plant−1) NUtE [g grain (gN)−1] HN 9827a 4.49b 36.8b LN 6710b 1.94a 57.9a Data are the means for the 2 years. Numbers followed by different letters indicate signiﬁcant differences (P < 0.05). TABLE 2 \| Effect of nitrogen (N) supply on grain yield, N accumulation at maturity and N utilization efﬁciency (NUtE). Data are the means for the 2 years. Numbers followed by different letters indicate signiﬁcant differences (P < 0.05). TABLE 3 \| Effect of nitrogen (N) supply on net photosynthetic rate (Pn), speciﬁc leaf N (SLN) and photosynthetic N use efﬁciency (PNUE) in maize ear-leaves during the grain-ﬁlling stage. N treatments Pn µmol CO2 m−2s−1 SLN g m−2 PNUE µmol CO2 g N−2s−1 HN 31.6a 2.48a 12.98c LN 30.5a 1.55c 19.69a Pn was measured using a light intensity of 1600 µmol m−2 s−1. Measurements were made on ear-leaves between 09:00 and 12:00. Data are the means for the 2 years. Numbers followed by different letters indicate signiﬁcant differences (P < 0.05). TABLE 4 \| Effect of nitrogen (N) supply on the contents of N compounds in maize ear-leaves during the grain-ﬁlling stage. N treatment Parameter HN LN Nitrate mg m−2 22b 26a Soluble protein mg m−2 885a 447b PEPC mg m−2 85a 39b PPDK mg m−2 68a 33b Rubisco mg m−2 355a 170b NTH mg m−2 775a 452b Nb mg m−2 107a 101a Nh mg m−2 668a 351b Ncw mg m−2 243a 158b Na mg m−2 112a 55b TABLE 3 \| Effect of nitrogen (N) supply on net photosynthetic rate (Pn), speciﬁc leaf N (SLN) and photosynthetic N use efﬁciency (PNUE) in maize ear-leaves during the grain-ﬁlling stage. N treatments Pn µmol CO2 m−2s−1 SLN g m−2 PNUE µmol CO2 g N−2s−1 HN 31.6a 2.48a 12.98c LN 30.5a 1.55c 19.69a Pn was measured using a light intensity of 1600 µmol m−2 s−1. Measurements were made on ear-leaves between 09:00 and 12:00. Data are the means for the 2 years. Effect of N Supply on Chlorophyll (Chl) and Chlorophyll Fluorescence Numbers followed by different letters indicate signiﬁcant differences (P < 0.05). Soluble protein included PEPC, PPDK, and Rubisco. Abbreviations were listed in Table 1. May 2016 \| Volume 7 \| Article 699 Frontiers in Plant Science \| www.frontiersin.org 5 Within-Leaf Nitrogen Allocation Mu et al. that the proportions of Rubisco, PEPC, and PPDK in soluble protein were, respectively, about 35, 8, and 6% in leaves of maize seedlings grown under optimal conditions. At near-optimal growth conditions in our study, Rubisco, PEPC, and PPDK constituted approximately 40, 10, and 8% of soluble protein and 14.5, 3.5, and 2.8% of total leaf N, respectively (Table 4; Figure 3). Under LN treatment, the contents of Rubisco, PEPC, and PPDK per unit leaf area were reduced by 52, 54, and 51%, respectively (Table 4; Figure 4). In addition, their corresponding proportions of total leaf N declined by 24, 28, and 23% under low N condition (Figure 3). These responses are not consistent with what found in the developed young leaves at seedling stage. In these leaves, low N availability preferentially reduced PEPC, followed by PPDK, with less eﬀect on Rubisco. As a result, the proportion of Rubisco to PEPC and PPDK increases concomitantly (Sugiyama et al., 1984; Sugiharto et al., 1990). The reason for the inconsistency may be that, in young leaves at seedling stage, the nitrogen- dependent changes in the amount of Rubisco, PEPC, and PPDK in plants is mainly due to changes in the rates of protein synthesis (Sugiharto et al., 1990). While in the senescing leaves such as the ear-leaf in grain ﬁlling phase in the present study, change of these proteins should be mainly due to the breakdown of these proteins (Feller et al., 2008; Masclaux-Daubresse et al., 2010). In fact, in the old leaves of maize seedlings, N starvation reduced Rubisco, PEPC, and PPDK to a similar extent (Sugiharto et al., 1990). The above data suggest that, in N-deﬁcient functional leaves at seedling stage, maize maintains photosynthesis system probably by downregulating CO2-trapping mediated by PEPC and PPDK but maintaining CO2 conversion mediated by Rubisco. This is supported by Usuda (1984) who found that Rubisco but not PEPC may be rate-limiting factors for photosynthesis in maize seedling. Similarly, Usuda et al. (1984) ﬁnd that, among ten C4 plants, photosynthetic rate was strongly correlated to Rubisco but not PEPC activity. Effect of N Supply on Chlorophyll (Chl) and Chlorophyll Fluorescence (1984) found FIGURE 1 \| Effect of nitrogen (N) supply on the percentages of NTH/speciﬁc leaf N (SLN), Nh/NTH and Nb/NTH of ear-leaf in maize. Bars denote the SE of the mean. ns, not signiﬁcant (P > 0.05); ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, respectively. HN corresponds to a N application of 180 kg ha−1; LN indicates that no N was applied. FIGURE 1 \| Effect of nitrogen (N) supply on the percentages of NTH/speciﬁc leaf N (SLN), Nh/NTH and Nb/NTH of ear-leaf in maize. Bars denote the SE of the mean. ns, not signiﬁcant (P > 0.05); ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, respectively. HN corresponds to a N application of 180 kg ha−1; LN indicates that no N was applied. the amount of Rubisco, PEPC, and PPDK might not be the limiting factors for maintaining photosynthesis. Other factors, FIGURE 2 \| Effect of nitrogen (N) supply on the cell wall biomass fraction of ear-leaf in maize. Bars denote the SE of the mean. ns, not signiﬁcant (P > 0.05); ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, respectively. HN corresponds to a N application of 180 kg ha−1; LN indicates that no N was applied. FIGURE 1 \| Effect of nitrogen (N) supply on the percentages of NTH/speciﬁc leaf N (SLN), Nh/NTH and Nb/NTH of ear-leaf in maize. Bars denote the SE of the mean. ns, not signiﬁcant (P > 0.05); ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, respectively. HN corresponds to a N application of 180 kg ha−1; LN indicates that no N was applied. FIGURE 2 \| Effect of nitrogen (N) supply on the cell wall biomass fraction of ear-leaf in maize. Bars denote the SE of the mean. ns, not signiﬁcant (P > 0.05); ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, respectively. HN corresponds to a N application of 180 kg ha−1; LN indicates that no N was applied. FIGURE 1 \| Effect of nitrogen (N) supply on the percentages of NTH/speciﬁc leaf N (SLN), Nh/NTH and Nb/NTH of ear-leaf in maize. Bars denote the SE of the mean. ns, not signiﬁcant (P > 0.05); ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, respectively. HN corresponds to a N application of 180 kg ha−1; LN indicates that no N was applied. Effect of N Supply on Chlorophyll (Chl) and Chlorophyll Fluorescence While in the mature leaves in which a large amount of soluble proteins (including Rubisco, PEPC, and PPDK) accumulate (Table 4, Sugiharto et al., 1990; Lawlor, 2002), the amount of Rubisco, PEPC, and PPDK might not be the limiting factors for maintaining photosynthesis. Other factors, thylakoids in their studied maize plants. There are two types of thylakoid N, namely that associated with bioenergetics such as the electron transport chain and photophosphorylation, and N involved in the light-harvesting complex. Further analysis indicated that the distribution of N between these two thylakoid categories diﬀered between N treatments. In particular, relatively more N of thylakoid was allocated into biogenetics than into light harvesting under LN compared with the HN treatment (Figures 1 and 4). In fact, absolute N content devoted to bioenergetics was similar under the LN and HN treatment (Table 4). This proved that leaf prioritization for stabilization of electron transfer photophosphorylation under low-N stress and thus maximization of quantum yield (Khamis et al., 1990; Lu and Zhang, 2000; Lu et al., 2001; Tóth et al., 2002; Antal et al., 2010). This is supported by the ﬁnding that 8PSII and ETR were not diﬀerent between N treatments (Figure 6). On the other hand, a reduction in the allocation of N to the light-harvesting complex should help control excess electron production. In C3 plants, Hikosaka and Terashima (1995) used models to calculate the nitrogen allocation among photosynthetic components at diﬀerent incident photon ﬂux density and N levels. They found that the relative amount of N in Rubisco decreases with decreases in leaf N, while that in electron transport components, coupling factor and Calvin cycle enzymes increases (apart from Rubisco, PSI, and PSII). And the relative amount of N partitioned into Chl-protein complexes (PSI, PSII, and LHCII) remained almost constant with decreasing leaf N. Low N supply caused a relative reduction in both soluble protein per unit leaf-N content (from 2.23 to 1.80 g g−1 N) and the percentage of total soluble protein-N in total leaf N (from 36% to 30%; Figure 3). This latter value is in good agreement with the results of Makino et al. (2003), who reported that 33% of leaf N was allocated to soluble protein. Among soluble proteins, Rubisco, PEPC, and PPDK are key enzymes involved in C4 photosynthesis. Sugiyama et al. Effect of N Supply on Chlorophyll (Chl) and Chlorophyll Fluorescence FIGURE 2 \| Effect of nitrogen (N) supply on the cell wall biomass fraction of ear-leaf in maize. Bars denote the SE of the mean. ns, not signiﬁcant (P > 0.05); ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, respectively. HN corresponds to a N application of 180 kg ha−1; LN indicates that no N was applied. FIGURE 1 \| Effect of nitrogen (N) supply on the percentages of NTH/speciﬁc leaf N (SLN), Nh/NTH and Nb/NTH of ear-leaf in maize. Bars denote the SE of the mean. ns, not signiﬁcant (P > 0.05); ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, respectively. HN corresponds to a N application of 180 kg ha−1; LN indicates that no N was applied. May 2016 \| Volume 7 \| Article 699 Frontiers in Plant Science \| www.frontiersin.org 6 Within-Leaf Nitrogen Allocation Mu et al. such as electron transfer and quantum yield as suggested above might become essential for CO2 assimilation. conversion, carbon capture and conversion, possibly by fast developing genetic engineering technologies (Ort et al., 2015). During grain ﬁlling phase in ﬁeld grown maize, N is continuously remobilized from the leaves and transported to grains for protein synthesis. In such a situation, the current results suggest that suﬃcient N allocation into bioenergetics is essential to maintain leaf photosynthesis with reducing SLN. That means, under normal N supplies, the amount of Rubisco, PEPC, and PPDK might be beyond what is required for achieving the potential Pn (Sugiyama et al., 1984; Tazoe et al., 2005; Uribelarrea et al., 2009). Therefore, if grain protein is not a concern, N partitioning into Chl, light-harvesting proteins, and soluble protein (including Rubisco, PEPC, and PPDK) etc., can be substantially reduced to improve N utilization eﬃciency. On the other hand, the current data also imply that, to further increase the potential of leaf photosynthesis at optimal growth condition, genetic modiﬁcation of Rubisco, PEPC, and PPDK should focus more on their activity, but not their synthesis. For example, Uribelarrea et al. (2009) found a positive linear correlation between Rubisco initial activity and net photosynthesis in maize at grain ﬁlling stage. Ghannoum et al. (2005) concluded that superior N-use eﬃciency of NADP- ME relative to NAD-ME grasses is related to faster Rubisco turn over. Further studies are required to verify these hypotheses. g Yin et al. Effect of N Supply on Chlorophyll (Chl) and Chlorophyll Fluorescence (2011) found that maize plants grown under low N had lower bundle-sheath conductance (gbs) for CO2, which contributed to their lower CO2 leakage and photorespiration. The lower gbs value under low N was presumably associated with an increase in wall thickness of the bundle-sheath cells (Leegood, 2002; Yin et al., 2011). In the present study, the fraction of cell wall biomass was increased by low-N stress, probably because of enhanced carbohydrate accumulation in leaves and allocation to cell walls (Schlüter et al., 2012). Although Ncw decreased under LN treatment, the percentage of Ncw in total leaf N remained unchanged (Table 4; Figure 3). This result suggests that N allocation to cell walls is preserved to maintain normal cell wall properties under LN conditions, thus possibly contributing to a decrease in gbs and maintenance of Pn in bundle sheath cells to the greatest extent possible. Leaf proteins and cell walls together constituted 72 and 78% of leaf N in LN and HN-grown plants, respectively (Figure 3). The remaining N would have included nucleic acids and defensive compounds such as alkaloids, cyanogenic glycosides, and nicotine (Onoda et al., 2004; Takashima et al., 2004). Nucleic acids account for approximately 5–15% of leaf N (Chapin et al., 1986; Chapin, 1989; Evans, 1989b), while defensive compounds constitute up to about 5% (Höft et al., 1996; Burns et al., 2002). N investment into these N components was maintained under low- N conditions (Figure 3), suggesting these components are crucial for normal physiological activity under low-N stress. CONCLUSION HN corresponds to a N application of 180 kg ha−1; LN indicates that no N was applied. Mu et al. Mu et al. Within-Leaf Nitrogen Allocation FIGURE 4 \| The change of N contents in photosynthetic apparatus caused by LN treatment. (A) The percentage together with the arrows indicate the reduction of N in different photosynthetic apparatus under LN compared to HN treatment. (B) The allocation of N between Nh and Nb within the thylakoid under LN d HN Abb i i li d i T bl 1 FIGURE 4 \| The change of N contents in photosynthetic apparatus caused by LN treatment. (A) The percentage together with the arrows indicate the reduction of N in different photosynthetic apparatus under LN compared to HN treatment. (B) The allocation of N between Nh and Nb within the thylakoid under LN and HN treatment. Abbreviations were listed in Table 1. FIGURE 4 \| The change of N contents in photosynthetic apparatus caused by LN treatment. (A) The percentage together with the arrows indicate the reduction of N in different photosynthetic apparatus under LN compared to HN treatment. (B) The allocation of N between Nh and Nb within the thylakoid under LN and HN treatment. Abbreviations were listed in Table 1. FIGURE 6 \| Effect of nitrogen (N) supply on the actual quantum yield of PSII photochemistry (8PSII) and electron transport rate (ETR) of ear-leaves in maize. Bars denote the SE of the mean. ns, not signiﬁcant (P > 0.05); ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, respectively. HN corresponds to a N application of 180 kg ha−1; LN indicates that no N was applied. FIGURE 6 \| Effect of nitrogen (N) supply on the actual quantum yield of PSII photochemistry (8PSII) and electron transport rate (ETR) of ear-leaves in maize. Bars denote the SE of the mean. ns, not signiﬁcant (P > 0.05); ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, respectively. HN corresponds to a N application of 180 kg ha−1; LN indicates that no N was applied. FIGURE 5 \| Effect of nitrogen (N) supply on the content of chlorophyll (Chl), Chl a, Chl b, and Chl a/b ratio of ear-leaf in maize. Bars denote the SE of the mean. ns, not signiﬁcant (P > 0.05); ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, respectively. CONCLUSION Optimization of N allocation within maize leaves may be an adaptive mechanism to maximize N utilization for photosynthesis, thus increasing grain yield per unit of plant N during the grain-ﬁlling stage under a low-N environment. Although ear-leaf SLN was reduced by 38% under LN treatment, Pn was hardly aﬀected and PNUE was increased by 54%. Under low-N stress, maize plants tended to invest relatively more N into bioenergetics to sustain electron transport. In Yield increase during Green Revolution is largely driven by the increases of the portion of biomass partition into grains. Most recently, it is supposed that redesigning photosynthesis is essential to sustainably meet global food and bioenergy demand, that is, increase photosynthesis with less input of land, water, nutrients etc. (Evans, 2013; Ort et al., 2015). New models are proposed to increase the eﬃciency of light capture, light energy FIGURE 3 \| Effect of nitrogen (N) supply on the percentage of different N components in total ear-leaf N. Exploded slices were signiﬁcantly different between high and low N supply at P < 0.05. FIGURE 3 \| Effect of nitrogen (N) supply on the percentage of different N components in total ear-leaf N. Exploded slices were signiﬁcantly different between high and low N supply at P < 0.05. May 2016 \| Volume 7 \| Article 699 Frontiers in Plant Science \| www.frontiersin.org 7 Mu et al. Within-Leaf Nitrogen Allocation FIGURE 4 \| The change of N contents in photosynthetic apparatus caused by LN treatment. (A) The percentage together with the arrows indicate the reduction of N in different photosynthetic apparatus under LN compared to HN treatment. (B) The allocation of N between Nh and Nb within the thylakoid under LN and HN treatment. Abbreviations were listed in Table 1. FIGURE 5 \| Effect of nitrogen (N) supply on the content of chlorophyll (Chl), Chl a, Chl b, and Chl a/b ratio of ear-leaf in maize. Bars denote the SE of the mean. ns, not signiﬁcant (P > 0.05); ∗P < 0.05, ∗∗P < 0.01, FIGURE 6 \| Effect of nitrogen (N) supply on the actual quantum yield of PSII photochemistry (8PSII) and electron transport rate (ETR) of ear-leaves in maize. Bars denote the SE of the mean. ns, not signiﬁcant (P > 0.05); ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, respectively. REFERENCES 53, 789–799. doi: 10.1093/jexbot/53.370.789 Chapin, F. S. III. (1989). The cost of tundra plant structures: evaluation of concepts and currencies. Am. Nat. 133, 1–19. doi: 10.1086/284898 Ghannoum, O., Evans, J. R., Chow, W. S., Andrews, T. J., Conroy, J. P., and Von Caemmerer, S. (2005). Faster Rubisco is the key to superior nitrogen-use eﬃciency in NADP-malic enzyme relative to NAD-malic enzyme C4 grasses. Plant Physiol. 137, 638–650. doi: 10.1104/pp.104.054759 and currencies. Am. Nat. 133, 1–19. doi: 10.1086/284898 Chapin, F. S. III Shaver, G. R., and Kedrowski, R. A. (1986). Environmental controls over carbon, nitrogen and phosphorus fractions in Eriophorum vaginatum in Alaskan tussock tundra. J. Ecol. 74, 167–195. Chen, X., Chen, F., Chen, Y., Gao, Q., Yang, X., Yuan, L., et al. (2013). Modern maize hybrids in Northeast China exhibit increased yield potential and resource use eﬃciency despite adverse climate change. Global Change Biol. 19, 923–936. doi: 10.1111/gcb.12093 Grassi, G., Colom, M. R., and Minotta, G. (2001). Eﬀects of nutrient supply on photosynthetic acclimation and photoinhibition of one-year-old foliage of Picea abies. Physiol. Plant. 111, 245–254. doi: 10.1034/j.1399-3054.2001.1110217.x Hawkesford, M. J. (2012). Improving Nutrient use Eﬃciency in Crops. New York City, NY: Wiely. doi: 10.1002/9780470015902.a0023734 Chen, Y., Xiao, C., Chen, X., Li, Q., Zhang, J., Chen, F., et al. (2014). Characterization of the plant traits contributed to high grain yield and high grain nitrogen concentration in maize. Field Crops Res. 159, 1–9. doi: 10.1016/j.fcr.2014.01.002 Hikosaka, K. (2014). Optimal nitrogen distribution within a leaf canopy under direct and diﬀuse light. Plant Cell Environ. 37, 2077–2085. doi: 10.1111/pce.12291 Ding, L., Wang, K. J., Jiang, G. M., Biswas, D. K., Xu, H., Li, L. F., et al. (2005a). Eﬀects of nitrogen deﬁciency on photosynthetic traits of maize hybrids released in diﬀerent years. Ann. Bot. 96, 925–930. doi: 10.1093/aob/mci244 Hikosaka, K., and Terashima, I. (1995). A model of the acclimation of photosynthesis in the leaves of C3 plants to sun and shade with respect to nitrogen use. Plant Cell Environ. 18, 605–618. doi: 10.1111/j.1365- 3040.1995.tb00562.x Ding, L., Wang, K. J., Jiang, G. M., Liu, M. Z., Niu, S. L., and Gao, L. M. (2005b). Post-anthesis changes in photosynthetic traits of maize hybrids released in diﬀerent years. Field Crops Res. 93, 108–115. doi: 10.1016/j.fcr.2004. 09.008 Hirel, B., Le Gouis, J., Ney, B., and Gallais, A. (2007). REFERENCES Dwyer, L. M., and Stewart, D. W. (1986). Eﬀect of leaf age and position on net photosynthetic rates in maize (Zea mays L.). Agric. For. Meteorol. 37, 29–46. doi: 10.1016/0168-1923(86)90026-2 Antal, T., Mattila, H., Hakala-Yatkin, M., Tyystjärvi, T., and Tyystjärvi, E. (2010). Acclimation of photosynthesis to nitrogen deﬁciency in Phaseolus vulgaris. Planta 232, 887–898. doi: 10.1007/s00425-010-1227-5 Echarte, L., Rothstein, S., and Tollenaar, M. (2008). The response of leaf photosynthesis and dry matter accumulation to nitrogen supply in an older and a newer maize hybrid. Crop Sci. 48, 656–665. doi: 10.2135/cropsci2007.06.0366 Antonietta, M., Fanello, D. D., Acciaresi, H. A., and Guiamet, J. J. (2014). Senescence and yield responses to plant density in stay green and earlier- senescing maize hybrids from Argentina. Field Crops Res. 155, 111–119. doi: 10.1016/j.fcr.2013.09.016 Evans, J. R. (1989a). Photosynthesis and nitrogen relationships in leaves of C3 plants. Oecologia 78, 9–19. doi: 10.1007/BF00377192 Evans, J. R. (1989b). “The allocation of protein nitrogen in the photosynthetic apparatus: costs, consequences and control,” in Photosynthesis, ed. W. R. Brigs (New York. NY: Alan R. Liss, Inc.), Birch, C. J., Vos, J., and Van der Putten, P. (2003). Plant development and leaf area production in contrasting cultivars of maize grown in a cool temperate environment in the ﬁeld. Eur. J. Agron. 19, 173–188. doi: 10.1016/S1161- 0301(02)00034-5 Evans, J. R. (2013). Improving photosynthesis. Plant Physiol. 162, 1780–1793. doi: 10.1104/pp.113.219006 Brown, R. H. (1978). A diﬀerence in N use eﬃciency in C3 and C4 plants and its implications in adaptation and evolution. Crop Sci. 18, 93–98. doi: 10.2135/cropsci1978.0011183X001800010025x Feller, U., Iwona Anders, I., and Mae, T. (2008). Rubiscolytics: fate of Rubisco after its enzymatic function in a cell is terminated. J. Exp. Bot. 59, 1615–1624. doi: 10.1093/jxb/erm242 Fukayama, H., Tsuchida, H., Agarie, S., Nomura, M., Onodera, H., Ono, K., et al. (2001). Signiﬁcant accumulation of C4-speciﬁc pyruvate, orthophosphate dikinase in a C3 plant, rice. Plant Physiol. 127, 1136–1146. doi: 10.1104/pp.010641 Burns, A. E., Gleadow, R. M., and Woodrow, I. E. (2002). Light alters the allocation of nitrogen to cyanogenic glycosides in Eucalyptus cladocalyx. Oecologia 133, 288–294. doi: 10.1007/s00442-002-1055-9 Cataldo, D. A., Maroon, M., Schrader, L. E., and Youngs, V. L. (1975). Rapid colorimetric determination of nitrate in plant tissue by nitration of salicylic acid. Commun. Soil Sci. Plant Anal. 6, 71–80. doi: 10.1080/00103627509366547 Gastal, F., and Lemaire, G. (2002). N uptake and distribution in crops: an agronomical and ecophysiological perspective. J. Exp. Bot. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fpls.2016.00699 AUTHOR CONTRIBUTIONS FIGURE S1 \| Effect of nitrogen (N) supply on the amounts of phosphoenolpyruvate carboxylase (PEPC), pyruvate orthophosphate dikinase (PPDK), and the Rubisco large subunit (LSU) in maize ear-leaves as revealed by sodium dodecyl sulfate-polyacrylamide gels electrophoresis (Tazoe et al., 2005). Leaves were sampled during the grain-ﬁlling stage in maize grown under 0 and 180 kg ha−1 supplied N. Four biological replicates are shown per N treatment. FIGURE S1 \| Effect of nitrogen (N) supply on the amounts of phosphoenolpyruvate carboxylase (PEPC), pyruvate orthophosphate dikinase (PPDK), and the Rubisco large subunit (LSU) in maize ear-leaves as revealed by sodium dodecyl sulfate-polyacrylamide gels electrophoresis (Tazoe et al., 2005). Leaves were sampled during the grain-ﬁlling stage in maize grown under 0 and 180 kg ha−1 supplied N. Four biological replicates are shown per N treatment. XM collected the samples, analyzed the samples, and drafted the manuscript. QC made a contribution to acquisition and analysis of the work. FC and LY made a contribution to design of the work. GM made a contribution to design of the work, analysis and revise the manuscript. ACKNOWLEDGMENT allocated to Rubisco, PEPC, and PPDK declined 24, 28, and 23%, respectively, under LN treatment. These suggested that these proteins are present in even greater excess for photosynthesis under normal N supply. Our ﬁndings suggest that PNUE and whole-plant N utilization eﬃciency can be increased in maize by optimizing N partitioning within leaves. allocated to Rubisco, PEPC, and PPDK declined 24, 28, and 23%, respectively, under LN treatment. These suggested that these proteins are present in even greater excess for photosynthesis under normal N supply. Our ﬁndings suggest that PNUE and whole-plant N utilization eﬃciency can be increased in maize by optimizing N partitioning within leaves. We gratefully acknowledge the ﬁnancial support of the National Science Foundation of China (Nos. 31272233 and 31421092). CONCLUSION HN corresponds to a N application of 180 kg ha−1; LN indicates that no N was applied. to soluble protein were reduced by 49 and 19%, respectively, suggesting that a large portion of soluble protein served as N storage reservoirs to be later remobilized to grain for protein synthesis in HN treatment. Among the three major enzymes—Rubisco, PEPC, and PPDK —the percentage of leaf N contrast, Chl and light-harvesting proteins were reduced to control excess electron production. Under low N, total soluble protein per unit leaf area and the proportion of N allocated May 2016 \| Volume 7 \| Article 699 Frontiers in Plant Science \| www.frontiersin.org 8 Mu et al. Within-Leaf Nitrogen Allocation REFERENCES A mathematical model of the shape of corn leaves. Can. J. Plant Sci. 61, 1009–1011. doi: 10.4141/cjps 81-151 Makino, A., Sakuma, H., Sudo, E., and Mae, T. (2003). Diﬀerences between maize and rice in N-use eﬃciency for photosynthesis and protein allocation. Plant Cell Physiol. 44, 952–956. doi: 10.1093/pcp/pcg113 Makino, A., Shimada, T., Takumi, S., Kaneko, K., Matsuoka, M., Shimamoto, K., et al. (1997). Does decrease in ribulose-1, 5-bisphosphate carboxylase by antisense RbcS lead to a higher N-use eﬃciency of photosynthesis under conditions of saturating CO2 and light in rice plants? Plant Physiol. 114, 483–491. Schlüter, U., Mascher, M., Colmsee, C., Scholz, U., Bräutigam, A., Fahnenstich, H., et al. (2012). Maize source leaf adaptation to nitrogen deﬁciency aﬀects not only nitrogen and carbon metabolism but also control of phosphate homeostasis. Plant Physiol. 160, 1384–1406. doi: 10.1104/pp.112.204420 Plant Physiol. 160, 1384–1406. doi: 10.1104/pp.112.204420 Schmitt, M. R., and Edwards, G. E. (1981). Photosynthetic capacity and nitrogen use eﬃciency of maize, wheat, and rice: a comparison between C3 and C4 photosynthesis. J. Exp. Bot. 32, 459–466. doi: 10.1093/jxb/32.3.459 Masclaux-Daubresse, C., Daniel-Vedele, F., Dechorgnat, J., Chardon, F., Gauﬁchon, L., and Suzuki, A. (2010). Nitrogen uptake, assimilation and remobilization in plants: challenges for sustainable and productive agriculture. Ann. Bot. 105, 1141–1157. doi: 10.1093/aob/mcq028 Sinclair, T. R., and Horie, T. (1989). Leaf nitrogen, photosynthesis, and crop radiation use eﬃciency: a review. Crop Sci. 29, 90–98. doi: 10.2135/cropsci1989.0011183X002900010023x McCullough, D. E., Aguilera, A., and Tollenaar, M. (1994). N uptake, N partitioning, and photosynthetic N-use eﬃciency of an old and a new maize hybrid. Can. J. Plant Sci. 74, 479–484. doi: 10.4141/cjps94-088 Snir, N., and Neumann, P. M. (1997). Mineral nutrient supply, cell wall adjustment and the control of leaf growth. Plant Cell Environ. 20, 239–246. doi: 10.1046/j.1365-3040.1997.d01-57.x Mi, G., Chen, F., Wu, Q., Lai, N., Yuan, L., and Zhang, F. (2010). Ideotype root architecture for eﬃcient nitrogen acquisition by maize in intensive cropping systems. Sci. China Life Sci. 53, 1369–1373. doi: 10.1007/s11427-010-4097-y Sugiharto, B., Miyata, K., Nakamoto, H., Sasakawa, H., and Sugiyama, T. (1990). Regulation of expression of carbon-assimilating enzymes by nitrogen in maize leaf. Plant Physiol. 92, 963–969. doi: 10.1104/pp.92.4.963 Moll, R. H., Kamprath, E. J., and Jackson, W. A. (1982). Analysis and interpretation of factors which contribute to eﬃciency of nitrogen utilization. Agron. J. 74, 562–564. doi: 10.2134/agronj1982.00021962007400030037x Sugiyama, T. (1973). Puriﬁcation, molecular, and catalytic properties of pyruvate phosphate dikinase from the maize leaf. REFERENCES The challenge of improving nitrogen use eﬃciency in crop plants: towards a more central role for genetic variability and quantitative genetics within integrated approaches. J. Exp. Bot. 58, 2369–2387. doi: 10.1093/jxb/erm097 Drouet, J., and Bonhomme, R. (1999). Do variations in local leaf irradiance explain changes to leaf nitrogen within row maize canopies? Ann. Bot. 84, 61–69. doi: 10.1006/anbo.1999.0890 Höft, M., Verpoorte, R., and Beck, E. (1996). Growth and alkaloid contents in leaves of Tabernaemontana pachysiphon Stapf (Apocynaceae) as inﬂuenced May 2016 \| Volume 7 \| Article 699 Frontiers in Plant Science \| www.frontiersin.org 9 Mu et al. Within-Leaf Nitrogen Allocation by light intensity, water and nutrient supply. Oecologia 107, 160–169. doi: 10.1007/BF00327899 by light intensity, water and nutrient supply. Oecologia 107, 160–169. doi: 10.1007/BF00327899 Paponov, I. A., and Engels, C. (2003). Eﬀect of nitrogen supply on leaf traits related to photosynthesis during grain ﬁlling in two maize genotypes with diﬀerent N eﬃciency. J. Plant Nutr. Soil Sci. 166, 756–763. doi: 10.1002/jpln.200320339 Jovanovic, Z., Djakovic, T., Stikic, R., Prokic, L., and Sukalovic, V. H. (2004). Eﬀect of N deﬁciency on leaf growth and cell wall peroxidase activity in contrasting maize genotypes. Plant Soil 265, 211–223. doi: 10.1007/s11104-005-0503-9 Paponov, I. A., Sambo, P., Presterl, T., Geiger, H. H., and Engels, C. (2005). Grain yield and kernel weight of two maize genotypes diﬀering in nitrogen use eﬃciency at various levels of nitrogen and carbohydrate availability during ﬂowering and grain ﬁlling. Plant Soil 272, 111–123. doi: 10.1007/s11104-004- 4211-7 KavanovA, M., Lattanzi, F. A., and Schnyder, H. (2008). Nitrogen deﬁciency inhibits leaf blade growth in Lolium perenne by increasing cell cycle duration and decreasing mitotic and post-mitotic growth rates. Plant Cell Environ. 31, 727–737. doi: 10.1111/j.1365-3040.2008.01787.x Peng, L., Ma, J., Chi, W., Guo, J., Zhu, S., Lu, Q., et al. (2006). Low PSII accumulation1 is involved in eﬃcient assembly of photosystem II in Arabidopsis thaliana. Plant Cell 18, 955–969. doi: 10.1105/tpc.105.037689 Khamis, S., Lamaze, T., Lemoine, Y., and Foyer, C. (1990). Adaptation of the photosynthetic apparatus in maize leaves as a result of nitrogen limitation relationships between electron transport and carbon assimilation. Plant Physiol. 94, 1436–1443. doi: 10.1104/pp.94.3.1436 Pons, T. L., and Westbeek, M. H. (2004). Analysis of diﬀerences in photosynthetic nitrogen-use eﬃciency between four contrasting species. Physiol. Plant. 122, 68–78. Kosgey, J. R., Moot, D. J., Fletcher, A. L., and McKenzie, B. A. (2013). REFERENCES Dry matter accumulation and post-silking N economy of ‘stay-green’maize (Zea mays L.) hybrids. Eur. J. Agron. 51, 43–52. doi: 10.1016/j.eja.2013.07.001 Poorter, H., and Evans, J. R. (1998). Photosynthetic nitrogen-use eﬃciency of species that diﬀer inherently in speciﬁc leaf area. Oecologia 116, 26–37. doi: 10.1007/s004420050560 Lawlor, D. W. (2002). Carbon and nitrogen assimilation in relation to yield: mechanisms are the key to understanding production systems. J. Exp. Bot. 53, 773–787. doi: 10.1093/jexbot/53.370.773 Porra, R. J., Thompson, W. A., and Kriedemann, P. E. (1989). Determination of accurate extinction coeﬃcients and simultaneous equations for assaying chlorophylls a and b extracted with four diﬀerent solvents: veriﬁcation of the concentration of chlorophyll standards by atomic absorption spectroscopy. Biochim. Biophys. Acta 975, 384–394. doi: 10.1016/S0005-2728(89)80347-0 Leegood, R. C. (2002). C4 photosynthesis: principles of CO2 concentration and prospects for its introduction into C3 plants. J. Exp. Bot. 53, 581–590. doi: 10.1093/jexbot/53.369.581 Lu, C., and Zhang, J. (2000). Photosynthetic CO2 assimilation, chlorophyll ﬂuorescence and photoinhibition as aﬀected by nitrogen deﬁciency in maize plants. Plant Sci. 151, 135–143. doi: 10.1016/S0168-9452(99)00207-1 Radin, J. W., and Boyer, J. S. (1982). Control of leaf expansion by nitrogen nutrition in sunﬂower plants role of hydraulic conductivity and turgor. Plant Physiol. 69, 771–775. doi: 10.1104/pp.69.4.771 Ramalho, J. C., Pons, T. L., Groeneveld, H. W., and Nunes, M. A. (1997). Photosynthetic responses of Coﬀea arabica leaves to a short-term high light exposure in relation to N availability. Physiol. Plant. 101, 229–239. doi: 10.1111/j.1399-3054.1997.tb01841.x Lu, C., Zhang, J., Zhang, Q., Li, L., and Kuang, T. (2001). Modiﬁcation of photosystem II photochemistry in nitrogen deﬁcient maize and wheat plants. J. Plant Physiol. 158, 1423–1430. doi: 10.1078/0176-1617-00501 Lynch, J. P. (2013). Steep, cheap and deep: an ideotype to optimize water and N acquisition by maize root systems. Ann. Bot. 112, 347–357. doi: 10.1093/aob/mcs293 Sage, R. F., and Pearcy, R. W. (1987). The nitrogen use eﬃciency of C3 and C4 plants II. Leaf nitrogen eﬀects on the gas exchange characteristics of Chenopodium album (L.) and Amaranthus retroﬂexus (L.). Plant Physiol. 84, 959–963. doi: 10.1104/pp.84.3.954 Makino, A., Mae, T., and Ohira, K. (1986). Colorimetric measurement of protein stained with Coomassie Brilliant Blue R on sodium dodecyl sulfate- polyacrylamide gel electrophoresis by eluting with formamide. Agric. Biol. Chem. 50, 1911–1912. doi: 10.1271/bbb1961.50.1911 Sage, R. F., and Pearcy, R. W. (2000). The Physiological Ecology of C4 Photosynthesis. Berlin: Springer, 497–532. Sanderson, J. B., Daynard, T. B., and Tollenaar, M. (1981). REFERENCES Biochemistry 12, 2862–2868. doi: 10.1021/bi00739a014 Moore, S. (1968). Amino acid analysis: aqueous dimethyl sulfoxide as solvent for the ninhydrin reaction. J. Biol. Chem. 243, 6281–6283. Sugiyama, T., Mizuno, M., and Hayashi, M. (1984). Partitioning of nitrogen among ribulose-1, 5-bisphosphate carboxylase/oxygenase, phosphoenolpyruvate carboxylase, and pyruvate orthophosphate dikinase as related to biomass productivity in maize seedlings. Plant Physiol. 75, 665–669. doi: 10.1104/pp.75.3.665 Muchow, R. C., and Sinclair, T. R. (1994). Nitrogen response of leaf photosynthesis and canopy radiation use eﬃciency in ﬁeld-grown maize and sorghum. Crop Sci. 34, 721–727. doi: 10.2135/cropsci1994.0011183X003400030022x Onoda, Y., Hikosaka, K., and Hirose, T. (2004). Allocation of nitrogen to cell walls decreases photosynthetic nitrogen-use eﬃciency. Funct. Ecol. 18, 419–425. doi: 10.1111/j.0269-8463.2004.00847.x pp Takashima, T., Hikosaka, K., and Hirose, T. (2004). Photosynthesis or persistence: nitrogen allocation in leaves of evergreen and deciduous Quercus species. Plant Cell Environ. 27, 1047–1054. doi: 10.1111/j.1365-3040.2004. 01209.x Ort, D. R., Merchant, S. S., Alric, J., Barkan, A., Blankenship, R. E., Bock, R., et al. (2015). Redesigning photosynthesis to sustainably meet global food and bioenergy demand. Proc. Natl. Acad. Sci. U.S.A. 112, 8529–8536. doi: 10.1073/pnas.1424031112 Tazoe, Y., Noguchi, K. O., and Terashima, I. (2005). Eﬀects of growth light and nitrogen nutrition on the organization of the photosynthetic apparatus in leaves May 2016 \| Volume 7 \| Article 699 Frontiers in Plant Science \| www.frontiersin.org 10 Mu et al. Within-Leaf Nitrogen Allocation of a C4 plant, Amaranthus cruentus. Plant Cell Environ. 29, 691–700. doi: 10.1111/j.1365-3040.2005.01453.x of a C4 plant, Amaranthus cruentus. Plant Cell Environ. 29, 691–700. doi: 10.1111/j.1365-3040.2005.01453.x in maize (Zea mays L.). Field Crops Res. 93, 64–73. doi: 10.1016/j.fcr.2004. 09.013 j Tollenaar, M., and Lee, E. A. (2006). Dissection of physiological processes underlying grain yield in maize by examining genetic improvement and heterosis. MAYDICA 51, 399. Wang, Y., Mi, G., Chen, F., Zhang, J., and Zhang, F. (2005). Response of root morphology to nitrate supply and its contribution to nitrogen accumulation in maize. J. Plant Nutri. 27, 2189–2202. doi: 10.1081/PLN-2000 34683 Tóth, V. R., Mészáros, I., Veres, S., and Nagy, J. (2002). Eﬀects of the available nitrogen on the photosynthetic activity and xanthophyll cycle pool of maize in ﬁeld. J. Plant Physiol. 159, 627–634. doi: 10.1078/0176-1617-0640 Yin, X., Sun, Z., Struik, P. C., and Gu, J. (2011). Evaluating a new method to estimate the rate of leaf respiration in the light by analysis of combined gas exchange and chlorophyll ﬂuorescence measurements. J. Exp. Bot. Frontiers in Plant Science \| www.frontiersin.org REFERENCES 62, 3489–3499. doi: 10.1093/jxb/err038 Uedan, K., and Sugiyama, T. (1976). Puriﬁcation and characterization of phosphoenolpyruvate carboxylase from maize leaves. Plant Physiol. 57, 906– 910. doi: 10.1104/pp.57.6.906 Zhang, L., Paakkarinen, V., van Wijk, K. J., and Aro, E. (1999). Co-translational assembly of the D1 protein into photosystem II. J. Biol. Chem. 274, 16062– 16067. doi: 10.1074/jbc.274.23.16062 Uribelarrea, M., Crafts-Brandner, S. J., and Below, F. E. (2009). Physiological N response of ﬁeld-grown maize hybrids (Zea mays L.) with divergent yield potential and grain protein concentration. Plant Soil 316, 151–160. doi: 10.1007/s11104-008-9767-1 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. Usuda, H. (1984). Variations in the photosynthesis rate and activity of photosynthetic enzymes in maize leaf tissue of diﬀerent ages. Plant Cell Physiol. 25, 1297–1301. Usuda, H., Ku, M. S. B., and Edwards, G. E. (1984). Rates of photosynthesis relative to activity of photosynthetic enzymes, chlorophyll and soluble protein content among ten C4 species. Austral. J. Plant Physiol. 11, 509–517. doi: 10.1071/PP9840509 Copyright © 2016 Mu, Chen, Chen, Yuan and Mi. 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. Vos, J., Van Der Putten, P., and Birch, C. J. (2005). Eﬀect of nitrogen supply on leaf appearance, leaf growth, leaf nitrogen economy and photosynthetic capacity May 2016 \| Volume 7 \| Article 699 Frontiers in Plant Science \| www.frontiersin.org 11
https://openalex.org/W3200822761	https://europepmc.org/articles/pmc8471309?pdf=render	English	null	Electrospinning Mechanism of Nanofiber Yarn and Its Multiscale Wrapping Yarn	Polymers	2,021	cc-by	17,308	Article Electrospinning Mechanism of Nanoﬁber Yarn and Its Multiscale Wrapping Yarn Yajing Shi 1, Huimin Zhuang 1, Yu Lin 1, Dongdong Lu 2, Shengbin Cao 3 and Lvtao Zhu 4 Taohai Yan 1,, Yajing Shi 1, Huimin Zhuang 1, Yu Lin 1, Dongdong Lu 2, Shengbin Cao 3 an 1 Fujian Key Laboratory of Novel Functional Textile Fibers and Materials, Minjiang University, Fuzhou 350108, China; 2146@mju.edu.cn (Y.S.); 2563@mju.edu.cn (H.Z.); 2502@mju.edu.cn (Y.L.) 2 Key Lab for Sport Shoes Upper Materials of Fujian Province Fujian Huafeng New Material Co Ltd 1 Fujian Key Laboratory of Novel Functional Textile Fibers and Materials, Minjiang University, F h 350108 Chi 2146@ j d (YS ) 2563@ j d (H Z ) 2502@ j d (YL ) 1 Fujian Key Laboratory of Novel Functional Textile Fibers and Materials, Minjiang University, Fuzhou 350108, China; 2146@mju.edu.cn (Y.S.); 2563@mju.edu.cn (H.Z.); 2502@mju.edu.cn (Y.L.) 2 Key Lab for Sport Shoes Upper Materials of Fujian Province, Fujian Huafeng New Material Co., Ltd. Putian 351199, China; dongdong.lu@huafeng-cn.com j y y j g y Fuzhou 350108, China; 2146@mju.edu.cn (Y.S.); 2563@mju.edu.cn (H.Z.); 2502@mju.edu.cn (Y.L.) 2 Key Lab for Sport Shoes Upper Materials of Fujian Province, Fujian Huafeng New Material Co., Ltd., Putian 351199, China; dongdong.lu@huafeng-cn.com j ( ) j ( ) j ( ) 2 Key Lab for Sport Shoes Upper Materials of Fujian Province, Fujian Huafeng New Material Co., Ltd Putian 351199, China; dongdong.lu@huafeng-cn.com 3 School of Materials Science, Shanghai Dianji University, Shanghai 200003, China; caosb@sdju.edu.cn 4 College of Textile Science and Engineering, Zhejiang Sci-Tech University, Hangzhou 310018, China; zhult@zstu.edu.cn C d h @ j d T l 86 0591 8376 0411 3 School of Materials Science, Shanghai Dianji University, Shanghai 200003, China; caosb@sdju.edu.cn 4 College of Textile Science and Engineering, Zhejiang Sci-Tech University, Hangzhou 310018, China; zhult@zstu.edu.cn School of Materials Science, Shanghai Dianji University, Shanghai 200003, China; caosb@sdju.edu.cn 4 College of Textile Science and Engineering, Zhejiang Sci-Tech University, Hangzhou 310018, China; zhult@zstu.edu.cn * Correspondence: thyan@mju.edu.cn; Tel.: +86-0591-8376-0411 * Correspondence: thyan@mju.edu.cn; Tel.: +86-0591-8376-0411 Abstract: To analyze the feasibility of electrospinning nanoﬁber yarn using a wrapping yarn forming device, electrospun nanoﬁber-wrapped yarns and multiscale yarns were prepared by self-made equipment. The relationship between the surface morphology and properties of yarn and its prepa- ration process was studied. The process parameters were adjusted, and it was found that some nanoﬁbers formed Z-twisted yarns, while others showed exposed cores. Citation: Yan, T.; Shi, Y.; Zhuang, H.; Lin, Y.; Lu, D.; Cao, S.; Zhu, L. Electrospinning Mechanism of Nanoﬁber Yarn and Its Multiscale Wrapping Yarn. Polymers 2021, 13, 3189. https://doi.org/10.3390/ polym13183189 Keywords: electrospinning; nanoﬁbers yarn; multiscale yarn; mechanical properties; mechanism Academic Editor: Andrea Ehrmann 1. Background Electrospun nanoﬁber materials have shown great application prospects in the ﬁeld of functional textiles due to their ultra-ﬁne ﬁber size, ﬂexible material selectivity, struc- tural controllability, and easy surface functionalization [1]; however, they generally have weak mechanical properties, and their nanoﬁber aggregates have poor structural stability, which severely restricts their practical applications as functional textiles [2,3]. In recent years, how to achieve the high-efﬁciency and controllable preparation of micro-nano ﬁber assemblies with stable structures, excellent mechanical properties, and long-term func- tions has become a popular research topic [4,5]. Yarns constructed based on electrospun nanoﬁbers display the typical anisotropic structural characteristics of traditional yarns [6]. The ﬁbers are aligned and closely contacted to form ultraﬁne capillary cavities and con- tinuous channels [7]. This can overcome the inherent defects of the membrane structure nanoﬁber aggregates and has broad application prospects in biomedicine [8–10], smart wearables [11,12], sensing [13,14], functional textiles [15,16], and other ﬁelds, therefore promoting the industrial applications of electrospinning technology [17–19]. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. 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/). Article Electrospinning Mechanism of Nanoﬁber Yarn and Its Multiscale Wrapping Yarn To analyze the forming mechanism of electrospun nanoﬁber-wrapped yarn, the concept of winding displacement difference in the twisted yarn core A was introduced. The formation of nanoﬁber-wrapped structural yarns was discussed using three values of A. The starting point of each twist was the same position when A = 0 with a constant corner angle β. However, the oriented nanoﬁber broke or was pulled out from the gripping point when it was twisted, and it appeared disordered. The forming process of electrospun nanoﬁber-wrapped yarn displayed some unique phenomena, including the emission of directional nanoﬁbers during collection, ﬁber non-continuity, and twist angle non-uniformity. The conclusions of this research have theoretical and practical value to guide the industrial preparation of nanoﬁber yarns and their wrapped yarns. polymers polymers polymers 2. Introduction Fiber and yarn are the main materials used in the traditional textile industry, but only ﬁbers with a diameter larger than several microns can be processed using existing textile https://www.mdpi.com/journal/polymers Polymers 2021, 13, 3189. https://doi.org/10.3390/polym13183189 Polymers 2021, 13, 3189 2 of 20 2 of 20 processing techniques [20]. Electrospinning can prepare ﬁbers with diameters ranging from several nanometers to several micrometers; however, when the diameter reaches the nanometer level, the fracture strength and wear resistance of the ﬁbers are relatively poor, making them difﬁcult to process using traditional textile techniques [21]. According to the speciﬁc application requirements in textiles and apparel, microelectronic devices, and composite materials, it is necessary to form oriented nanoﬁber bundles and twisted yarns from them [22]. If nanoﬁbers can be used in traditional textile methods such as knitting and weaving, yarns can be prepared from them [23]. This process involves collecting nanoﬁbers into a certain orientation, followed by twisting the ﬁber bundles to improve the yarn strength and collective properties [24]. There are various methods for preparing nanoﬁber bundles with speciﬁc orientations. One is to use an auxiliary electrode method, including a spacer conductive plate collection device and a disk collection device to collect oriented nanoﬁbers between parallel grounded double disks. This method is simple and effective, and the ﬁber orientation degree is high. The other method involves using the mutual attraction of oppositely charged ﬁbers. Nega- tive and positive spinning nozzles emit ﬁbers with opposite charges to form ﬁber bundles at certain locations because of their charge attraction, which produces continuous oriented nanoﬁbers; however, these ﬁber bundles are relatively ﬁne [25]. Thirdly, continuous ori- ented nanoﬁbers can be obtained by drawing oriented nanoﬁbers with a zero-high-speed drum, but the degree of orientation is difﬁcult to control, and the ﬁbers are easily stretched and broken. There is another nanoﬁber collection method using a solution, in which ﬁbers are collected into bundles, and the ﬁbers within the bundles are oriented [26,27]. The ﬁber bundles are usually twisted mechanically or using airﬂow. The mechanical twisting method is highly controllable, simple, and reliable, and the yarn quality is good. This is the method used to produce most nanoﬁber yarns. Airﬂow twisting requires the use of professional nozzles and corresponding airﬂow twisting devices. The mechanism and process are relatively complex, and the twisting effect can change depending on the airﬂow parameters. 3.2.1. Self-Made Electrospinning Nanoﬁber Yarn Forming Device 3.2.1. Self-Made Electrospinning Nanofiber Yarn Forming Device Th l t i i fib f i d i i h 3.2.1. Self-Made Electrospinning Nanoﬁber Yarn Forming Device 3.2.1. Self-Made Electrospinning Nanofiber Yarn Forming Device Th l i i fib f i d i i h The electrospinning nanoﬁber yarn forming device is shown in Figure 1a. The spinning solution was transported to a single needle through a pipe conveying ﬂuid by an injection pump. The polymer formed nanoﬁbers under the action of a high voltage electric ﬁeld, and orientated nanoﬁbers formed between the grounding disk and the grounding ring. The rotation of the metal disk twists the oriented nanoﬁbers to form nanoﬁber yarns. The electrospinning nanoﬁber-wrapped yarn forming device is shown in Figure 1b. Figure 1b has a larger core yarn unwinding system (13) than Figure 1a, which is marked in red. The 75D/36F non-twisted polyester multiﬁlament unwound from the bobbin was used as the core yarn and passed through the center of the disk and the center of the ring, in turn. The rotating metal disk twisted the oriented nanoﬁbers and wrapped the nanoﬁbers on the core yarn. The ﬁber covered the structural yarn, and the formed covered yarn was ﬁnally wound on the ﬁnished bobbin by a winding device. The electrospinning nanofiber yarn forming device is shown in Figure 1a. The spin- ning solution was transported to a single needle through a pipe conveying fluid by an injection pump. The polymer formed nanofibers under the action of a high voltage electric field, and orientated nanofibers formed between the grounding disk and the grounding ring. The rotation of the metal disk twists the oriented nanofibers to form nanofiber yarns. The electrospinning nanofiber-wrapped yarn forming device is shown in Figure 1b. Fig- ure 1b has a larger core yarn unwinding system (13) than Figure 1a, which is marked in red. The 75D/36F non-twisted polyester multifilament unwound from the bobbin was used as the core yarn and passed through the center of the disk and the center of the ring, in turn. The rotating metal disk twisted the oriented nanofibers and wrapped the nano- fibers on the core yarn. The fiber covered the structural yarn, and the formed covered yarn was finally wound on the finished bobbin by a winding device. (a) (b) Figure 1. Schematic diagram of the forming device for electrospinning nanofiber yarn. (a),electrospinning nanofiber yarn forming device.(b),electrospinning nanofiber-wrapped yarn forming device. 2. Introduction p In contrast to electrospun pure nanoﬁber yarn, multiscale composite yarn is composed of traditional microﬁbers and electrospun nanoﬁbers according to some structure rules. A nanoﬁber-wrapped core yarn or yarn wrapped on a core yarn may be a disordered or oriented nanoﬁber, and the oriented nanoﬁber is generally wrapped on the core yarn in a twisting manner. Zhou used electrospinning to collect nanoﬁbers on parallel-aligned monoﬁlaments and then aggregated and twisted monoﬁlaments with nanoﬁbers on the sur- face [28]. The nanoﬁbers wrapped on the core yarn were disordered. Oriented nanoﬁbers are mainly twisted by rotating hollow cylinders, disks, bell mouths, water bath vortexes, and air vortexes. Liu rotated a core yarn in the axial direction and collected oriented nanoﬁbers between two parallel grounded aluminum sheets [29]. The nanoﬁbers were twisted and wound on the surface to obtain an oriented wrapped yarn. Scardino used air vortex twisting to coat the surface of a core yarn with electrospun nanoﬁbers. The prepared nanoﬁber-wrapped yarn also had a certain degree of orientation. Niu passed a core yarn through two hollow cylinders that were grounded relative to one another, and oriented electrospun nanoﬁbers were collected between them [30]. One of the cylinders was used to twist the nanoﬁbers on the core yarn, and the twisting orientation of the nanoﬁbers was more apparent. pp Multiscale yarn is made of a variety of ﬁbers with different scales, which can fully in- tegrate the structural characteristics and performance advantages of traditional microﬁbers and electrospun nanoﬁbers. This allows them to make up for each other’s disadvantages while giving full play to the advantages of the textile yarn structure [31]. The weavability of nanoﬁbers can be improved by preparing nanoﬁber yarns, and high-value-added func- tional textiles can be produced by combining the structural and functional advantages of conventional yarns and nano yarns. A composite yarn with this functional structure can be used without touching the surface of functional materials, such as fragrance ﬁnishing, energy storage, and temperature regulation. No studies have previously investigated the Polymers 2021, 13, 3189 3 of 20 nctional as fra- 3 of 20 nctional as fra- wrapping mechanism of nanoﬁber-wrapped yarns. In this paper, a self-made oriented nanoﬁber-wrapped yarn forming electrospinning device was designed. The nanoﬁbers were twisted and wrapped on the core yarn by a disk. Electric ﬁeld simulations were carried out on the device. 2. Introduction The formation mechanism of the wrapped nanoﬁber structure was studied and veriﬁed. made oriented nanofiber-wrapped yarn forming electrospinning device was designed. The nanofibers were twisted and wrapped on the core yarn by a disk. Electric field simu- lations were carried out on the device. The formation mechanism of the wrapped nano- fiber structure was studied and verified. 3 Materials and Methods 3.2.1. Self-Made Electrospinning Nanoﬁber Yarn Forming Device 3.2.1. Self-Made Electrospinning Nanofiber Yarn Forming Device Th l t i i fib f i d i i h 1—High voltage DC power, 2—Syringe, 3— Micro-injection pump, 4—Infusion tube, 5—Needle tube, 6—Motor, 7—Receiving disk, 8—Ring, 9—Nanofiber, 10— Ground electrode, 11—Winding system, 12—Core yarn, 13—Unwinding system Figure 1. Schematic diagram of the forming device for electrospinning nanoﬁber yarn. (a) electrospinning nanoﬁber yarn forming device. (b) electrospinning nanoﬁber-wrapped yarn forming device. 1—High voltage DC power, 2—Syringe, 3—Micro-injection pump, 4—Infusion tube, 5—Needle tube, 6—Motor, 7—Receiving disk, 8—Ring, 9—Nanoﬁber, 10—Ground electrode, 11—Winding system, 12—Core yarn, 13—Unwinding system. (b) ospinning nanofiber yarn (a) electrospinning na (a) S h ti di f th f i d i f l (b) (a) Figure 1. Schematic diagram of the forming device for electrospinning nanofiber yarn. (a),electrospinning nanofiber yarn forming device.(b),electrospinning nanofiber-wrapped yarn forming device. 1—High voltage DC power, 2—Syringe, 3— Micro-injection pump, 4—Infusion tube, 5—Needle tube, 6—Motor, 7—Receiving disk, 8—Ring, 9—Nanofiber, 10— Ground electrode, 11—Winding system, 12—Core yarn, 13—Unwinding system Figure 1. Schematic diagram of the forming device for electrospinning nanoﬁber yarn. (a) electrospinning nanoﬁber yarn forming device. (b) electrospinning nanoﬁber-wrapped yarn forming device. 1—High voltage DC power, 2—Syringe, 3—Micro-injection pump, 4—Infusion tube, 5—Needle tube, 6—Motor, 7—Receiving disk, 8—Ring, 9—Nanoﬁber, 10—Ground electrode, 11—Winding system, 12—Core yarn, 13—Unwinding system. Figure 1. Schematic diagram of the forming device for electrospinning nanofiber yarn. (a),electrospinning nanofiber yarn forming device.(b),electrospinning nanofiber-wrapped yarn forming device. 1—High voltage DC power, 2—Syringe, 3— Micro-injection pump, 4—Infusion tube, 5—Needle tube, 6—Motor, 7—Receiving disk, 8—Ring, 9—Nanofiber, 10— Ground electrode, 11—Winding system, 12—Core yarn, 13—Unwinding system Figure 1. Schematic diagram of the forming device for electrospinning nanoﬁber yarn. (a) electrospinning nanoﬁber yarn forming device. (b) electrospinning nanoﬁber-wrapped yarn forming device. 1—High voltage DC power, 2—Syringe, 3—Micro-injection pump, 4—Infusion tube, 5—Needle tube, 6—Motor, 7—Receiving disk, 8—Ring, 9—Nanoﬁber, 10—Ground electrode, 11—Winding system, 12—Core yarn, 13—Unwinding system. 3. Materials and Methods 3.1. Main Materials and Equ 3.1. Main Materials and Equipment This process involves collec 3.1. Main Materials and Equipment This process involves collec This process involves collecting nanoﬁbers into a certain orientation, followed by twisting the ﬁber bundles to improve the yarn’s strength and collective properties. Poly- acrylonitrile (PAN) was purchased from Taicang Kelda Plastic Materials Co., Ltd. (Taicang, China), with a molecular weight of 150,000. N, N-dimethylformamide (DMF) was pur- chased from Guangdong Jinhua Chemical Reagent Co., Ltd. (Guangzhou, China) and was analytically pure. 75D/36F twistless polyester multiﬁlament was purchased from Fujian Jinjiang Technology Co., Ltd. (Jinjiang, China). High voltage DC power DW-P503 was purchased from Dongwen High Voltage Power (Tianjin, China) Co., Ltd. An LSP-10-18 micro-injection pump was purchased from Baoding Co., Ltd. (Baoding, China). p g y twisting the fiber bundles to improve the yarn’s strength and collective properties. Poly- acrylonitrile (PAN) was purchased from Taicang Kelda Plastic Materials Co., Ltd. (Tai- cang, China), with a molecular weight of 150,000. N, N-dimethylformamide (DMF) was purchased from Guangdong Jinhua Chemical Reagent Co., Ltd.(Guangzhou, China) and was analytically pure. 75D/36F twistless polyester multifilament was purchased from Fu- jian Jinjiang Technology Co., Ltd.(Jinjiang, China). High voltage DC power DW-P503 was purchased from Dongwen High Voltage Power (Tianjin, China) Co., Ltd. An LSP-10-18 micro-injection pump was purchased from Baoding Co., Ltd.(Baoding, China) 3.2.2. Preparation of Electrospinning Solution PAN powder with a mass fraction of 12% was added to DMF solvent and stirred at a constant temperature of 40 ◦C overnight. An electrospinning nanoﬁber yarn forming device was arranged in accordance with Figure 1. The speciﬁc parameters are as follows: Polymers 2021, 13, 3189 4 of 20 the receiving disk has a 10 cm radius and 1 mm thickness, the material was stainless steel, the hole diameter of the middle opening was 1.0 cm; the distances between the plane of the needle tip and disk center were 7 cm, 8 cm, 9 cm, 10 cm, and 11 cm. The inner diameter of the needle tube was 1 mm, the outer diameter was 1.4 mm, the length of the needle tube was 13 mm; the angle between the needle tube and disk was 45◦; the center of the ring and disk were horizontal, and the distances between the two were 3 cm, 4 cm, 5 cm, 6 cm, and 7 cm. The ring was made from stainless steel, the outer ring radius was 0.5 cm, the inner ring radius was 0.3 cm, the thickness was 2 mm. The voltage was 18 kV. The ﬂow rate of the pump was 0.6 mL/h, and the disk rotation speed was 40 r/min, 60 r/min, 80 r/min, 100 r/min, and 120 r/min. The spinning time was 15 min, the ambient temperature was 25 ◦C, and the ambient humidity was 65%. 3.2.3. Analytical Test Method The surface morphology of the electrospun nanoﬁber yarn and its wrapped yarn was studied by a JSM-6390 scanning electron microscope (SEM, JEOL, Beijing, China). The voltage was 15 kV, and the magniﬁcation was 200, 300, and 5000 times. The SEM images were analyzed using Adobe Acrobat 9 Pro software to calculate the average diameter of yarns and ﬁbers. The prepared yarn was subjected to a tensile testing standard ISO 2062:1993 using an Instron 3365 electronic strength tester (Micoforce, Shanghai, China), and the average was taken from 10 repeated measurements for each sample. 4.1. Electric Field Simulation Analysis of Electrospinning Nanoﬁber Yarn Forming Device To theoretically verify the feasibility of electrospinning nanoﬁber-wrapped structures using a yarn-making device, Maxwell analysis software was used to analyze the electro- static ﬁeld to determine the scalar potential distribution of electric ﬁelds generated by charge distribution or an applied potential. Because the movement of nanoﬁbers in an electrostatic ﬁeld is very complex, we simpliﬁed the electric ﬁeld model; therefore, the electric ﬁeld distribution formed between the nozzle, the receiving ring, and the receiving disk is the main factor to consider in this simulation. The three-dimensional space model established according to the experimental device is shown in Figure 2. The high voltage power supply, bracket, platform, and other devices that have little inﬂuence on the electrostatic ﬁeld were neglected, leaving only the single- needle nozzle and the receiving ring and disk portion, which greatly inﬂuence the working electric ﬁeld. The distance between the ring and disk was set to 9 cm, the angle between the needle and the disk was 45◦, the voltage was 18 kV, and the other parameters were the same as in Section 3.2.2. The needle was located in the lower right corner of Figure 2, which was marked as a short brown line. 5 of 23 gure 2. Spatial position model. Figure 2. Spatial position model. gure 2. Spatial position model. Figure 3 shows the potential distribution nephogram of the electric field and the nee- e tip portion of the single-needle nanofiber-wrapped structure yarn forming device un- er an applied voltage of 18 kV. The different colors represent the level of the electric otential, which increases from blue to red. It can be seen that the red area is most con- ntrated near the needle tip indicating the highest potential; the blue area near the re- Figure 2. Spatial position model. Figure 3 shows the potential distribution nephogram of the electric ﬁeld and the needle tip portion of the single-needle nanoﬁber-wrapped structure yarn forming device under an applied voltage of 18 kV. The different colors represent the level of the electric potential, which increases from blue to red. It can be seen that the red area is most concentrated ure 2. Spatial position model. Figure 2. Spatial position model. Figure 3 shows the potential distribution nephogram of the electric field and the nee- e tip portion of the single-needle nanofiber-wrapped structure yarn forming device un- r an applied voltage of 18 kV. 4.1. Electric Field Simulation Analysis of Electrospinning Nanoﬁber Yarn Forming Device As shown in Figure 4, the peak electric ﬁeld intensity appears near the needle, and the electric ﬁeld intensity vectors of the needle nearly all pointed to the receiving disk and ring, in accordance with experimental phenomena. EVIEW 6 of 23 Figure 4. Electric field simulation vector diagram. 4.2. SEM Image Analysis 4.2.1. Effect of Changing the Distance from the Ring to the Disk on the Surface Morphol- ogy of the Fiber When the distance from the ring to the disk was set to hp L , the distance zp L from the needle to the center of the disk is 9 cm, and the rotation speed n of the disk was 40 r/min. The SEM images in Figure 5 a, b, and c respectively show that the nanofiber yarn was prepared by changing L from 3 cm 5 cm and 7 cm The nanofibers formed yarn Figure 4. Electric ﬁeld simulation vector diagram. 4.2. SEM Image Analysis 4.2.1. Effect of Changing the Distance from the Ring to the Disk on the Surface Morphology of the Fiber When the distance from the ring to the disk was set to Lhp, the distance Lzp from the needle to the center of the disk is 9 cm, and the rotation speed n of the disk was 40 r/min. The SEM images in Figure 5a,b, and c respectively show that the nanoﬁber yarn was prepared by changing Lhp from 3 cm, 5 cm, and 7 cm. The nanoﬁbers formed yarn twisted in the z-direction, and the average twist angle decreased as Lhp increased (Figure 6). At a constant disk rotation speed, the number of twists per unit time was constant, and the Figure 3. Potential distribution nephogram. As shown in Figure 4, the peak electric field intensity appears near the needle, and the electric field intensity vectors of the needle nearly all pointed to the receiving disk and Figure 3. Potential distribution nephogram. As shown in Figure 4, the peak electric ﬁeld intensity appears near the needle, and the electric ﬁeld intensity vectors of the needle nearly all pointed to the receiving disk and ring, in accordance with experimental phenomena. VIEW 6 of 23 Figure 3. Potential distribution nephogram. Figure 3. Potential distribution nephogram. n nephogram. Figure 3. Potential distribution nephogram. 4.1. Electric Field Simulation Analysis of Electrospinning Nanoﬁber Yarn Forming Device p g Figure 4, the peak electric field intensity appears near the needle, and tensity vectors of the needle nearly all pointed to the receiving disk and h l h As shown in Figure 4, the peak electric ﬁeld intensity appears near the needle, and the electric ﬁeld intensity vectors of the needle nearly all pointed to the receiving disk and ring, in accordance with experimental phenomena. 6 of 23 ring, in accordance with experimental phenomena. Figure 4. Electric field simulation vector diagram. Figure 4. Electric ﬁeld simulation vector diagram. g p p Figure 4. Electric field simulation vector diagram. 4.2. SEM Image Analysis 4.2.1. Effect of Changing the Distance from the Ring to the Disk on the Surface Morphol- Figure 4. Electric ﬁeld simulation vector diagram. 4.2. SEM Image Analysis 4.2.1. Effect of Changing the Distance from the Ring to the Disk on the Surface Morphology of the Fiber ation vector diagram. Figure 4. Electric ﬁeld simulation vector diagram. l i 4.2. SEM Image Analysis alysis anging the Distance from the Ring to the Disk on the Surface Morphol- 4.2. SEM Image Analysis 4.2.1. Effect of Changing the Distance from the Ring to the Disk on the Surface Morphology of the Fiber alysis nging the Distance from the Ring to the Disk on the Surface Morphol- 4.2. SEM Image Analysis 4.2.1. Effect of Changing the Distance from the Ring to the Disk on the Surface Morphology of the Fiber alysis anging the Distance from the Ring to the Disk on the Surface Morphol- 4.2.1. Effect of Changing the Distance from the Ring to the Disk on the Surface Morphology of the Fiber anging the Distance from the Ring to the Disk on the Surface Morphol stance from the ring to the disk was set to hp L , the distance zp L from center of the disk is 9 cm, and the rotation speed n of the disk was 40 mages in Figure 5 a, b, and c respectively show that the nanofiber yarn changing hp L from 3 cm, 5 cm, and 7 cm. The nanofibers formed yarn When the distance from the ring to the disk was set to Lhp, the distance Lzp from the needle to the center of the disk is 9 cm, and the rotation speed n of the disk was 40 r/min. 4.1. Electric Field Simulation Analysis of Electrospinning Nanoﬁber Yarn Forming Device The different colors represent the level of the electric tential, which increases from blue to red. It can be seen that the red area is most con- Figure 3 shows the potential distribution nephogram of the electric ﬁeld and the needle tip portion of the single-needle nanoﬁber-wrapped structure yarn forming device under an applied voltage of 18 kV. The different colors represent the level of the electric potential, which increases from blue to red. It can be seen that the red area is most concentrated Figure 3 shows the potential distribution nephogram of the electric field and the nee- e tip portion of the single-needle nanofiber-wrapped structure yarn forming device un- r an applied voltage of 18 kV. The different colors represent the level of the electric tential, which increases from blue to red. It can be seen that the red area is most con- Figure 3 shows the potential distribution nephogram of the electric ﬁeld and the needle tip portion of the single-needle nanoﬁber-wrapped structure yarn forming device under an applied voltage of 18 kV. The different colors represent the level of the electric potential, which increases from blue to red. It can be seen that the red area is most concentrated Polymers 2021, 13, 3189 dle tip por der an ap 5 of 20 5 of 20 near the needle tip, indicating the highest potential; the blue area near the receiving disk and the ring were also concentrated, indicating the lowest potential. As the distance from the single-needle tip increased, the potential decreased, and the color changed. This veriﬁes the basic principle of electrospinning and veriﬁes the feasibility of the device from a theoretical basis. e needle tip, indicating the highest potential; the blue area near the re the ring were also concentrated, indicating the lowest potential. As the e single-needle tip increased, the potential decreased, and the color ifies the basic principle of electrospinning and verifies the feasibility of theoretical basis. Figure 3. Potential distribution nephogram. As shown in Figure 4, the peak electric field intensity appears near the needle, and the electric field intensity vectors of the needle nearly all pointed to the receiving disk and ring, in accordance with experimental phenomena. Figure 3. Potential distribution nephogram. 4.1. Electric Field Simulation Analysis of Electrospinning Nanoﬁber Yarn Forming Device rs 2021, 13, x FOR PEER REVIEW 7 of 23 (c) hp L = 7 cm (a) hp L = 3 cm (b) hp L = 5 cm (a) hp L = 3 cm (c) hp L = 7 cm (b) hp L = 5 cm Figure 5. SEM image of nanofiber yarns at different distances between the ring and disk. (a),SEM image of nanofiber yarns at hp L = 3 cm. (b)SEM image of nanofiber yarns at hp L = 5 cm. (c),SEM image of nanofiber yarns at hp L = 7 cm. Figure 5. SEM image of nanoﬁber yarns at different distances between the ring and disk. (a) SEM image of nanoﬁber yarns at Lhp= 3 cm. (b) SEM image of nanoﬁber yarns at Lhp = 5 cm. (c) SEM image of nanoﬁber yarns at Lhp = 7 cm. 2021, 13, x FOR PEER REVIEW 7 of 23 ure 6. The relationship between the ring-to-disk distance and the nanofiber yarn twist angle and nofiber diameter 3 4 5 6 7 20 25 30 35 40 45 Twist Angle Average Diameter Lhp/(cm) Twist Angle/(°) 0.24 0.26 0.28 0.30 0.32 Average Diameter/(μm) Figure 6. The relationship between the ring-to-disk distance and the nanoﬁber yarn twist angle and nanoﬁber diameter. 3 4 5 6 7 20 25 30 35 40 45 Twist Angle Average Diameter Lhp/(cm) Twist Angle/(°) 0.24 0.26 0.28 0.30 0.32 Average Diameter/(μm) e 6. The relationship between the ring-to-disk distance and the nanofiber yarn twist angle and fiber diameter Figure 6. The relationship between the ring-to-disk distance and the nanoﬁber yarn twist angle and nanoﬁber diameter. fiber diameter. . Effect of Changing the Distance between the Tip of the Needle and the Center of 4.2.2. Effect of Changing the Distance between the Tip of the Needle and the Center of the Disk on the Surface Morphology of the Fiber g g p Disk on the Surface Morphology of the Fiber The ring-to-disk distance hp L was 5 cm, and the rotation speed n of the disk was 40 n. The SEM images in Figure 7a, b, and c respectively show the nanofiber yarns pre- d by changing zp L at 7 cm, 9 cm, and 11 cm. Figure 7 a,b, and c show that the nano- rs formed z-twisted twisted yarns, and the average twist angle of the nanofiber yarns htly increased upon increasing zp L . 4.1. Electric Field Simulation Analysis of Electrospinning Nanoﬁber Yarn Forming Device The SEM images in Figure 5a,b, and c respectively show that the nanoﬁber yarn was prepared by changing Lhp from 3 cm, 5 cm, and 7 cm. The nanoﬁbers formed yarn twisted in the z-direction, and the average twist angle decreased as Lhp increased (Figure 6). At a constant disk rotation speed, the number of twists per unit time was constant, and the twisting of the nanoﬁber yarn theoretically remained unchanged. The diameters of the Polymers 2021, 13, 3189 6 of 20 Figure electrospinning nanoﬁber yarns in a, b, and c of Figure 5 were (88.0 ± 5.3) µm, (77.0 ± 3.9) µm, and (76.8 ± 3.0) µm, respectively. As the distance from the ring to the disk increased, the diameter of nanoﬁber yarns increased because the greater the twist angle β of the yarn with the same twist, the greater the twist of the ﬁber, resulting in a thicker yarn. Nanoﬁbers oriented between disks and rings tend to be stretched and reﬁned upon increasing the distance between them. electrospinning nanofiber yarns in a, b, and c of Figure 5 were (88.0 ± 5.3) µm, (77.0 ± 3.9) µm, and (76.8 ± 3.0) µm, respectively. As the distance from the ring to the disk in- creased, the diameter of nanofiber yarns increased because the greater the twist angle β of the yarn with the same twist, the greater the twist of the fiber, resulting in a thicker yarn. Nanofibers oriented between disks and rings tend to be stretched and refined upon increasing the distance between them. (a) hp L = 3 cm (b) hp L = 5 cm (c) hp L = 7 cm Figure 5. SEM image of nanofiber yarns at different distances between the ring and disk. (a),SEM image of nanofiber yarns at hp L = 3 cm. (b)SEM image of nanofiber yarns at hp L = 5 cm. (c),SEM image of nanofiber yarns at hp L = 7 cm. Figure 5. SEM image of nanoﬁber yarns at different distances between the ring and disk. (a) SEM image of nanoﬁber yarns at Lhp= 3 cm. (b) SEM image of nanoﬁber yarns at Lhp = 5 cm. (c) SEM image of nanoﬁber yarns at Lhp = 7 cm. 4.1. Electric Field Simulation Analysis of Electrospinning Nanoﬁber Yarn Forming Device Effect of Changing the Disk Rotation Speed on the Fiber Surface Morphology g Eff f Ch h k S d h b S f h l 4.2.3. Effect of Changing the Disk Rotation Speed on the Fiber Surface Morphology . Effect of Changing the Disk Rotation Speed on the Fiber Surface Morphology The ring-to-disk distance hp L was 5 cm, and the distance zp L from the needle to disk center was 9 cm. The images a, b, and c in Figure 9 respectively show the SEM ges of nanofiber yarns prepared by changing the rotational speed n of the disk (40 n, 80 r/min, and 120 r/min). It can be seen from a, b, and c of Figure 9 that the nano- rs formed a z-twisted twisted yarn, and as the disk rotation speed increased, the aver- twist angle of the nanofiber yarn first increased and then decreased, but this was not ous in Figure 10. This feature is consistent with the theoretical analysis below. There o significant relationship between the twist angle of the nanofiber yarn and the disk tion speed, only between the ring-to-disk spacing and the position of the nanofibers he disk. Interestingly, when the disk rotation speed increased, in addition to more ely arranged nanofibers in the nanofiber yarn, some of the nanofibers mutually ad- d due to the slow evaporation of the solvent. In a, b, and c of Figure 9, the average ofiber yarn diameter at disk rotation speeds of 40 r/min, 80 r/min, and 120 r/min were ± 3 9) µm (106 5 ± 4 83) µm (93 0 ± 4 36) µm respectively There is no obvious The ring-to-disk distance Lhp was 5 cm, and the distance Lzp from the needle to the disk center was 9 cm. The images a, b, and c in Figure 9 respectively show the SEM images of nanoﬁber yarns prepared by changing the rotational speed n of the disk (40 r/min, 80 r/min, and 120 r/min). It can be seen from a, b, and c of Figure 9 that the nanoﬁbers formed a z-twisted twisted yarn, and as the disk rotation speed increased, the average twist angle of the nanoﬁber yarn ﬁrst increased and then decreased, but this was not obvious in Figure 10. This feature is consistent with the theoretical analysis below. 4.1. Electric Field Simulation Analysis of Electrospinning Nanoﬁber Yarn Forming Device The difference between the twist angles was not e, but a slight increase was observed in Figure 8. The diameters of nanofiber yarns pared with zp L = 7 cm, 9 cm, and 11 cm were (80 ± 7.81) µm, (77 ± 3.9) µm, and 4 ± 5.43) µm, respectively. The nanofiber yarn diameter decreased farther away from center of the disk. Fewer oriented nanofibers between the ring and the disk formed The ring-to-disk distance Lhp was 5 cm, and the rotation speed n of the disk was 40 r/min. The SEM images in Figure 7a, b, and c respectively show the nanoﬁber yarns prepared by changing Lzp at 7 cm, 9 cm, and 11 cm. Figure 7a,b, and c show that the nanoﬁbers formed z-twisted twisted yarns, and the average twist angle of the nanoﬁber yarns slightly increased upon increasing Lzp. The difference between the twist angles was not large, but a slight increase was observed in Figure 8. The diameters of nanoﬁber yarns prepared with Lzp = 7 cm, 9 cm, and 11 cm were (80 ± 7.81) µm, (77 ± 3.9) µm, and (48.4 ± 5.43) µm, respectively. The nanoﬁber yarn diameter decreased farther away from the center of the disk. Fewer oriented nanoﬁbers between the ring and the disk formed when the needle was farther away, and there were more non-oriented ﬁbers; however, less-oriented nanoﬁbers were twisted, resulting in a ﬁner twisted yarn. The farther the needle was from the disk center, the smaller the average diameter of the nanoﬁbers in Polymers 2021, 13, 3189 7 of 20 ormed wever 7 of 20 ormed e e Figure 8 because at greater distances, the nanoﬁbers had more time to stretch in the electric ﬁeld, and their average diameter decreased. needle was from the disk center, the smaller the average diameter of the nanofibers in Figure 8 because at greater distances, the nanofibers had more time to stretch in the elec- tric field, and their average diameter decreased. (a) zp L =7 cm (c) zp L =11 cm (b) zp L =9 cm (a) zp L =7 cm (b) zp L =9 cm (c) zp L =11 cm Figure 7. SEM images of nanofiber yarns at different distances between the needle tip and the disk center.(a),SEM image of nanofiber yarns at zp L =7 cm. (b)SEM image of nanofiber yarns at zp L =9 cm. 4.1. Electric Field Simulation Analysis of Electrospinning Nanoﬁber Yarn Forming Device (c),SEM image of nanofiber yarns at zp L =11 cm Figure 7. SEM images of nanoﬁber yarns at different distances between the needle tip and the disk center. (a) SEM image of nanoﬁber yarns at Lzp = 7 cm. (b) SEM image of nanoﬁber yarns at Lzp = 9 cm. (c) SEM image of nanoﬁber yarns at Lzp = 11 cm. rs 2021, 13, x FOR PEER REVIEW 8 of 23 (a) zp L =7 cm (c) zp L =11 cm (b) zp L =9 cm Figure 7. SEM images of nanofiber yarns at different distances between the needle tip and the disk center.(a),SEM image of nanofiber yarns at zp L =7 cm. (b)SEM image of nanofiber yarns at zp L =9 cm. (c),SEM image of nanofiber yarns at zp L =11 cm Figure 7. SEM images of nanoﬁber yarns at different distances between the needle tip and the disk center. (a) SEM image of nanoﬁber yarns at Lzp = 7 cm. (b) SEM image of nanoﬁber yarns at Lzp = 9 cm. (c) SEM image of nanoﬁber yarns at Lzp = 11 cm. 2021, 13, x FOR PEER REVIEW 8 of 23 7 8 9 10 11 26 28 30 32 34 36 38 Twist Angle Average Diameter Lzp/(cm) Twist Angle/(°) 0.22 0.24 0.26 0.28 0.30 0.32 0.34 Average Diameter/(μm) ure 8. The relationship between the distance from the needle tip to disk center and the nanofiber n twist angle and nanofiber diameter. 7 8 9 10 11 26 28 30 32 34 36 38 Twist Angle Average Diameter Lzp/(cm) Twist Angle/(°) 0.22 0.24 0.26 0.28 0.30 0.32 0.34 Average Diameter/(μm) Figure 8. The relationship between the distance from the needle tip to disk center and the nanoﬁber yarn twist angle and nanoﬁber diameter. re 8. The relationship between the distance from the needle tip to disk center and the nanofiber twist angle and nanofiber diameter Figure 8. The relationship between the distance from the needle tip to disk center and the nanoﬁber yarn twist angle and nanoﬁber diameter. twist angle and nanofiber diameter. Eff f Ch i h Di k R i S d h Fib S f M h l 4.2.3. Effect of Changing the Disk Rotation Speed on the Fiber Surface Morphology twist angle and nanofiber diameter. 4.2.3. diameter. 4.2.4. Electrospun Nanoﬁber-Wrapped Structural Yarns after Changing Lzp 4. Electrospun Nanofiber-Wrapped Structural Yarns after Changing The ring-to-disk distance hp L was 5 cm, the ring-to-disk center penetrated the core n of a 75D/36F twistless polyester multifilament, the disk rotation speed n was 80 n, and the spinning time was 5 min. The SEM images in Figure 11a, b, and c show the trospun nanofiber-wrapped structural yarns prepared by changing the distance zp L m the tip to the center of the disk from 7 cm, 9 cm, to 11 cm, respectively. It can be seen m Figure 11a that the nanofiber formed a z-direction twisted yarn with a twist angle of ut 19.11°. The twist angle of the nanofiber yarns was 38.19° under the same condition hout the core yarn. This confirms the basic yarn theory that the twist angles of the rs in each layer of the spinning yarns were different. The nanofiber-wrapped struc- l yarns in Figure 11b,c do not show significant twist directions, and the nanofibers e disordered because zp L was relatively large, and there were fewer oriented nano- rs between the ring and the disk. The spinning time was also shorter, the oriented zp L The ring-to-disk distance Lhp was 5 cm, the ring-to-disk center penetrated the core yarn of a 75D/36F twistless polyester multiﬁlament, the disk rotation speed n was 80 r/min, and the spinning time was 5 min. The SEM images in Figure 11a–c show the electrospun nanoﬁber-wrapped structural yarns prepared by changing the distance Lzp from the tip to the center of the disk from 7 cm, 9 cm, to 11 cm, respectively. It can be seen from Figure 11a that the nanoﬁber formed a z-direction twisted yarn with a twist angle of about 19.11◦. The twist angle of the nanoﬁber yarns was 38.19◦under the same condition without the core yarn. This conﬁrms the basic yarn theory that the twist angles of the ﬁbers in each layer of the spinning yarns were different. The nanoﬁber-wrapped structural yarns in Figure 11b,c do not show signiﬁcant twist directions, and the nanoﬁbers were disordered because Lzp was relatively large, and there were fewer oriented nanoﬁbers between the ring and the disk. The spinning time was also shorter, the oriented nanoﬁbers and some of the nanoﬁbers directly injected into the yarns overlapped each other, resulting in a disordered arrangement between the nanoﬁbers. 4.1. Electric Field Simulation Analysis of Electrospinning Nanoﬁber Yarn Forming Device There is no signiﬁcant relationship between the twist angle of the nanoﬁber yarn and the disk rotation speed, only between the ring-to-disk spacing and the position of the nanoﬁbers in the disk. Interestingly, when the disk rotation speed increased, in addition to more closely arranged nanoﬁbers in the nanoﬁber yarn, some of the nanoﬁbers mutually adhered due to the slow evaporation of the solvent. In a, b, and c of Figure 9, the average nanoﬁber yarn diameter at disk rotation speeds of 40 r/min, 80 r/min, and 120 r/min were (77 ± 3.9) µm, (106.5 ± 4.83) µm, (93.0 ± 4.36) µm, respectively. There is no obvious change in the average diameters of nanoﬁber yarns. The results show that in addition to satisfying the basic Polymers 2021, 13, 3189 8 of 20 n were bvious 8 of 20 n were b iou spinning principles and rules, there are many variables in when electrospinning nanoﬁbers. The average nanoﬁber diameter decreased upon increasing the disk rotation speed. satisfying the basic spinning principles and rules, there are many variables in when elec trospinning nanofibers. The average nanofiber diameter decreased upon increasing the disk rotation speed. spinning principles and rules, there are many variables in when electrospinning nanoﬁbers. The average nanoﬁber diameter decreased upon increasing the disk rotation speed. satisfying the basic spinning principles and rules, there are many variables in when elec trospinning nanofibers. The average nanofiber diameter decreased upon increasing the disk rotation speed. (a) n = 40 r/min ers 2021, 13, x FOR PEER REVIEW (c) n = 120 r/min 9 of 23 (b) n = 80 r/min (a) n = 40 r/min (b) n = 80 r/min (c) n = 120 r/min Figure 9. SEM images of nanofiber yarns at different distances between the needle tip and disk center.(a),SEM image of nanofiber yarns at n = 40 r/min. (b)SEM image of nanofiber yarns at n = 80 r/min. (c),SEM image of nanofiber yarns at n = 120 r/min. Figure 9. SEM images of nanoﬁber yarns at different distances between the needle tip and disk center. (a) SEM image of nanoﬁber yarns at n = 40 r/min. (b) SEM image of nanoﬁber yarns at n = 80 r/min. (c) SEM image of nanoﬁber yarns at n = 120 r/min. 4.1. Electric Field Simulation Analysis of Electrospinning Nanoﬁber Yarn Forming Device s 2021, 13, x FOR PEER REVIEW 9 of 23 (c) n = 120 r/min 9 (a) n = 40 r/min FOR PEER REVIEW (b) n = 80 r/min Figure 9. SEM images of nanofiber yarns at different distances between the needle tip and disk center.(a),SEM image of nanofiber yarns at n = 40 r/min. (b)SEM image of nanofiber yarns at n = 80 r/min. (c),SEM image of nanofiber yarns at n = 120 r/min. Figure 9. SEM images of nanoﬁber yarns at different distances between the needle tip and disk center. (a) SEM image of nanoﬁber yarns at n = 40 r/min. (b) SEM image of nanoﬁber yarns at n = 80 r/min. (c) SEM image of nanoﬁber yarns at n = 120 r/min. ure 10. The relationship between disk rotation speed and nanofiber yarn twist angle and nano- r diameter 40 60 80 100 120 25 30 35 40 45 50 55 60 Twist Angle Average Diameter n (r/min) Twist Angle (°) 0.22 0.23 0.24 0.25 0.26 0.27 0.28 0.29 0.30 0.31 Average Diameter (μm) Figure 10. The relationship between disk rotation speed and nanoﬁber yarn twist angle and nanoﬁber diameter. 40 60 80 100 120 25 30 35 40 45 50 55 60 Twist Angle Average Diameter n (r/min) Twist Angle (°) 0.22 0.23 0.24 0.25 0.26 0.27 0.28 0.29 0.30 0.31 Average Diameter (μm) e 10. The relationship between disk rotation speed and nanofiber yarn twist angle and nano- diameter Figure 10. The relationship between disk rotation speed and nanoﬁber yarn twist angle and nanoﬁber diameter. diameter. 4.2.4. Electrospun Nanoﬁber-Wrapped Structural Yarns after Changing Lzp diameter. 4.2.4. Electrospun Nanoﬁber-Wrapped Structural Yarns after Changing Lzp diameter. 4.2.4. Electrospun Nanoﬁber-Wrapped Structural Yarns after Changing Lzp Many oriented nanoﬁbers were also observed in Figure 11b,c, and their alignments were oriented. 9 of 20 m, 0.26 r also Polymers 2021, 13, 3189 (a) zp L =7 cm (b) zp L =9 cm (c) zp L =11 cm Figure 11. SEM images of electrospun nanofiber-wrapped yarns. (a),SEM image of electrospun nanofiber-wrapped yarns at zp L =7 cm. (b)SEM image of electrospun nanofiber-wrapped yarns at zp L =9 cm. (c),SEM image of electrospun nano- fib d tL 11 Figure 11. SEM images of electrospun nanoﬁber-wrapped yarns. (a) SEM image of electrospun nanoﬁber-wrapped yarns at Lzp = 7 cm. (b) SEM image of electrospun nanoﬁber-wrapped yarns at Lzp = 9 cm. (c) SEM image of electrospun nanoﬁber-wrapped yarns at Lzp = 11 cm. (c) zp L =11 cm (b) zp L =9 cm (c) zp L =11 cm (a) zp L =7 cm (b) zp L =9 cm Figure 11. SEM images of electrospun nanofiber-wrapped yarns. (a),SEM image of electrospun nanofiber-wrapped yarns at zp L =7 cm. (b)SEM image of electrospun nanofiber-wrapped yarns at zp L =9 cm. (c),SEM image of electrospun nano- fib d L 11 Figure 11. SEM images of electrospun nanoﬁber-wrapped yarns. (a) SEM image of electrospun nanoﬁber-wrapped yarns at Lzp = 7 cm. (b) SEM image of electrospun nanoﬁber-wrapped yarns at Lzp = 9 cm. (c) SEM image of electrospun nanoﬁber-wrapped yarns at Lzp = 11 cm. 4.3. Mechanical Performance Analysis The mechanical tests return the breaking strength, which must be converted into the fracture strength as follows σ = Fb/So. The cross-sectional area of the sample was meas- ured from the SEM images, and the diameter is converted according to the area formula. 4.3.1. Change the Electrospinning Nanofiber Yarn under zp L The distance zp L of the needle from the center of the disk was 9 cm and the rota- tional speed n of the disk was 40 r/min. Figure 12 shows the mechanical properties of L Figure 11a–c show that when Lzp = 7 cm, the nanoﬁber-wrapped core yarn was relatively uniform, and no core exposure was observed. However, in b and c of Figure 11, the exposed core was completely visible through the surface of the nanoﬁbers. The core yarn was composed of many untwisted monoﬁlaments. diameter. 4.2.4. Electrospun Nanoﬁber-Wrapped Structural Yarns after Changing Lzp The Figure 11a–c show that at a certain spinning time, as Lzp increased, the effect of the nanoﬁber wrapping structure became increasingly worse, and the wrapping surface became smaller. This is also because, at greater distances, there were fewer oriented nanoﬁbers between the ring and the disk, and the nanoﬁber yarn diameter decreased as Lzp increased. However, we can also infer that as the spinning time of the electrospun nanoﬁber-wrapped structure yarns was extended from 5 min to 15 min, more oriented nanoﬁbers were wrapped on the core yarn, and the wrapping effect was better. electrospun nanofiber yarns prepared using ring-to-disk distances hp L of 3 cm, 4 cm, 5 cm, 6 cm, and 7 cm. Figure 11 shows that the breaking strength of nanofiber yarns pre- pared was the highest (18.51 ± 1.31) MPa, when hp L = 7 cm, and the elongation at break was the highest 15.11%, which is more suitable for traditional weaving processing. The fracture strength and elongation at break of the nanofiber yarns increased gradually, mainly because the length of the oriented nanofibers between the rings and disks in- creased upon increasing the distance which strengthened the orientation degree of mac It can also be seen from Figure 11a–c that the nanoﬁbers were arranged parallel and adhered to each other. This phenomenon conﬁrms that the individual oriented nanoﬁbers were pushed together during twisting and bonded as the solvent evaporated. In panels Figure 11a–c, for electrospun nanoﬁber-wrapped structural yarns prepared at Lzp = 7 cm, 9 cm, and 11 cm, the corresponding nanoﬁbers have an average diameter of 0.28 µm, 0.26 µm, and 0.25 µm, respectively. As Lzp increased, the average nanoﬁber diameter also decreased. p g romolecules along the nanofiber dire f h f b Th ll f h d 4.3. Mechanical Performance Analysis of the fibers. Theoretically, if the distance between the needle tip and the center of the disk exceeds a certain limit, few nanofibers will be collected by the device due to the large receiving distance, and yarn will not form. The mechanical tests return the breaking strength, which must be converted into the fracture strength as follows σ = Fb/So. The cross-sectional area of the sample was measured from the SEM images, and the diameter is converted according to the area formula. 4.3.1. Change the Electrospinning Nanoﬁber Yarn under Lzp 4.3.1. Change the Electrospinning Nanoﬁber Yarn under Lzp The distance Lzp of the needle from the center of the disk was 9 cm and the rotational speed n of the disk was 40 r/min. Figure 12 shows the mechanical properties of electrospun nanoﬁber yarns prepared using ring-to-disk distances Lhp of 3 cm, 4 cm, 5 cm, 6 cm, and 7 cm. Figure 11 shows that the breaking strength of nanoﬁber yarns prepared was the highest (18.51 ± 1.31) MPa, when Lhp = 7 cm, and the elongation at break was the highest 15.11%, which is more suitable for traditional weaving processing. The fracture strength and elongation at break of the nanoﬁber yarns increased gradually, mainly because the length of the oriented nanoﬁbers between the rings and disks increased upon increasing the distance, which strengthened the orientation degree of macromolecules along the nanoﬁber direction, and thus improved the mechanical properties of the ﬁbers. Theoretically, if the distance between the needle tip and the center of the disk exceeds a certain limit, few nanoﬁbers will be collected by the device due to the large receiving distance, and yarn will not form. 10 of 20 23 Polymers 2021, 13, 3189 mers 2021, 13, x FOR PEER ure 12. Mechanical properties of nanofiber yarns at different distances between the ring and the k 3 4 5 6 7 12 13 14 15 16 17 18 19 20 Breaking Strength Elongation At Break Lhp (cm) Breaking Strength (MPa) 6 8 10 12 14 16 18 Elongation At Break (%) Figure 12. Mechanical properties of nanoﬁber yarns at different distances between the ring and the disk. re 12. Mechanical properties of nanofiber yarns at different distances between the ring and the Figure 12. Mechanical properties of nanoﬁber yarns at different distances between the ring and the disk. The ring-to-disk distance hp L = 5 cm, and the rotational speed n of the disk is 40 n. Figure 13 shows the mechanical properties of electrospun nanofiber yarns prepared n the distance between needle and disk center zp L = 7 cm, 8 cm, 9 cm, 10 cm, and 11 The breaking strength of the nanofiber yarn was the highest (16.81 ± 1.89) MPa, when = 8 cm, but its breaking elongation did not reach a maximum. 4.3.1. Change the Electrospinning Nanoﬁber Yarn under Lzp As zp L between the dle and the disk center increased, the fracture strength of the yarn increased first and n decreased, and the elongation at break always increased. As zp L increased, the nan- ers had sufficient time to stretch and draw in the electric field, and the macromolecular ntation in the nanofibers was consistent with the axial direction, so that the fracture h f h i d h h L l fib The ring-to-disk distance Lhp = 5 cm, and the rotational speed n of the disk is 40 r/min. Figure 13 shows the mechanical properties of electrospun nanoﬁber yarns prepared when the distance between needle and disk center Lzp = 7 cm, 8 cm, 9 cm, 10 cm, and 11 cm. The breaking strength of the nanoﬁber yarn was the highest (16.81 ± 1.89) MPa, when Lzp = 8 cm, but its breaking elongation did not reach a maximum. As Lzp between the nee- dle and the disk center increased, the fracture strength of the yarn increased ﬁrst and then decreased, and the elongation at break always increased. As Lzp increased, the nanoﬁbers had sufﬁcient time to stretch and draw in the electric ﬁeld, and the macromolecular orienta- tion in the nanoﬁbers was consistent with the axial direction, so that the fracture strength of the yarn increased; however, when Lzp was large, some nanoﬁbers were scattered outside the twisted area, and no oriented nanoﬁbers formed between the disk and the ring. Thus, too few ﬁbers were formed in the nanoﬁber yarn, which decreased the fracture strength. 12 of 23 g y zp g ed outside the twisted area, and no oriented nanofibers formed between the disk and ring. Thus, too few fibers were formed in the nanofiber yarn, which decreased the cture strength. ure 13. Mechanical properties of nanofiber yarns at different distances between the needle tip disk center. The ring-to-disk distance hp L = 5 cm and the distance zp L between the needle and center of the disk was 9 cm. Figure 14 shows the mechanical properties of electrospun ofiber yarns prepared when the rotational speed n of the disk was 40 r/min, 60 r/min, /min, 100 r/min and 120 r/min. 4.3.1. Change the Electrospinning Nanoﬁber Yarn under Lzp Figure 13 shows that the highest breakage strength of 7 8 9 10 11 12 13 14 15 16 17 18 19 Breaking Strength Elongation At Break Lzp (cm) Breaking Strength (MPa) 9 10 11 12 13 14 15 16 17 Elongation At Break (%) Figure 13. Mechanical properties of nanoﬁber yarns at different distances between the needle tip and disk center. The ring-to-disk distance Lhp = 5 cm and the distance Lzp between the needle and the center of the disk was 9 cm. Figure 14 shows the mechanical properties of electrospun nanoﬁber yarns prepared when the rotational speed n of the disk was 40 r/min, 60 r/min, 80 r/min, 100 r/min and 120 r/min. Figure 13 shows that the highest breakage strength of e 13. Mechanical properties of nanofiber yarns at different distances between the needle tip disk center Figure 13. Mechanical properties of nanoﬁber yarns at different distances between the needle tip and disk center. The ring-to-disk distance hp L = 5 cm and the distance zp L between the needle and enter of the disk was 9 cm. Figure 14 shows the mechanical properties of electrospun fiber yarns prepared when the rotational speed n of the disk was 40 r/min, 60 r/min, The ring-to-disk distance Lhp = 5 cm and the distance Lzp between the needle and the center of the disk was 9 cm. Figure 14 shows the mechanical properties of electrospun nanoﬁber yarns prepared when the rotational speed n of the disk was 40 r/min, 60 r/min, 80 r/min, 100 r/min and 120 r/min. Figure 13 shows that the highest breakage strength of The ring-to-disk distance hp L = 5 cm and the distance zp L between the needle and center of the disk was 9 cm. Figure 14 shows the mechanical properties of electrospun ofiber yarns prepared when the rotational speed n of the disk was 40 r/min, 60 r/min, The ring-to-disk distance Lhp = 5 cm and the distance Lzp between the needle and the center of the disk was 9 cm. Figure 14 shows the mechanical properties of electrospun nanoﬁber yarns prepared when the rotational speed n of the disk was 40 r/min, 60 r/min, 80 r/min, 100 r/min and 120 r/min. 4.3.1. Change the Electrospinning Nanoﬁber Yarn under Lzp Figure 13 shows that the highest breakage strength of Polymers 2021, 13, 3189 11 of 20 n nanoﬁber yarns prepared at n = 120 r/min was (21.87 ± 2.29) MPa, and the highest elonga- tion at break was 16.55%. The fracture strength and elongation at break of nanoﬁber yarns tended to gradually improve at a disk rotation speed of 120 r/min or lower. The rotating metal disk twisted the oriented nanoﬁbers. Increasing the disk rotation speed increased the degree of nanoﬁber twisting, and the bonding between the nanoﬁbers tightened, which increased the fracture strength and elongation at break of the nanoﬁber yarns. As the disk rotation speed increased, the mechanical properties of the nanoﬁber yarns did not always improve. Theoretically, there is a limit, and when this limit is exceeded, the mechanical properties will decrease. Because the rotation speed was too high, the nanoﬁbers broke, and a yarn did not form. ofiber yarns prepared at n = 120 r/min was (21.87 ±2.29) MPa, and the highest elonga- at break was 16.55%. The fracture strength and elongation at break of nanofiber yarns ed to gradually improve at a disk rotation speed of 120 r/min or lower. The rotating l disk twisted the oriented nanofibers. Increasing the disk rotation speed increased degree of nanofiber twisting, and the bonding between the nanofibers tightened, h increased the fracture strength and elongation at break of the nanofiber yarns. As disk rotation speed increased, the mechanical properties of the nanofiber yarns did always improve. Theoretically, there is a limit, and when this limit is exceeded, the hanical properties will decrease. Because the rotation speed was too high, the nano- s broke, and a yarn did not form. ure 14. Mechanical properties of nanofiber yarns at different disk speeds. 40 60 80 100 120 14 16 18 20 22 24 Breaking Strength Elongation At Break n (r/min) Breaking Strength (MPa) 8 10 12 14 16 18 Elongation At Break (%) Figure 14. Mechanical properties of nanoﬁber yarns at different disk speeds. 40 60 80 100 120 14 16 18 20 22 24 Breaking Strength Elongation At Break n (r/min) Breaking Strength (MPa) 8 10 12 14 16 18 Elongation At Break (%) 14 M h i l ti f fib t diff t di k d Figure 14. Mechanical properties of nanoﬁber yarns at different disk speeds. e 14. g p W a i Me ha i u de S e ifi A u tio 5.1. Wrapping Mechanism under Speciﬁc Assumptions W a i Me ha i u de S e ifi A u tio 5.1. Wrapping Mechanism under Speciﬁc Assumptions Wrapping Mechanism under Specific Assumptions It is assumed that the ring aperture is exactly the same as the diameter of the core n, and the inner and outer diameters of the ring are the same. After this simplification, ording to the electric field nephogram in Figure 3, one of the two ends of the oriented ofibers is on the ring, and the other end is at any point on the disk. The distance from endpoint to the center of the disk is d0 (mm), and the length of the oriented nanofibers qL (mm). At the same time, the core yarn may be wound up at a winding speed of v m/s). The twist angle is β (°), the disk rotation speed is n (r/min), the core yarn is as- med to be a cylinder with a diameter d (mm), the ring diameter is D1 (mm), the disk It is assumed that the ring aperture is exactly the same as the diameter of the core yarn, and the inner and outer diameters of the ring are the same. After this simpliﬁcation, according to the electric ﬁeld nephogram in Figure 3, one of the two ends of the oriented nanoﬁbers is on the ring, and the other end is at any point on the disk. The distance from the endpoint to the center of the disk is d0 (mm), and the length of the oriented nanoﬁbers is Lq (mm). At the same time, the core yarn may be wound up at a winding speed of v (mm/s). The twist angle is β (◦), the disk rotation speed is n (r/min), the core yarn is assumed to be a cylinder with a diameter d (mm), the ring diameter is D1 (mm), the disk diameter is D2 (mm), and the disk aperture is D0 (mm), and the twisting time is t (s). med to be a cylinder with a diameter d (mm), the ring diameter is D1 (mm), the disk meter is D2 (mm), and the disk aperture is D0 (mm), and the twisting time is t (s). It is assumed that oriented nanofibers were formed at a certain fixed position be- en the ring and the disk and that they do not break during twisting. 4.3.1. Change the Electrospinning Nanoﬁber Yarn under Lzp Mechanical properties of nanofiber yarns at different disk speeds. 4.3.2. Electrospun Nanoﬁber-Wrapped Structural Yarns after Changing Lzp p The ring-to-disk distance Lhp = 5 cm, the ring-to-disk center penetrated the core yarn of the 75D/36F twistless polyester multiﬁlament, the disk rotation speed n = 80 r/min, and the spinning time was 5 min. The fracture strength of electrospun nanoﬁber-wrapped structural yarns prepared at c Lzp = 7 cm, 8 cm, 9 cm, 10 cm, and 11 cm are shown in Figure 15, and the elongation at break is shown in Figure 14. It can be seen from Figure 15 that as the distance from the tip of the needle to the center of the disk increased, the fracture strength ﬁrst increased and then decreased. The elongation at break decreased, but the fracture strength and elongation at break of the nanoﬁber-wrapped yarns were substantially higher than those of the core yarn. The reason is that the oriented nanoﬁbers were wrapped on the surface of the core yarn by twisting, and the multiﬁlaments in the core yarn increased due to friction and cohesion. These nanoﬁbers were basically submicron ﬁbers. The twisted-oriented nanoﬁbers enhanced the core yarn. After the twistless yarns broke, the wrapped nanoﬁbers that were not pulled off outside the core yarn continued to stretch; therefore, the fracture strength and elongation at break of the yarns after wrapping with nanoﬁber were improved. However, when the distance from the tip of the needle to the disk center was 11 cm, there were fewer oriented nanoﬁbers between the ring and the disk, which led to a low coverage rate of the nanoﬁber-wrapped yarns. The yarn strength and elongation at break were much lower than when Lzp = 9 cm. 12 of 20 he he Polymers 2021, 13, 3189 re 15. Mechanical properties of nanofiber-wrapped yarns at different distances from the tip of needle to the disk center 7 8 9 10 11 24 26 28 30 32 34 36 38 40 42 Breaking Stength Elongation At Break Lzp(cm) Breaking Stength (MPa) 15 20 25 30 35 40 45 50 55 Elongation At Break (%) Figure 15. Mechanical properties of nanoﬁber-wrapped yarns at different distances from the tip of the needle to the disk center. e 15. Mechanical properties of nanofiber-wrapped yarns at different distances from the tip of eedle to the disk center Figure 15. 4.3.1. Change the Electrospinning Nanoﬁber Yarn under Lzp Mechanical properties of nanoﬁber-wrapped yarns at different distances from the tip of the needle to the disk center. eedle to the disk center. 5. The Nanoﬁber-Wrapped Yarn Formation Mechanism he Nanofiber-Wrapped Yarn Formation Mechanism The formation of electrospun nanofiber-wrapped structural yarns occurs via the for- on of many oriented nanofibers between the disk and the ring. The rotation of the drives the oriented nanofibers to coat the core yarn at an angle. Its schematic diagram The formation of electrospun nanoﬁber-wrapped structural yarns occurs via the formation of many oriented nanoﬁbers between the disk and the ring. The rotation of the disk drives the oriented nanoﬁbers to coat the core yarn at an angle. Its schematic diagram is shown in Figure 16. 14 of 23 own in Figure 16. ure 16. Schematic diagram of the formation of electrospun nanofiber-wrapped yarns. Figure 16. Schematic diagram of the formation of electrospun nanoﬁber-wrapped yarns. ure 16. Schematic diagram of the formation of electrospun nanofiber-wrapped yarns. Figure 16. Schematic diagram of the formation of electrospun nanoﬁber-wrapped yarns. g p W a i Me ha i u de S e ifi A u tio 5.1. Wrapping Mechanism under Speciﬁc Assumptions The twisted nan- bers are inclined, and the greater the twisting degree, the greater the incline. Therefore, inclination angle of the nanofibers on the core yarn—twist angle β, i.e., the angle be- e the a ofibe u fa e a d the ya a i (Fi u e 17) a be al ulated by the follo It is assumed that oriented nanoﬁbers were formed at a certain ﬁxed position between the ring and the disk and that they do not break during twisting. The twisted nanoﬁbers are inclined, and the greater the twisting degree, the greater the incline. Therefore, the inclination angle of the nanoﬁbers on the core yarn—twist angle β, i.e., the angle between the nanoﬁber surface and the yarn axis (Figure 17), can be calculated by the following Formula (1): xis (Figure 17), can be calculated by the follow- tan β = πd L (1) xis (Figure 17) tan β = πd L (1) 13 of 20 w- 13 of 20 w- Polymers 2021, 13, 3189 Figure 17. Schematic diagram of the nanoﬁbers twisted on the core yarn. EW 15 of 23 Figure 17. Schematic diagram of the nanoﬁbers twisted on the core yarn. W 15 of 23 Figure 17. Schematic diagram of the nanoﬁbers twisted on the core yarn. ure 17. Schematic diagram of the nanofibers twisted on the core yarn. d β π t (1) In the formula, β is the twist angle (◦), d is the core yarn diameter (mm), and L is the nanoﬁber displacement along the core yarn axis when twisted at a ﬁxed angle, β. At this time, 100 The distance the core yarn moves in the axial direction in a twist, i.e., the distance the core yarn is wound during a twist is λ. L d β π n = (1) L = 100 Ttex (2) n vL n L v 60 = = (3) (2) In the formula, β is the twist angle (°), d is the core yarn diameter (mm), and L is the In the formula, Ttex is the twist of nanoﬁbers, twist/10 cm. n n 60 (3) ofiber displacement along the core yarn axis when twisted at a fixed angle, β. At this e, The distance the core yarn moves in the axial direction in a twist, i.e., the distance the core yarn is wound during a twist is λ. g p W a i Me ha i u de S e ifi A u tio 5.1. Wrapping Mechanism under Speciﬁc Assumptions 60 In the formula, v is the core yarn winding speed, and n is the disk rotation speed. tex 100 T L = (2) λ = v L n 60 = 60vL n (3) of the core yarn wound in one twist turn is the differ- g distance λ in one twist turn and the displacement L axial direction of the core yarn in one twist turn. (3) t L In the formula, tex T is the twist of nanofibers, twist/10 cm. In the formula, v is the core yarn winding speed, and n is the disk rotation speed. L λ A − = (4) , tex , / The displacement difference A of the core yarn wound in one twist turn is the differ- ence between the core yarn winding distance λ in one twist turn and the displacement L of the nanoﬁber moving along the axial direction of the core yarn in one twist turn. ( ) A can adopt one of three different values: A = 0, A > 0, and A < 0. (1) A =0, A = λ −L (4) n vL 60 =  60 n v = A = λ −L n vL 60 = v (4) A can adopt one of three different values: A = 0, A > 0, and A < 0. (1) A = 0, 60 L n 60 The winding speed is equal to the displacement of nanofibers movin ction of the core yarn in a given twisting time i e the starting point λ = L = 60vL n ⇒v = n 60 given twisting time, i.e., the starting point of each twist is The winding speed is equal to the displacement of nanoﬁbers moving along the axial direction of the core yarn in a given twisting time, i.e., the starting point of each twist is the same. g p W a i Me ha i u de S e ifi A u tio 5.1. Wrapping Mechanism under Speciﬁc Assumptions At this time, vn d n v d L d β π π π 60 60 tan = = = (5) tan β = πd L = πd v n 60 = 60πd vn (5) v 60 he disk twisting is shown in Figure 18, where (5) A schematic diagram of the disk twisting is shown in Figure 18, where d d tan β = d0 −d 2 Lhp (6) hp L d β 2 tan 0 − = (6) tan β = d0 −d 2 Lhp hp L d β 2 tan 0 = Figure 18 S he ati dia a of di k t i ti Figure 18. Schematic diagram of disk twisting. tan β = d0 −d 2 Lhp hp L d β 2 tan 0 = (6) (6) Figure 18. Schematic diagram of disk twisting. Polymers 2021, 13, 3189 14 of 20 14 of 20 In this case, the twist angle β is constant, which can be applied to the traditional twist coefﬁcient Formula (7). dT tgβ = πdTtex 100 (7) tgβ = πdTtex 100 (7) The twisting of the nanoﬁber yarns on a cylindrical core yarn is somewhat similar to winding the yarns on a cylindrical bobbin at a constant winding angle. The difference between the two is that winding yarns is continuous because the yarns can be continuously supplied, while oriented nanoﬁbers twisted on a core yarn are discontinuous. Oriented nanoﬁbers are formed between the ring and the disk. During twisting, the oriented nanoﬁbers are wrapped on the core yarn, while the two ends of the oriented nanoﬁbers remain between the ring and the disk. The twist angle of the same oriented nanoﬁber remains constant when twisted. The greater the twist angle β of the yarns with the same twist, the greater the twist of the ﬁbers and the thicker the yarns. Figures 5 and 6 verify Formula (7) and explain its trend. Some researchers have used the edge of a disk to collect oriented nanofibers and then used the disk to twist the nanofibers to form yarns. By increasing the rotational speed of the disk, the twist angle increased accordingly. This study also verified Formulas (5) and (7) [32]. g p W a i Me ha i u de S e ifi A u tio 5.1. Wrapping Mechanism under Speciﬁc Assumptions g g y y ( ) ( ) The length of the wrapping of each twisted nanoﬁbers is given by: L0 = L cos β = πd sin β (8) (8) when L0 > L, the oriented nanoﬁbers are stretched during twisting. It is assumed that the elongation at break of the nanoﬁbers is ε, the value of which depends on the nature of the polymer used in the electrospinning solution and the electrospinning process parameters. When the oriented nanoﬁbers are twisted the number of twists is m and the length when L0 > L, the oriented nanoﬁbers are stretched during twisting. It is assumed that the elongation at break of the nanoﬁbers is ε, the value of which depends on the nature of the polymer used in the electrospinning solution and the electrospinning process parameters. When the oriented nanoﬁbers are twisted, the number of twists is m, and the length of the oriented nanoﬁbers is Lq, then: polymer used in the electrospinning solution and the electrospinning process parameters. When the oriented nanoﬁbers are twisted, the number of twists is m, and the length of the oriented nanoﬁbers is Lq, then: p y p g p g p p When the oriented nanoﬁbers are twisted, the number of twists is m, and the length of the oriented nanoﬁbers is Lq, then: mL0 > Lq(1 + ε , i.e., m L cos β = m πd sin β > Lq(1 + ε) At this time, the oriented nanoﬁbers will break, and they will no longer be twisted on the core yarn by a twist angle β. The nanoﬁbers will wrap on the core yarn with the oriented nanoﬁbers irregularly formed behind them during twisting. Therefore, only some of the oriented nanoﬁbers that are twisted on the core yarn form a stable twist angle β, and most irregularly wrap on the core yarn. Some nanoﬁbers are directly deposited on the core yarn, and they are also irregular. These inferences are consistent with the experimental phenomena in this paper. There are many oriented nanoﬁbers on the same layer that are irregularly wrapped on the core yarn. The phenomenon in which some nanoﬁbers are twisted on the yarns in an irregular form in Figure 8a, b and c also veriﬁes these laws. (2) A > 0 Here, there are two situations. g p W a i Me ha i u de S e ifi A u tio 5.1. Wrapping Mechanism under Speciﬁc Assumptions The ﬁrst is L < λ ≤Lq(1 + ε) cosβ, and the second one is λ > Lq(1 + ε) cosβ. Here, there are two situations. The ﬁrst is L < λ ≤Lq(1 + ε) cosβ, and the second one is λ > Lq(1 + ε) cosβ. q( β When L < λ ≤Lq(1 + ε) cosβ, the corresponding phenomenon and conclusion are the same as A = 0. When λ > Lq(1 + ε) cosβ, the nanoﬁbers that are not twisted are broken by the high-speed winding core yarn during twisting, so the nanoﬁber-wrapped structural yarns cannot form. In the above two situations where A > 0, it is assumed that the twist angle is ﬁxed. The actual situation is that the twist angle changes, and the twist angle β’ has the following characteristics: d tan β′ = d0 −d 2 Lhp −vt (9) tan β′ = d0 −d 2 Lhp −vt (9) Polymers 2021, 13, 3189 15 of 20 15 of 20 Formula (10) shows that the twist angle β’ changes continuously over time, and the Formula (10) shows that the twist angle β’ changes continuously over time, and the twist angle β’ constantly increases. The maximum twist angle β0’ has the following formula: ( ) g β g y twist angle β’ constantly increases. The maximum twist angle β0’ has the following formula: tan β′ 0 = d0 −d 2 Lhp −R 60m n 0 vdt (10) (10) (3) A < 0 During a twist, tan β′′ = ππd L (11) (11) At this time, according to the disk twisting diagram in Figure 16: tan β′′ = d0 −d Lhp −∑n 1(Lx −λx) (12) (12) In the formula, x is the xth twist. In the formula, x is the xth twist. Formula (12) shows that β” is a variable, and as the number of twists increases, the twist angle becomes larger. During a twist, L in Formula (12) becomes smaller as the number of twists is increased. Formula (8) shows that the wrapping length formula of each twisted nanoﬁber is as follows. g p W a i Me ha i u de S e ifi A u tio 5.1. Wrapping Mechanism under Speciﬁc Assumptions L d L0 = L cos β′′ = πd sin β′′ (13) (13) When oriented nanoﬁbers are twisted, the twisting number is m, and the length of the oriented nanoﬁbers is Lq, then mL0 > Lq(1 + ε , i.e., m L cos β′′ = m πd sin β′′ > Lq(1 + ε) At this time, the oriented nanoﬁbers will break, and the nanoﬁbers are no longer twisted on the core yarn by a twist angle, β′. The nanoﬁbers will wrap on the core yarn with the oriented nanoﬁbers irregularly formed behind them during twisting. Therefore, only some of the oriented nanoﬁbers that are twisted on the core yarn will form a stable twist angle β, and most will wrap on the core yarn in an irregular form. Some nanoﬁbers will directly deposit on the core yarn, and this part of the nanoﬁbers is also irregular. However, when the ﬁber head end is twisted to a certain degree, the nanoﬁbers will be pulled out from the grip of the disk and twisted into the yarn body by the next layer of nanoﬁbers in an irregular shape. 5.2. Wrapping Mechanism under Non-Speciﬁc Conditions 5.2.1. Inner and Outer Diameters of the Ring Are Not Ignored 5.2.1. Inner and Outer Diameters of the Ring Are Not Ignored All the above inferences and conclusions are based on the assumption that the ring aperture and the core yarn diameter are identical, as are the inner and outer diameters of the ring. The actual situation is that the ring aperture and the core yarn diameter are different, and the inner diameters and the outer diameter of the ring are also different. According to the electric ﬁeld vector diagram in Figure 4, one of the two ends of the oriented nanoﬁbers may be located at any point between the inner and outer diameters of the ring, and the other end may be located at any point on the non-central hole of the disk. The distance between the nanoﬁber at one end of the ring and the center of the core yarn is x1, and the distance between the end of the disk and the center of the core yarn is x2. The twisting of oriented nanoﬁbers is shown in Figure 19. 16 of 20 2. The Polymers 2021, 13, 3189 Figure 19. Oriented nanoﬁber twisting schematic. Figure 19. Oriented nanofiber twisting schematic. When the oriented nanofiber is twisted, it will always contact the core yarn, and when it is twisted further, it will wrap the core yarn at a twist angle β. The position at hi h th t i ti b i i h b i Fi 19 Th t i ti t t iti i d When the oriented nanoﬁber is twisted, it will always contact the core yarn, and when it is twisted further, it will wrap the core yarn at a twist angle β. The position at which the twisting begins is shown by p0 in Figure 19. The twisting start position is described as follows: X L by p0 in Figure 19. The twisting start position is de- hp1 1 L L X X = (14) X1 X2 = Lhp1 Lhp2 (14) Lhp1 + Lhp2 = Lhp (15) d d h l y p0 in Figure 19. 5.2.1. Inner and Outer Diameters of the Ring Are Not Ignored The twisting start position is de- X1 X2 = Lhp1 Lhp2 (14) de- (14) 14) (15) hp1 1 L X = (14) Lhp1 + Lhp2 = Lhp (15) hp2 2 L X (14) hp hp2 hp1 L L L = + (15) I th L t i th i i t t t d t i ti d t Th i In the Lhp1 segment, since the ring is not rotated, twisting does not occur. The principle of twisting in the Lhp2 segment is the same, in which it is assumed that the ring diameter and core yarn diameter are identical, and the inner and the outer diameters of the ring are also the same; therefore, the inferences and conclusions apply equally. In the hp1 L segment, since the ring iple of twisting in the L segment is 5.2.2. Randomly Fixed Nanoﬁber ends ciple of twisting in the hp2 L segment is the same, in which it is assumed that the ring diameter and core yarn diameter are identical, and the inner and the outer diameters of he ring are also the same; therefore, the inferences and conclusions apply equally. 5.2.2. Randomly Fixed Nanofiber ends The above inferences and conclusions are based on the assumption that oriented nan- ofibers are formed at a fixed position between the ring and the disk, i.e., the environment The above inferences and conclusions are based on the assumption that oriented nanoﬁbers are formed at a ﬁxed position between the ring and the disk, i.e., the environment in which oriented nanoﬁbers is ﬁxed. The actual situation is relatively complicated. One of the two ends of an oriented nanoﬁber may be located at any point between the inner and outer diameters of the ring, while the other end can be at any point on the non-central hole of the disk. The twisting process mainly involves disk twisting, so only the position of the nanoﬁbers on the disk affects the twist angle. p g n which oriented nanofibers is fixed. The actual situation is relatively complicated. One of the two ends of an oriented nanofiber may be located at any point between the inner and outer diameters of the ring, while the other end can be at any point on the non-central hole of the disk. The twisting process mainly involves disk twisting, so only the position of the nanofibers on the disk affects the twist angle. The distance xi between the nanofibers at the disk and the center of the core yarn is a The distance xi between the nanoﬁbers at the disk and the center of the core yarn is a random variable, which can be a ﬁnite number or inﬁnity and can be listed one by one, and its twist angle is one or several ﬁnite or inﬁnite intervals. This random variable xi is a typical discrete random variable. The probability corresponding to xi is p(xi), and the sum of the products is the mathematical expectation of the discrete random variable xi, denoted as E(x). 5.2.3. Coreless Yarn The above inference is also applicable in the case of a coreless yarn, i.e., in the case where the core yarn diameter d = 0 (Figure 18). At this time, d0 in Figure 18 is the distance from the ﬁber endpoint on the disk to the center of the disk. Formula (6) becomes tan β = d0 Lhp , Formula (8) becomes L0 = L cos β, Formula (9) be- comes tan β′ = d0 Lhp−vt, Formula (10) becomes tan β′ 0 = d0 Lhp−R 60m n 0 vdt , Formula (13) becomes L0 = L cos β′′ . L0 = L cos β′′ . L0 = L cos β′′ . 5.3. Analysis of the Uniqueness of the Electrospinning Nanoﬁber Wrapping Process 5.3. Analysis of the Uniqueness of the Electrospinning Nanoﬁber Wrapping Process 5.3.1. Dispersion of Oriented Nanoﬁber Collection Process 5.3.1. Dispersion of Oriented Nanoﬁber Collection Process Electrospun nanoﬁbers move spirally in an electric ﬁeld, and their trajectory con- tinually changes. The collection of oriented nanoﬁbers between the ring and the disk is guided by the electric ﬁeld of the grounding ring and the disk, and oriented nanoﬁbers are deposited between the ring and the disk. These oriented nanoﬁbers are only some of the ﬁbers formed by electrospinning (Figure 20). Some nanoﬁbers escape directly into other non-collecting device areas, and a small portion of the nanoﬁbers are directly sprayed onto the core yarn due to probability. This is caused by the uncertainty of the nanoﬁber movement during electrospinning when the electric ﬁeld is stretched. Although the electric ﬁeld of the grounding ring and the disk will form oriented nanoﬁbers between them, the nanoﬁbers are easily affected by the surrounding environment and their intermolecular forces during stretching in the electric ﬁeld; thus, their motion trajectories are unpredictable. Some ﬁbers that are stretched out in the electric ﬁeld escape from the ring and the disk receiving electrode. They escape outside of the receiving range and become ﬂying ﬂowers. This is not the same principle of macro-spinning methods, such as melt spinning. Although all involve polymer stretching, ﬁbers produced by melt spinning are always precisely con- trolled because both ends are ﬁxed during stretching; however, electrospinning stretching only involves limited guidance from the electrodes, machinery, and ﬂuids. VIEW 20 of 23 Figure 20. Schematic diagram showing the formation of oriented nanofibers. Figure 20. Schematic diagram showing the formation of oriented nanoﬁbers. Figure 20. In the hp1 L segment, since the ring iple of twisting in the L segment is 5.2.2. Randomly Fixed Nanoﬁber ends y ndom variable, which can be a finite number or infinity and can be listed one by one, The value of the discrete random variable x is: andom variable, which can be a finite number or infinity The value of the discrete random variable x is: , y y , nd its twist angle is one or several finite or infinite intervals. This random variable ix is typical discrete random variable The probability corresponding to xi is p(xi) and the x1, x2, x3, . . . , xn, p(x1),p(x2),p(x3),...,p(xn) the probability that x corresponds to the value, then: products is the mathematical expectation of the discrete random var E(x) E(x)= x1∗p(x1)+x2∗p(x2)+x3∗p(x3)+ . . . + xn∗p(xn)= ∑ ∞ k=1 xkpk ( ) The value of the discrete random variable x is: In other words, the mathematical expectation E(x) of the random variable of the distance between the nanoﬁbers on the disk and the center of the core yarn can be used instead of d0 in Figure 18 under the assumption that oriented nanoﬁbers are formed at a ﬁxed displacement between the ring and disk. The reasoning and formula are the same. Because of the random variables, there is no obvious change in the twist angles. In Figures 5, 7 and 9, the twist angles of nanoﬁbers in the same layer are not constant, large Polymers 2021, 13, 3189 17 of 20 17 of 20 or small, which veriﬁes the randomness of this phenomenon. The average twist angles of nanoﬁbers in each layer also veriﬁes the existence of this mathematical expectation law. or small, which veriﬁes the randomness of this phenomenon. The average twist angles of nanoﬁbers in each layer also veriﬁes the existence of this mathematical expectation law. 5.2.3. Coreless Yarn Schematic diagram showing the formation of oriented nanofibers. Figure 20. Schematic diagram showing the formation of oriented nanoﬁbers. 5 3 2 Discontinuity of Fiber Length Direction during Mechanical Twisting 5.3.2. Discontinuity of Fiber Length Direction during Mechanical Twisting 5.3.2. Discontinuity of Fiber Length Direction during Mechanical Twisting Figure 20 shows a schematic diagram of the mechanical twisting principle. Oriented nanofibers formed between the disk and the ring are wrapped on the core yarn at an angle with the disk rotation. It can be seen from Figure 20 that the formation of oriented nano- fibers is intermittent, and the formation of the first oriented nanofiber is followed by the formation of the second oriented nanofibers, and so on. During twisting, the first nano- fiber is formed, and the second nanofiber is formed after the disk is rotated for a time (t), and the angle φ between the first and second nanofibers follows the Formula (16): Figure 20 shows a schematic diagram of the mechanical twisting principle. Oriented nanoﬁbers formed between the disk and the ring are wrapped on the core yarn at an angle with the disk rotation. It can be seen from Figure 20 that the formation of oriented nanoﬁbers is intermittent, and the formation of the ﬁrst oriented nanoﬁber is followed by the formation of the second oriented nanoﬁbers, and so on. During twisting, the ﬁrst nanoﬁber is formed, and the second nanoﬁber is formed after the disk is rotated for a time (t), and the angle ϕ between the ﬁrst and second nanoﬁbers follows the Formula (16): (16) (16) ω φ = ϕ = ωt ω φ = ϕ = ωt Polymers 2021, 13, 3189 18 of 20 18 of 20 where ω is the disk rotation speed. where ω is the disk rotation speed. p Formula (18) shows that oriented nanoﬁbers have intermittent formation, but also have a certain angular relationship with each other during mechanical twisting. The discon- tinuity of ﬁber length direction is caused not only by the intermittent formation of oriented nanoﬁbers, but also by nanoﬁber breakage described by the formula:mL0 > Lq(1 + ε . q The mechanical twisting and wrapping process is obviously different from the wrapped structure, in which the outer wrapping yarn is generally continuously wrapped around the core yarn under the action of machinery and ﬂuid. 5.3.3. Non-Uniformity of the Twist Angles during Mechanical Twisting and Wrapping Process The analysis in Sections 5.1 and 5.2 showed that the twist angle remained constant during twisting only under certain conditions. In the ﬁrst case where A > 0, A = 0, the nanoﬁbers are not broken and are ﬁxed in a certain layer in the yarns. The oriented nanoﬁbers are formed at a ﬁxed position between the disk and the ring. This constant twist angle under only certain conditions is identical to the traditional spinning process. Nanoﬁbers have different twist angles in different yarn layers, which is consistent with the characteristics of traditional spinning. In the second case where A > 0 (Section 5.1), the nanoﬁbers not twisted by the disk rotation are broken by the core yarn. In this case, a twisted wrapping is not formed. In other cases, the nanoﬁbers can be wrapped after being broken. Under this condition, the other end of the broken nanoﬁbers is not held but ﬂuctuates irregularly with the surrounding environment (airﬂow, electric ﬁeld, inertia force, and friction force, etc.). Figure 20 is a schematic diagram of nanoﬁbers wrapped on the core yarn after breaking. The red line is the previous layer ﬁber, and the green line is a subsequent layer ﬁber. The broken ﬁber near the end of the disk becomes a ﬂying ﬂower and no longer participates in the twisting wrapping. After breaking, some ﬁbers near the end of the ring fraying outside the yarns, and some are twisted into yarns by the next layer of nanoﬁbers, and the structure of this part of nanoﬁbers is disordered. According to Section 5.2 and the electric ﬁeld simulation analysis, one of the two ends of the oriented nanoﬁbers may be located at any point between the inner and outer diameters of the ring, and the other end may be at any point of the non-central hole of the disk. This rule was conﬁrmed in the actual process of wrapping and twisting the yarns. Therefore, from the twist angle calculation Formulas (1), (5), and (7), we can see that the twist angle of nanoﬁbers is not constant, even within the same layer, and there is certain randomness during twisting and wrapping of the disk and ring. The random expectation E(x) can be introduced to represent the average twist angle. 5.2.3. Coreless Yarn However, the length of the nanoﬁbers during mechanical twisting wrapping herein is discontinuous. 5.3.3. Non-Uniformity of the Twist Angles during Mechanical Twisting and Wrapping Process 5.3.3. Non-Uniformity of the Twist Angles during Mechanical Twisting and Wrapping Process 6. Conclusions In this paper, a self-made device was used to form oriented nanoﬁbers between a disk and ring. By rotating the disk, the oriented nanoﬁbers were twisted into yarns with a speciﬁc angle, and electrospun nanoﬁber yarns and its multiscale wrapping yarns were prepared. The electric ﬁeld simulation analysis of the electrospinning nanoﬁber yarn forming was in agreement with experimental observations. During the preparation of electrospun nanoﬁber yarns, the average twist angle and diameter of the nanoﬁber yarns was correlated with changes in the ring-to-disk distance, the tip and the center of the disk, and the disk rotation speed. When preparing electrospun nanoﬁber-wrapped yarns, oriented nanoﬁbers were arranged in a speciﬁc direction. This paper also discussed the mechanism of the nanoﬁber-wrapped yarn in three cases, according to the displacement difference A of the core yarn within one twist. When A = 0, the starting point of each twist was the same. When the oriented nanoﬁber was twisted to a certain degree, it broke, or the ﬁber tip was pulled out and was no longer held. The nanoﬁbers no longer twisted on the core yarn at a twist angle β, and they irregularly wrapped the core yarn by the latter layer. Compared with traditional spinning processes, the electrospinning nanoﬁber Polymers 2021, 13, 3189 19 of 20 wrapping process has unique properties, such as the uncertainty of the oriented nanoﬁber collection process, the discontinuity of the ﬁber lengthwise direction during twisting, and the non-uniform twist angle. The research conclusion of this paper has guiding signiﬁcance for accurately controlling the mass production of electrospun micro- and nanoﬁbers. In this paper, the 24-h output of pure electrospun yarn was about 672 cm, and the output of electrospun micro/nanocore spun yarn was about 2016 cm. It also has certain industrial application value for high value-added tissue engineering, sensors, and other ﬁelds. If the single needle is replaced by multiple needled or needle-free electrospinning technology, the output will be much higher than that of traditional single-needle electro- spinning. The diameter distribution and yarn strength with the CV% were not analyzed because of software limitations. This paper only prepared micro/nanocore spun yarn using ordinary yarn as the inner layer and nanoﬁber as the outer layer, and it did not study the fabric prepared by this micro/nano-structured yarn or the functionality of this fabric. The research team will carry out further research concerning these limitations. 6. Conclusions Interestingly, we are studying the electrospun nanoﬁber-core spun yarn wrapped with a layer of ordinary yarn. This sandwich yarn will protect the nanoﬁbers in the middle layer, which can be processed by traditional weaving technology; thus, they will have broader potential applications in areas such as supercapacitors and functional textiles. Author Contributions: Conceptualization, T.Y. and Y.S.; methodology, H.Z.; software, Y.L.; valida- tion, T.Y., Y.L. and D.L.; formal analysis, T.Y. and Shengbin Cao; investigation, T.Y. and L.Z.; resources, T.Y.; data curation, T.Y.; writing—original draft preparation, T.Y. and H.Z.; writing—review and editing, T.Y. and Y.L.; visualization, Y.S.; supervision, S.C. and D.L.; project administration, L.Z. and Y.S.; funding acquisition, T.Y. and D.L. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by Fuzhou Science and Technology Achievement Transfer and Transformation Project, grant number 2020-GX-03, the Open Project Program of Key Lab for Sport Shoes Upper Materials of Fujian Province, Fujian Huafeng New Material Co.,Ltd., grant number SSUM217, SSUM215, and Science and Technology Project of Minjiang University, grant number MJY21022. Acknowledgments: Our deepest gratitude goes to the anonymous reviewers for their careful work and thoughtful suggestions that have helped improve this paper substantially. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. References 1. Li, P.; Liu, D.; Zhu, B.; Li, B.; Jia, X.; Wang, L.; Li, G.; Yang, X. Synchronous effects of multiscale reinforced and toughened CFRP composites by MWNTs-EP/PSF hybrid nanoﬁbers with preferred orientation. Compos. Part A Appl. Sci. Manuf. 2015, 68, 72–80. [CrossRef] 1. Li, P.; Liu, D.; Zhu, B.; Li, B.; Jia, X.; Wang, L.; Li, G.; Yang, X. Synchronous effects of multiscale reinforced and toughened CFRP composites by MWNTs-EP/PSF hybrid nanoﬁbers with preferred orientation. Compos. Part A Appl. Sci. Manuf. 2015, 68, 72–80. [CrossRef] [ ] 2. Yan, T.; Wang, Z.; Wang, Y.-Q.; Pan, Z.-J. Carbon/graphene composite nanoﬁber yarns for highly sensitive strain sensors. Mater. Des. 2018, 143, 214–223. [CrossRef] 3. Gao, H.; Asheghali, D.; Yadavalli, N.; Pham, M.T.; Nguyen, T.D.; Minko, S.; Sharma, S. Fabrication of core-sheath nanoyarn via touchspinning and its application in wearable piezoelectric nanogenerator. J. Text. Inst. 2020, 111, 906–915. [CrossRef] h l b l h ll f l h f l 4. Sensini, A.; Santare, M.H.; Eichenlaub, E.; Bloom, E.; Gotti, C.; Zucchelli, A.; Cristofolini, L. Tuning the Structure of Nylon 6,6 Electrospun Bundles to Mimic the Mechanical Performance of Tendon Fascicles. Front. Bioeng. Biotechnol. 2021, 9, 230. [CrossRef] [PubMed] 5. Yang, G.; Li, X.; He, Y.; Ma, J.; Ni, G.; Zhou, S. From nano to micro to macro: Electrospun hierarchically structured polymeric ﬁbers for biomedical applications. Prog. Polym. Sci. 2018, 81, 80–113. [CrossRef] 5. Yang, G.; Li, X.; He, Y.; Ma, J.; Ni, G.; Zhou, S. From nano to micro to macro: Electrospun hierarchically structured polymeric ﬁbers for biomedical applications. Prog. Polym. Sci. 2018, 81, 80–113. [CrossRef] pp g y [ ] 6. Yang, Y.; Zhao, Y.; Quan, Z.; Zhang, H.; Qin, X.; Wang, R.; Yu, J. An efﬁcient hybrid strategy for composite yarns of micro-/nano- ﬁbers. Mater. Des. 2019, 184, 108196. [CrossRef] pp g y 6. Yang, Y.; Zhao, Y.; Quan, Z.; Zhang, H.; Qin, X.; Wang, R.; Yu, J. An efﬁcient hybrid strategy for composite yarns of micro-/nano- ﬁbers. Mater. Des. 2019, 184, 108196. [CrossRef] 7. Zhong, S.; Zhang, Y.; Lim, C.T. Fabrication of Large Pores in Electrospun Nanoﬁbrous Scaffolds for Cellular Inﬁltration: A Review. Tissue Eng. Part B Rev. 2012, 18, 77–87. [CrossRef] [PubMed] 8. Qasim, S.B.; Zafar, M.S.; Najeeb, S.; Khurshid, Z.; Shah, A.H.; Husain, S.; Rehman, I.U. Electrospinning of chitosan-based solutions for tissue engineering and regenerative medicine. Int. J. Mol. Sci. 2018, 19, 407. References 2018, 28, 1801114. [CrossRef] 20. Yan, T.; Zhang, M.; Jiang, J.; Chen, N. Synthesis of DBSA-doped Polyaniline by Emulsion Polymerization and PANI/PLA Electrospun Fiber Membrane Conductivity. J. Text. Inst. 2019, 110, 274–281. [CrossRef] p y J 21. Valtera, J.; Kalous, T.; Pokorny, P. Fabrication of dual-functional composite yarns with a nanoﬁbrous envelope using high throughput AC needleless and collectorless electrospinning. Scientiﬁc reports 2019, 9, 1–10. [CrossRef] [PubMed] g p p g ﬁ p 22. Xu, T.C.; Han, D.H.; Zhu, Y.M.; Duan, G.G.; Liu, K.M.; Hou, H.Q. High Strength Electrospun Single Copolyacrylonitrile (coPAN) Nanoﬁbers with Improved Molecular Orientation by Drawing. Chin. J. Polym. Sci. 2021, 39, 174–180. [CrossRef] 23. Wu, S.H.; Zhang, C.; Qin, X. Progress in Electrospun Aligned Nanoﬁber Bundles and Yarns. Polym. Mater. Sci. Eng. 2014, 30, 182–186. 24. Maleki, H.; Gharehaghaji, A.A.; Dijkstra, P.J. Electrospinning of continuous poly (L-lactide) yarns: Effect of twist on the morphology, thermal properties and mechanical behavior. J. Mech. Behav. Biomed. Mater. 2017, 71, 231–237. 25. Yao, C.; Hui, Y.U.; Jia, Y.; Borisov, L. Inﬂuence of spinning parameters based on gap conductive plate polycaprotactonenanoﬁbers. J. Text. Res. 2016, 37, 11–15. 26. Ko, F.; Gogotsi, Y.; Ali, A.; Naguib, N.; Ye, H.; Yang, G.; Li, C.; Willis, P. Electrospinning of Continuous Carbon Nanotube-Filled Nanoﬁber Yarns. Adv. Mater. 2003, 15, 1161–1165. [CrossRef] 27. Yousefzadeh, M.; Latiﬁ, M.; Teo, W.E.; Tehran, M.A.; Ramakrishna, S. Producing continuous twisted yarn from well-aligned nanoﬁbers by water vortex. Polym. Eng. Sci. 2010, 51, 323–329. [CrossRef] 28. Zhou, F.L.; Gong, R.H.; Porat, I. Nano-wrapped hybrid yarns using electrospinning. Surface Wrap. Tech 28. Zhou, F.L.; Gong, R.H.; Porat, I. Nano-wrapped hybrid yarns using electrospinning. Surface Wrap. Technol. 2010, 204, 3459–3463. 29. Liu, C.; He, H.; Sun, R.; Yu, Q. Inﬂuence of Spinning Process on Covering Property of Core-Spun NanoﬁberYarn. Polym. Mater. Sci. Eng. 2016, 32, 82–86. , ; g, ; , pp y y g p g f p , , 29. Liu, C.; He, H.; Sun, R.; Yu, Q. Inﬂuence of Spinning Process on Covering Property of Core-Spun NanoﬁberYarn. Polym. Mater. Sci. Eng. 2016, 32, 82–86. g 30. Niu, H.; Gao, W.; Lin, T.; Wang, X.; Kong, L. Composite yarns fabricated from continuous needleless electrospunnanoﬁbers. Polym. Eng. Sci. 2014, 54, 1495–1502. y g 31. Katnam, K.B.; Dalﬁ, H.; Potluri, P. Towards balancing in-plane mechanical properties and impact damage tolerance of composite laminates using quasi-UD woven fabrics with hybrid warp yarns. Compos. Struct. References [CrossRef] g g g 9. Zafar, M.; Najeeb, S.; Khurshid, Z.; Vazirzadeh, M.; Zohaib, S.; Najeeb, B.; Sefat, F. Potential of Electrospun Nanoﬁbers for Biomedical and Dental Applications. Materials 2016, 9, 73. [CrossRef] [PubMed] 20 of 20 Polymers 2021, 13, 3189 10. Jiang, C.; Wang, K.; Liu, Y.; Zhang, C.; Wang, B. Using Wet Electrospun PCL/Gelatin/CNT Yarns to Fabricate Textile-Based Scaffolds for Vascular Tissue Engineering. ACS Biomater. Sci. Eng. 2021, 7, 2627–2637. [CrossRef] g g g 11. Zheng, Y.; Panatdasirisuk, W.; Liu, J.; Tong, A.; Xiang, Y.; Yang, S. Patterned, Wearable UV Indicators from Electrospun Photochromic Fibers and Yarns. Adv. Mater. Technol. 2020, 5, 2000564. [CrossRef] 12. Yang, E.; Xu, Z.; Chur, L.K.; Behroozfar, A.; Baniasadi, M.; Moreno, S.; Huang, J.; Gilligan, J.; Minary-Jolandan, M. Nanoﬁbrous Smart Fabrics from Twisted Yarns of Electrospun Piezopolymer. ACS Appl. Mater. Interfaces 2017, 9, 24220–24229. [CrossRef] Yang, E.; Xu, Z.; Chur, L.K.; Behroozfar, A.; Baniasadi, M.; Moreno, S.; Huang, J.; Gilligan, J.; Minary-Jolan S t F b i f T i t d Y f El t Pi l ACS A l M t I t f 2017 9 24220 p p y pp f , , [ ] 13. Wu, S.; Liu, P.; Zhang, Y.; Zhang, H.; Qin, X. Flexible and conductive nanoﬁber-structured single yarn sensor for smart wearable devices. Sens. Actuators B Chem. 2017, 252, 697–705. [CrossRef] 14. Busolo, T.; Szewczyk, P.K.; Nair, M.; Stachewicz, U.; Kar-Narayan, S. Triboelectric Yarns with Electrospun Functional Polymer Coatings for Highly Durable and Washable Smart Textile Applications. ACS Appl. Mater. Interfaces 2021, 13, 16876–16886. [CrossRef] 15. Dai, Z.; Yan, F.; Qin, M.; Yan, X. Fabrication of ﬂexible SiO2 nanoﬁbrous yarn via a conjugate electrospinning process. e-Polymers 2020, 20, 600–605. [CrossRef] 16. Qiu, Q.; Chen, S.; Li, Y.; Yang, Y.; Zhang, H.; Quan, Z.; Qin, X.; Wang, R.; Yu, J. Functional nanoﬁbers embedded into textiles for durable antibacterial properties. Chem. Eng. J. 2020, 384, 123241. [CrossRef] 17. Sensini, A.; Gualandi, C.; Focarete, M.L.; Belcari, J.; Zucchelli, A.; Boyle, L.; Reilly, G.C.; Kao, A.P.; Tozzi, G.; Cristofolini, L. Multiscale hierarchical bioresorbable scaffolds for the regeneration of tendons and ligaments. Biofabrication 2019, 11, 035026. [CrossRef] 18. Chen, R.; Liu, J.; Sun, Z.; Chen, D. Functional Nanoﬁbers with Multiscale Structure by Electrospinning. Nanofabrication 2018, 4, 17–31. [CrossRef] 19. Hou, L.; Wang, N.; Wu, J.; Cui, Z.; Jiang, L.; Zhao, Y. Bioinspired Superwettability Electrospun Micr Applications. Adv. Funct. Mater. References 2019, 225, 111083. [CrossRef] 32 Jin S ; Xin B ; Zheng Y Preparation and characterization of polysulfone amide nanoyarns by the dynamic rotating electrospinning y g 31. Katnam, K.B.; Dalﬁ, H.; Potluri, P. Towards balancing in-plane mechanical properties and impact da laminates using quasi-UD woven fabrics with hybrid warp yarns. Compos. Struct. 2019, 225, 111083 g q y p y p , , [ ] 32. Jin, S.; Xin, B.; Zheng, Y. Preparation and characterization of polysulfone amide nanoyarns by the dynamic rotating electrospinning method. Text. Res. J. 2019, 89, 52–62. [CrossRef]
https://openalex.org/W2051441438	https://periodicos.ufsc.br/index.php/biotemas/article/download/2175-7925.2013v26n4p179/25714	Portuguese	null	Anatomia descritiva da traqueia do macaco-prego (Sapajus apella)	Biotemas	2,013	cc-by	2,563	Luciano César Pereira Campos Leonel * Tânia Cristina Lima Rodrigo Lopes de Felipe Elaine Maria da Silva Gustavo Alexandre de Oliveira Silva Daniela Cristina de Oliveira Silva Roseâmely Angélica de Carvalho-Barros Zenon Silva Universidade Federal de Goiás Avenida Margon, 890, Margon II, CEP 75711-020, Catalão – GO, Brasil * Autor para correspondência enf.luciano@yahoo.com.br Submetido em 28/06/2013 Aceito para publicação em 16/07/2013 Submetido em 28/06/2013 Aceito para publicação em 16/07/2013 Resumo O estudo descritivo da anatomia de animais silvestres experimenta, nos dias atuais, inegável importância. O cerrado constitui um bioma complexo que abriga uma grande variedade de espécies, entre elas, o macaco-prego (Sapajus apella); foram utilizados quatro espécimes, comparando os resultados encontrados com a literatura humana e veterinária, já bem estabelecidas. Os espécimes foram fixados em solução aquosa de formol a 10%, dissecados, analisados descritivamente e fotografados. Os resultados evidenciam uma traqueia cuja extensão varia conforme a compleição física do animal, um número variável de anéis cartilaginosos incompletos, sendo o fechamento do tubo, na face dorsal, feito por tecido musculomembranoso. A parede musculomembranosa não é uniforme em toda a sua extensão, exibindo maior largura no terço médio-cranial. Então, a traqueia do S. apella não diverge muito daquela observada no homem e animais domésticos, sendo as variações, aparentemente, relacionadas às adaptações à compleição física do animal. Palavras-chave: Anatomia; Anatomia comparada; Anatomia veterinária; Traqueia Biotemas, 26 (4): 179-183, dezembro de 2013 ISSNe 2175-7925 Biotemas, 26 (4): 179-183, dezembro de 2013 ISSNe 2175-7925 Biotemas, 26 (4): 179-183, dezembro de 2013 ISSNe 2175-7925 179 http://dx.doi.org/10.5007/2175-7925.2013v26n4p179 Revista Biotemas, 26 (4), dezembro de 2013 Introdução é comprovada a extinção tanto de espécies exclusivas do território nacional, já cientificamente, bem conhecidas, como de espécies que sequer foram estudadas. O estudo comparativo-descritivo da anatomia de animais silvestres está em fase de franco crescimento, no Brasil, e em outras partes do mundo, principalmente agora quando o bioma natural de muitos grupos sofre múltiplas agressões, obrigando muitas espécies a migrarem em busca de maior segurança alimentar, reprodutiva e porque não, de sua própria sobrevivência, ou então, engrossando cada vez mais o rol das espécies em risco ou em processo de extinção. Assim sendo, face à conjuntura atual, a comunidade científica vem se preocupando cada vez mais, em estudar e desenvolver trabalhos que possam contribuir com a melhoria do conhecimento, assim como fornecer subsídios para a preservação de espécies ameaçadas. As variantes como biologia comportamental, fisiologia e morfologia comparativa são instrumentos de grande importância nessa nova abordagem de conhecimento (MOURA et al., 2012). O Sapajus apella é um primata de médio porte, cuja massa corporal varia até 5-6 kg, apresenta larga distribuição ao longo da América Central e do Sul e é bastante versátil, vivendo em matas, Caatinga e, principalmente, áreas de restingas e cerrados. É um primata que se adapta com razoável facilidade, inclusive se reproduzindo em cativeiro sem maiores problemas. Muito embora possa ser considerado, talvez o primata não humano mais inteligente do novo mundo, ainda é quase desconhecido, no que se refere à sua Anatomia. O Cerrado é um bioma complexo, que abriga uma grande variedade de espécies, incluindo muitos mamíferos em risco de extinção. Não obstante há espécies que adotaram o cerrado como lar, depois que tiveram seus habitats alterados ou destruídos, principalmente, por ação humana. Segundo Aguiar e Camargo (2004), este bioma corresponde à aproximadamente 21% do território nacional, com uma área de 1,8 milhões de km², porém ao longo dos anos, com sua intensa exploração e destruição acredita-se que nos tenha restado apenas 43% de sua área original (MEDEIROS, 2012). Por outro lado, há tempos vivenciamos situações de conflito entre o homem e a natureza, mesmo quando se tenta educar a sociedade no sentido de utilizar os recursos naturais de forma sustentável. Abstract Descriptive anatomy of trachea in the tufted capuchin monkey (Sapajus apella). The descriptive study of wild animals’ anatomy enjoys an undeniable importance nowadays. Cerrado constitutes a complex biome which is home to a wide variety of species, among them the tufted capuchin monkey (Sapajus apella); four specimens were used, comparing the results found to the human and veterinary literature, which are already well- established. The specimens were fixed in a 10% formalin aqueous solution, dissected, analyzed in a descriptive Revista Biotemas, 26 (4), dezembro de 2013 L. C. P. C. Leonel et al. 180 way, and photographed. The results show a trachea whose length varies according to the animal’s physique, a variable number of incomplete cartilage rings, and the tube closure, in the dorsal face, occurs by means of musculomembranous tissue. The musculomembranous wall isn’t uniform throughout its length, showing the greatest width at the middle cranial third. So, the trachea in S. apella doesn’t differ much from that observed in the human being and domestic animals, and the variations are, seemingly, related to adaptations to the animal’s physique. Key words: Anatomy; Comparative anatomy; Trachea; Veterinary anatomy Revista Biotemas, 26 (4), dezembro de 2013 Resultados macaco-prego (S. apella) mostra importância sob os aspectos anatômicos, fisiológicos e evolutivos, sendo o enfoque desta pesquisa comparar a Anatomia da traqueia do macaco-prego (S. apella) com a do homem, vez que ambos os grupos são primatas, porém filogeneticamente em posições diferentes, embora próximas e a Anatomia do Homem já se encontra bem estabelecida. Com base nas dissecações verifica-se que a traqueia do S. apella é um tubo aerífero cilíndrico, porém, um pouco achatado dorso-ventralmente que se estende desde a borda caudal da cartilagem cricóide (Figura 1a; 2a) até a origem dos brônquios principais esquerdo e direito. A traqueia deste animal é constituída por 29 a 30 anéis cartilaginosos incompletos (Figura 1d), os quais na porção dorsal são formados, não por cartilagem, mas por uma parede musculomembranosa, o ‘músculo traqueal’ (Figura 2b). A largura desta parede não é uniforme em toda a extensão da traqueia, mostrando-se mais larga no terço médio-cranial. Introdução Para Benite e Mamede (2008), a proposta de educação ambiental representa uma forma de interação entre ciências, e a mesma torna- se indispensável nas novas políticas de uma sociedade sustentável, gerando muito mais que preocupações, mas também a sensibilização e direcionamento nas tomadas de decisões para estratégias de conservação. Um dos fatores que explica a perda de áreas do cerrado é, principalmente, a disponibilidade de partes que ainda não foram exploradas comercialmente, uma vez que houve o esgotamento das áreas cultiváveis na porção central e sul do bioma, além deste bioma oferecer condições físicas que permitem o avanço da pastagem e demais culturas agrícolas (BOCCHIGLIERI et al., 2010). Conhecer a biologia de animais do cerrado, entre eles o Sapajus apella, faz parte das contribuições que norteiam a organização e implantação de programas de proteção e preservação. O sistema respiratório do É fato que a exploração indiscriminada da fauna e da flora brasileira tem, ao longo dos tempos, produzido sérias consequências, não apenas para o meio ambiente natural, mas também para o Homem que usufrui dele. Já Anatomia descritiva da traqueia do Sapajus apella 181 FIGURA 1: Fotomacrografia da traqueia do macaco-prego (Sapajus apella). Vista ventral: a-borda caudal da cartilagem cricóidea; b – anel traqueal; c – ligamentos anulares; d – anéis incompletos, e – anel bifurcado, f – brônquio principal esquerdo, g – brônquio principal direito, (– 20% : 11,5 cm). Material e Métodos A presente pesquisa foi aprovada pelo Comitê de Ética em Pesquisa da Universidade Federal de Goiás com o número do protocolo 045/12. Neste estudo foram utilizados quatro espécimes de macaco-prego (S. apella). A amostra, composta por três machos e uma fêmea foi doada pelo IBAMA – MG, após óbito por causas variadas. Cada espécime foi fixado com solução aquosa de formol a 10% e nela conservado até o processamento (dissecação) para análise. Depois de fixados, os animais foram dissecados e analisados à luz de técnicas usuais em Anatomia Macroscópica. Alguns dos anéis componentes da traqueia são ramificados (Figura 1e) e outros não. Na porção caudal o músculo traqueal é muito estreito, porque as extremidades dos anéis tendem a ser muito próximas, porém, não se unem. Na extremidade cranial a traqueia está conectada à cartilagem cricóide, por meio da membrana cricotraqueal e cada um dos anéis cartilaginosos da traqueia unem-se aos outros por meio do ligamento anular (Figura 1c). Em sua extremidade caudal a traqueia divide-se em dois ramos: o Brônquio Principal Esquerdo (Figura 1f) e Brônquio Principal Direito (Figura 1g). FIGURA 1: Fotomacrografia da traqueia do macaco-prego (Sapajus apella). Vista ventral: a-borda caudal da cartilagem cricóidea; b – anel traqueal; c – ligamentos anulares; d – anéis incompletos, e – anel bifurcado, f – brônquio principal esquerdo, g – brônquio principal direito, (– 20% : 11,5 cm). FIGURA 1: Fotomacrografia da traqueia do macaco-prego (Sapajus apella). Vista ventral: a-borda caudal da cartilagem cricóidea; b – anel traqueal; c – ligamentos anulares; d – anéis incompletos, e – anel bifurcado, f – brônquio principal esquerdo, g – brônquio principal direito, (– 20% : 11,5 cm). Revista Biotemas, 26 (4), dezembro de 2013 Revista Biotemas, 26 (4), dezembro de 2013 182 L. C. P. C. Leonel et al. a da traqueia do macaco-prego (Sapajus apella). Vista dorsal: a – cartilagem cricóidea; b – músculo traqueal, m brônquios principais, (– 20% : 17,3 cm). Revista Biotemas, 26 (4), dezembro de 2013 Referências grande variação quanto ao número dos mesmos. Assim, no cão doméstico há 42 a 46 anéis; 38 a 48 no gato, 32 a 36 em suínos, 48 a 60 em equinos e ruminantes (HARE, 2008). No Saimiri sciureus, segundo Pinheiro et al. (2012), o número de anéis varia entre 30 e 36 nos animais jovens e 28 a 32 nos adultos. A grande variabilidade relativa ao número de anéis traqueais nos diferentes grupos sugere a ausência de associação entre o número de anéis, quer seja inter ou intra-específica. Além disso, animais com pescoço mais curto podem exibir um número maior de anéis e vice-versa. AGUIAR, L. M. S.; CAMARGO, R. B.; MARINHO-FILHO, J. A diversidade biológica do Cerrado. In: AGUIAR, L. M. S.; AGUIAR, L. M. S.; CAMARGO, R. B.; MARINHO-FILHO, J. A diversidade biológica do Cerrado. In: AGUIAR, L. M. S.; CAMARGO, A. J. A. (Ed.). Cerrado: ecologia e caracterização. Planaltina: Embrapa Cerrados; Brasília. 2004. p. 17-40. CAMARGO, A. J. A. (Ed.). Cerrado: ecologia e caracterização. Planaltina: Embrapa Cerrados; Brasília. 2004. p. 17-40. BENITES, M.; MAMEDE, S. B. Mamíferos e aves como instrumentos de educação e conservação ambiental em corredores de biodiversidade do Cerrado, Brasil. Mastozoologia Neotropical, Mendoza, v. 15, n. 2, p. 261-271, 2008. BENITES, M.; MAMEDE, S. B. Mamíferos e aves como instrumentos de educação e conservação ambiental em corredores BOCCHIGLIERI, A.; MENDONÇA, A. F.; HENRIQUES, R. P. B. Composição e diversidade de mamíferos de médio e grande porte no Cerrado do Brasil central. Biota Neotropical, Campinas, v. 10, n. 3, p. 169-176, 2010. HARE, W. C. D. Sistema respiratório do carnívoro. In: GETTY, R. (Ed.). Sisson & Grossman: anatomia dos animais domésticos. Vol. 2. 5. ed. Rio de Janeiro: Guanabara Koogan, 2008. p. 1465-1480. Por outro lado, a largura dos anéis é igualmente um aspecto variável na espécie estudada, alguns podem ser ramificados e anastomoses podem ocorrer entre os ramos. Os resultados desta pesquisa corroboram com a descrição encontrada na literatura de Raven (1950), em gorila, de Testut e Latarjet (1979), em humanos e a de Hare (2008), em animais domésticos. HILL, W. C. O. Primates comparative anatomy and taxonomy II Haplorhini: Tarsioidea. Edinburgh: University Press, 1955. 189 p. HILL, W. C. O. Primates comparative anatomy and taxonomy V Cebidae. Edinburgh: University Press, 1962. 414 p. HILL, W. C. O. Primates comparative anatomy and taxonomy III Pithecoidea Platyrrhini. Edinburgh: University Press, 1957. 165 p. Discussão Lorisoidae, nos quais é constituída por anéis em formato de ‘C’ (HILL, 1955). Semelhante descrição é feita em humanos (TESTUT; LATARJET, 1979), e em animais domésticos (HARE, 2008). A traqueia do macaco-prego, conforme observações verificadas neste trabalho está constituída por anéis cartilaginosos incompletos, cujo número varia entre 29 a 30, mas esse número não está relacionado ao comprimento do pescoço, tão pouco está associado ao porte do animal. O formato em ‘C’ descrito por Testut e Latarjet (1979), em humanos e por Hill (1955) em Lorisoidea não foi observado no S. apella, vez que as extremidades livres dos semianéis encontram-se muito próximas uma da outra, por vezes exibindo apenas um filete de membrana. A literatura compulsada neste artigo indica consideráveis divergências nesse aspecto, no gorila há descrições de 17 anéis (RAVEN, 1950). Já o gênero Aloautta exibe 14 a 15 anéis, Brachyteles e Lagothrix, 22 anéis e Ateles 25 anéis (HILL, 1962). Uma análise acurada das peças anatômicas utilizadas nesta pesquisa revela que a traqueia do macaco-prego é um tubo aproximadamente cilíndrico, levemente achatado dorso-ventralmente, que se estende desde a borda distal de cartilagem cricóide até o interior do tórax, findando em uma bifurcação que dá origem aos brônquios primários direito e esquerdo. Esses achados estão de acordo com as observações dos estudos de Testut e Latarjet (1979), em humanos, e Hare (2008), em carnívoros domésticos. No S. apella, a traqueia ocupa posição mediana, ventralmente ao esôfago e dorsalmente aos músculos ventrais do pescoço por, aproximadamente, toda a sua extensão, mas nos segmentos caudais está um pouco deslocada para a direita, empurrada pela aorta. Esses resultados não concordam com as citações de Raven (1950), quando descreve a traqueia do gorila, no qual a mesma é deslocada à esquerda do esôfago ao nível de T1 e à direita no mediastino superior. Enquanto isso, a traqueia encontra-se posicionada no plano sagital, em A literatura descreve a traqueia humana com um número aproximado de 15 a 20 anéis (TESTUT; LATARJET, 1979), igualmente, em outros táxons ocorre Anatomia descritiva da traqueia do Sapajus apella Anatomia descritiva da traqueia do Sapajus apella 183 Referências No gorila foram descritas duas placas bifurcadas (RAVEN, 1950), ocorre ainda bifurcação em Lorisoidea e Pithecoidea, alguns anéis são bifurcados e a sua descontinuidade é maior cranialmente em relação aos segmentos caudais (HILL, 1955). Em Hapalideos alguns anéis são bifurcados (HILL, 1957). A largura e o número de anéis ao longo de toda a extensão da traqueia variam consideravelmente de espécie para espécie (TESTUT; LATARJET, 1979; HARE, 2008), mas nossas observações em macaco-prego sugerem variabilidade inclusive intra-específica. MEDEIROS, K. M. O planejamento ambiental e exploratório no bioma do Cerrado. Revista Facitec, Taguatinga, v. 1, n. 1, s/ paginação, 2007. MOURA, C. E. B.; ALBUQUERQUE, J. F. G.; MAGALHÃES, M. S.; SILVA, N. B.; OLIVEIRA, M. F.; PAPA, P. C. Análise comparativa da origem do plexo braquil de catetos (Tayassu tajacu). Pesquisa Veterinária Brasileira, Brasília, v. 27, n. 09, p. 357-362, 2007. PINHEIRO, L. L.; LIMA, A. R.; MUNIZ, J. A. P. C.; IMBELONI, A.; FIORETO, E. T.; FONTES, R. F.; CABRAL, R.; BRANCO, E. Anatomy and morphometric aspects of the trachea of Saimiri sciureus Linnaues, 1758: knowledge for emergency procedures. Anais da Academia Brasileira de Ciências, Rio de Janeiro, v. 84, n. 4, p. 973-977, 2012. Anais da Academia Brasileira de Ciências, Rio de Janeiro, v. 84, n. 4, p. 973-977, 2012. A estrutura anatômica da traqueia do S. apella não apresenta grandes divergências em relação à morfologia da traqueia de humanos e de animais domésticos. RAVEN, H. C. The Anatomy of the Gorilla. New York: Columbia University Press, 1950. 190 p. TESTUT, L.; LATARJET, A. Aparato de la repiracion y de la fonacion. In: TESTUT, L.; LATARJET, A. (Ed.). Tratado de anatomia humana. São Paulo: Ed. Salvat, 1979. p. 881-1021. Revista Biotemas, 26 (4), dezembro de 2013
https://openalex.org/W4200358872	https://figshare.com/ndownloader/files/31746491	English	null	Data_Sheet_1.PDF	null	2,021	cc-by	4,649	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 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 B C D E F G H I J K L YEAR N° SEX DIAGNOSIS AGE LAB CODE CH NUMBER CLINICAL DIAGNOSIS ORIGIN CITY RESULTS ALLELE WEIGHT CGG REPETITION 1 M 11 ADN 092 442724 Fragile X Quito ND 300 27 2 M 5 ADN 127 386256 Fragile X Pasaje ND 300 27 3 M 9 ADN 128 344450 Fragile X Guayaquil ND 300 27 4 M 6 ADN 162 Fragile X Quito ND 300 27 5 M 9 ADN 186 Fragile X Quito ND 300 27 6 M 9 ADN 194 453974 Fragile X Quito, Carapungo ND 350 43 7 M 8 ADN 217 353692 Fragile X Quito ND 350 43 8 M 11 ADN 223 Fragile X Loja ND 300 27 9 M 9 ADN 228 457250 Fragile X Quito ND 350 43 10 M 13 ADN 238 458355 Fragile X Quito D 11 M 6 ADN 250 460331 Fragile X Quito ND 300 27 12 M 7 ADN 268 377128 Develomental and Language Delay Esmeraldas ND 300 27 13 M 3 ADN 278 463979 Fragile X and Language Delay Guayaquil ND 320 33 14 M 2 ADN 275 430220 Fragile X Quitumbe ND 325 35 15 M 10 ADN 340 470456 ADHD Guayas ND 294 24 16 M 2 ADN 369 473369 Fragile X, developmental delay Chillogallo ND 325 35 17 M 12 ADN 372 473890 Fragile X Lago Agrio ND 325 35 18 M 14 ADN 383 475481 Intelectual disability Quito ND 325 35 19 M 3 ADN 391 475930 Fragile X Esmeraldas ND 285 21 20 M 21 ADN 433 191019 Intelectual disability Quito ND 285 21 21 M 1 ADN 444 481600 Fragile X Manta ND 272 18 22 M 7 ADN 445 481717 Fragile X Quito ND 309 30 23 M 7 ADN 468 482976 Develomental and Language Delay Manta ND 309 30 24 M 5 ADN 490 484145 Intelectual disability Guayaquil ND 262 14 25 M 2 ADN 523 485987 Intelectual disability Quito D 26 M 8 ADN 528 486388 Intelectual disability Ciudadela del Ejército ND 269 16 27 M 14 ADN 540 487097 Intelectual disability Sevilla Sucumbíos ND 257 12 28 M 13 ADN 552 336881 Fragile X Quito ND 265 15 29 M 11 ADN 553 487825 Intelectual disability Fragile X Quito ND 269 16 30 M 6 ADN 618 491224 Intelectual disability Quevedo ND 314 31 31 M 15 ADN 681 494504 Motor delay Guayaquil ND 315 31 32 M 3 ADN 702 495633 Intelectual disability Quito - Carcelen ND 325 34 33 M 13 ADN 704 436764 Other Congenital Malformations not classified Ambato ND 320 33 34 M 16 ADN 732 294626 Other Congenital Malformations not classified Quito ND 290 23 35 M 19 ADN 745 314697 Other Congenital Malformations not classified Quito ND 313 31 36 M 4 ADN 791 501056 Intelectual disability Quito ND 325 35 37 M 7 ADN 807 424350 ASD Quito D 38 M 14 ADN 810 501706 Other Congenital Malformations not classified Quito ND 334 38 39 M 10 ADN 832 502500 Other Congenital Malformations not classified Quito ND 296 25 40 M 14 ADN 835 502672 Other Congenital Malformations not classified Quito ND 296 25 41 M 13 ADN 842 502751 Other Congenital Malformations not classified Quito ND 318 33 42 M 13 ADN 843 502753 Other Congenital Malformations not classified Quito ND 321 34 43 M 8 ADN 849 502898 Other Congenital Malformations not classified Quito ND 321 34 44 M 13 ADN 857 503179 Other Congenital Malformations not classified Quito ND 297 26 45 M 11 ADN 863 503261 Other Congenital Malformations not classified Quito ND 297 26 46 M 12 ADN 865 503237 Other Congenital Malformations not classified Quito ND 307 29 47 M 2 ADN 869 480959 Other Congenital Malformations not classified Quito ND 306 29 48 M 9 ADN 898 503863 Other Congenital Malformations not classified Quito ND 302 27 49 M 24 ADN 899 462520 Epilepsy Quito ND 297 26 50 M 14 ADN 907 504056 Other Congenital Malformations not classified Quito ND 300 27 51 M 5 ADN 911 504157 Other Congenital Malformations not classified Quito ND 300 27 52 M 6 ADN 920 504579 Intelectual disability Quito ND 277 19 53 M 10 ADN 928 504841 Other Congenital Malformations not classified Quito ND 301 27 54 M 12 ADN 930 504577 Other Congenital Malformations not classified Quito ND 284 21 55 M 9 ADN 945 504812 Other Congenital Malformations not classified Quito ND 289 23 56 M 9 ADN 946 505294 Fragile X Quito D 57 M 13 ADN 951 505759 Other Congenital Malformations not classified Quito ND 298 26 58 M 9 ADN 962 444305 ADHD Quito ND 299 26 59 M 5 ADN 978 452772 Fragile X Sangolquí D 60 M 16 ADN 987 507117 Other Congenital Malformations not classified Carchi ND 318 32 61 M 12 ADN 1046 509264 Other Congenital Malformations not classified Quito ND 333 37 62 M 14 ADN 1047 509280 Other Congenital Malformations not classified Quito ND 271 16 63 M 10 ADN 1048 509382 Other Congenital Malformations not classified Pedro Vicente Maldonado ND 296 25 64 M 20 ADN 1051 229350 Other Congenital Malformations not classified Quito ND 288 22 65 M 4 ADN 1064 468821 Develomental and Language Delay ND 285 22 66 M 13 ADN 1077 510244 Other Congenital Malformations not classified ND 303 28 67 M 5 ADN 1078 510200 ADHD ND 303 28 68 M 13 ADN 1100 509822 Fragile X ND 304 28 69 M 15 ADN 1103 428031 Other Congenital Malformations not classified ND 304 28 70 M 4 ADN 1112 489784 ASD ND 314 31 71 M 15 ADN 1117 313743 ADHD ND 325 35 72 M 12 ADN 1132 510397 Other Congenital Malformations not classified ND 293 24 73 M 6 ADN 1139 508546 Other Congenital Malformations not classified ND 290 23 74 M 2 ADN 1154 511372 ASD ND 307 29 75 M 7 ADN 1102 511057 Fragile X ND 316 32 76 M 17 ADN 1177 512550 Other Congenital Malformations not classified ND 306 29 77 M 9 ADN 1179 433568 ASD ND 304 29 78 M 6 ADN 1184 511446 Other Congenital Malformations not classified ND 294 25 79 M 2 ADN 1198 512629 ASD ND 273 18 80 M 17 ADN 1199 512904 Other Congenital Malformations not classified ND 305 28 81 M 6 ADN 1225 513410 Language delay ND 305 28 82 M 13 ADN 1226 513461 Fragile X ND 291 24 83 M 2 ADN 1231 495874 Macrocephaly lia ND 291 24 84 M 3 ADN 1234 510361 Other Congenital Malformations not classified ND 311 30 85 M 7 ADN 1238 513676 Fragile X ND 291 23 86 M 8 ADN 1243 513739 Fragile X ND 296 25 87 M 15 ADN 1251 323328 Intelectual Disability ND 314 31 88 M 7 ADN 1253 513962 Fragile X ND 314 31 89 M 2 ADN 1255 492911 Language delay ND 310 30 90 M 10 ADN 1258 513959 Fragile X ND 309 30 91 M 5 ADN 1270 514190 Other Congenital Malformations not classified ND 278 19 92 M ADN 1274 514239 Fragile X D 93 M 9 ADN 1278 514360 Other Congenital Malformations not classified ND 314 31 94 M 9 ADN 1287 513457 Intelectual Disability ND 290 23 95 M 24 ADN 1303 514815 Fragile X ND 295 25 96 M 7 ADN 1305 514819 Fragile X D 97 M 9 ADN 1306 514818 Fragile X D 98 M 13 ADN 1311 514854 Fragile X D 99 M 16 ADN 1312 514855 Fragile X D 100 M 3 ADN 1318 510067 ASD ND 325 35 101 M 9 ADN 1319 514937 Other Congenital Malformations not classified ND 325 35 102 M 1 ADN 1322 513875 Other Congenital Malformations not classified ND 322 34 103 M 0 ADN 1364 515581 Fragile X ND 322 34 104 M 10 ADN 1372 468312 ASD ND 294 25 105 M 8 ADN 1375 515640 Fragile X ND 294 25 106 M 1 ADN 1386 515927 Developmental delay ND 309 29 107 M 16 ADN 1398 513693 ASD ND 305 29 108 M 22 ADN 1411 516188 ASD ND 343 41 109 M 20 ADN 1412 516189 ASD ND 310 30 110 M 17 ADN 1415 516264 Fragile X D 111 M 17 ADN 1416 516263 Fragile X D 112 M 16 ADN 1418 516298 Intelectual Disability ND 286 22 113 M 1 ADN 1422 516338 Epilepsy ND 292 24 114 M 1 ADN 1428 516412 Other Congenital Malformations not classified ND 298 26 115 M 2 ADN 1429 495718 ASD ND 298 26 116 M 3 ADN 1467 517008 ASD ND 289 23 117 M 1 ADN 1476 503877 Motor delay ND 310 30 118 M 7 ADN 1484 517371 Fragile X D 119 M 2 ADN 1486 517552 Fragile X ND 307 29 120 M 11 ADN 1506 396307 Intelectual Disability ND 277 19 2017 2016 2011 2012 2013 2014 2015 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 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 B C D E F G H I J K L YEAR N° SEX DIAGNOSIS AGE LAB CODE CH NUMBER CLINICAL DIAGNOSIS ORIGIN CITY RESULTS ALLELE WEIGHT CGG REPETITION 1 M 11 ADN 092 442724 Fragile X Quito ND 300 27 2 M 5 ADN 127 386256 Fragile X Pasaje ND 300 27 3 M 9 ADN 128 344450 Fragile X Guayaquil ND 300 27 4 M 6 ADN 162 Fragile X Quito ND 300 27 5 M 9 ADN 186 Fragile X Quito ND 300 27 6 M 9 ADN 194 453974 Fragile X Quito, Carapungo ND 350 43 7 M 8 ADN 217 353692 Fragile X Quito ND 350 43 8 M 11 ADN 223 Fragile X Loja ND 300 27 9 M 9 ADN 228 457250 Fragile X Quito ND 350 43 10 M 13 ADN 238 458355 Fragile X Quito D 11 M 6 ADN 250 460331 Fragile X Quito ND 300 27 12 M 7 ADN 268 377128 Develomental and Language Delay Esmeraldas ND 300 27 13 M 3 ADN 278 463979 Fragile X and Language Delay Guayaquil ND 320 33 14 M 2 ADN 275 430220 Fragile X Quitumbe ND 325 35 15 M 10 ADN 340 470456 ADHD Guayas ND 294 24 16 M 2 ADN 369 473369 Fragile X, developmental delay Chillogallo ND 325 35 17 M 12 ADN 372 473890 Fragile X Lago Agrio ND 325 35 18 M 14 ADN 383 475481 Intelectual disability Quito ND 325 35 19 M 3 ADN 391 475930 Fragile X Esmeraldas ND 285 21 20 M 21 ADN 433 191019 Intelectual disability Quito ND 285 21 21 M 1 ADN 444 481600 Fragile X Manta ND 272 18 22 M 7 ADN 445 481717 Fragile X Quito ND 309 30 23 M 7 ADN 468 482976 Develomental and Language Delay Manta ND 309 30 24 M 5 ADN 490 484145 Intelectual disability Guayaquil ND 262 14 25 M 2 ADN 523 485987 Intelectual disability Quito D 26 M 8 ADN 528 486388 Intelectual disability Ciudadela del Ejército ND 269 16 27 M 14 ADN 540 487097 Intelectual disability Sevilla Sucumbíos ND 257 12 28 M 13 ADN 552 336881 Fragile X Quito ND 265 15 29 M 11 ADN 553 487825 Intelectual disability Fragile X Quito ND 269 16 30 M 6 ADN 618 491224 Intelectual disability Quevedo ND 314 31 31 M 15 ADN 681 494504 Motor delay Guayaquil ND 315 31 32 M 3 ADN 702 495633 Intelectual disability Quito - Carcelen ND 325 34 33 M 13 ADN 704 436764 Other Congenital Malformations not classified Ambato ND 320 33 34 M 16 ADN 732 294626 Other Congenital Malformations not classified Quito ND 290 23 35 M 19 ADN 745 314697 Other Congenital Malformations not classified Quito ND 313 31 36 M 4 ADN 791 501056 Intelectual disability Quito ND 325 35 37 M 7 ADN 807 424350 ASD Quito D 38 M 14 ADN 810 501706 Other Congenital Malformations not classified Quito ND 334 38 39 M 10 ADN 832 502500 Other Congenital Malformations not classified Quito ND 296 25 40 M 14 ADN 835 502672 Other Congenital Malformations not classified Quito ND 296 25 41 M 13 ADN 842 502751 Other Congenital Malformations not classified Quito ND 318 33 42 M 13 ADN 843 502753 Other Congenital Malformations not classified Quito ND 321 34 43 M 8 ADN 849 502898 Other Congenital Malformations not classified Quito ND 321 34 44 M 13 ADN 857 503179 Other Congenital Malformations not classified Quito ND 297 26 45 M 11 ADN 863 503261 Other Congenital Malformations not classified Quito ND 297 26 46 M 12 ADN 865 503237 Other Congenital Malformations not classified Quito ND 307 29 47 M 2 ADN 869 480959 Other Congenital Malformations not classified Quito ND 306 29 48 M 9 ADN 898 503863 Other Congenital Malformations not classified Quito ND 302 27 49 M 24 ADN 899 462520 Epilepsy Quito ND 297 26 50 M 14 ADN 907 504056 Other Congenital Malformations not classified Quito ND 300 27 51 M 5 ADN 911 504157 Other Congenital Malformations not classified Quito ND 300 27 52 M 6 ADN 920 504579 Intelectual disability Quito ND 277 19 53 M 10 ADN 928 504841 Other Congenital Malformations not classified Quito ND 301 27 54 M 12 ADN 930 504577 Other Congenital Malformations not classified Quito ND 284 21 55 M 9 ADN 945 504812 Other Congenital Malformations not classified Quito ND 289 23 56 M 9 ADN 946 505294 Fragile X Quito D 57 M 13 ADN 951 505759 Other Congenital Malformations not classified Quito ND 298 26 58 M 9 ADN 962 444305 ADHD Quito ND 299 26 59 M 5 ADN 978 452772 Fragile X Sangolquí D 60 M 16 ADN 987 507117 Other Congenital Malformations not classified Carchi ND 318 32 61 M 12 ADN 1046 509264 Other Congenital Malformations not classified Quito ND 333 37 62 M 14 ADN 1047 509280 Other Congenital Malformations not classified Quito ND 271 16 63 M 10 ADN 1048 509382 Other Congenital Malformations not classified Pedro Vicente Maldonado ND 296 25 64 M 20 ADN 1051 229350 Other Congenital Malformations not classified Quito ND 288 22 65 M 4 ADN 1064 468821 Develomental and Language Delay ND 285 22 66 M 13 ADN 1077 510244 Other Congenital Malformations not classified ND 303 28 67 M 5 ADN 1078 510200 ADHD ND 303 28 68 M 13 ADN 1100 509822 Fragile X ND 304 28 69 M 15 ADN 1103 428031 Other Congenital Malformations not classified ND 304 28 70 M 4 ADN 1112 489784 ASD ND 314 31 71 M 15 ADN 1117 313743 ADHD ND 325 35 72 M 12 ADN 1132 510397 Other Congenital Malformations not classified ND 293 24 73 M 6 ADN 1139 508546 Other Congenital Malformations not classified ND 290 23 74 M 2 ADN 1154 511372 ASD ND 307 29 75 M 7 ADN 1102 511057 Fragile X ND 316 32 76 M 17 ADN 1177 512550 Other Congenital Malformations not classified ND 306 29 77 M 9 ADN 1179 433568 ASD ND 304 29 78 M 6 ADN 1184 511446 Other Congenital Malformations not classified ND 294 25 79 M 2 ADN 1198 512629 ASD ND 273 18 80 M 17 ADN 1199 512904 Other Congenital Malformations not classified ND 305 28 81 M 6 ADN 1225 513410 Language delay ND 305 28 82 M 13 ADN 1226 513461 Fragile X ND 291 24 83 M 2 ADN 1231 495874 Macrocephaly lia ND 291 24 84 M 3 ADN 1234 510361 Other Congenital Malformations not classified ND 311 30 85 M 7 ADN 1238 513676 Fragile X ND 291 23 86 M 8 ADN 1243 513739 Fragile X ND 296 25 87 M 15 ADN 1251 323328 Intelectual Disability ND 314 31 88 M 7 ADN 1253 513962 Fragile X ND 314 31 89 M 2 ADN 1255 492911 Language delay ND 310 30 90 M 10 ADN 1258 513959 Fragile X ND 309 30 91 M 5 ADN 1270 514190 Other Congenital Malformations not classified ND 278 19 92 M ADN 1274 514239 Fragile X D 93 M 9 ADN 1278 514360 Other Congenital Malformations not classified ND 314 31 94 M 9 ADN 1287 513457 Intelectual Disability ND 290 23 95 M 24 ADN 1303 514815 Fragile X ND 295 25 96 M 7 ADN 1305 514819 Fragile X D 97 M 9 ADN 1306 514818 Fragile X D 98 M 13 ADN 1311 514854 Fragile X D 99 M 16 ADN 1312 514855 Fragile X D 100 M 3 ADN 1318 510067 ASD ND 325 35 101 M 9 ADN 1319 514937 Other Congenital Malformations not classified ND 325 35 102 M 1 ADN 1322 513875 Other Congenital Malformations not classified ND 322 34 103 M 0 ADN 1364 515581 Fragile X ND 322 34 104 M 10 ADN 1372 468312 ASD ND 294 25 105 M 8 ADN 1375 515640 Fragile X ND 294 25 106 M 1 ADN 1386 515927 Developmental delay ND 309 29 107 M 16 ADN 1398 513693 ASD ND 305 29 108 M 22 ADN 1411 516188 ASD ND 343 41 109 M 20 ADN 1412 516189 ASD ND 310 30 110 M 17 ADN 1415 516264 Fragile X D 111 M 17 ADN 1416 516263 Fragile X D 112 M 16 ADN 1418 516298 Intelectual Disability ND 286 22 113 M 1 ADN 1422 516338 Epilepsy ND 292 24 114 M 1 ADN 1428 516412 Other Congenital Malformations not classified ND 298 26 115 M 2 ADN 1429 495718 ASD ND 298 26 116 M 3 ADN 1467 517008 ASD ND 289 23 117 M 1 ADN 1476 503877 Motor delay ND 310 30 118 M 7 ADN 1484 517371 Fragile X D 119 M 2 ADN 1486 517552 Fragile X ND 307 29 120 M 11 ADN 1506 396307 Intelectual Disability ND 277 19 2017 2016 2011 2012 2013 2014 2015 1 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 B C D E F G H I J K L 128 M 13 ADN 1602 519235 ASD ND 301 27 129 M 7 ADN 1615 473884 Intelectual Disability ND 319 33 130 M 2 ADN 1616 498441 Behavior Concerns ND 304 28 131 M 11 ADN 1625 3137468 ASD ND 304 28 132 M 14 ADN 1627 519527 Balanced Rearrangements and Structural Markers, not elsewhere classified ND 316 32 133 M 13 ADN 1637 519632 Other Congenital Malformations not classified ND 292 24 134 M 5 ADN 1638 488500 Developmental delay ND 322 34 135 M 5 ADN 1655 519907 Fragile X ND 292 24 136 M 16 ADN 1660 519989 Fragile X ND 289 23 137 M 3 ADN 1690 520518 Fragile X ND 298 26 138 M 7 ADN 1692 520585 Fragile X ND 298 26 139 M 15 ADN 1694 520597 Other Congenital Malformations not classified ND 295 25 140 M 0 ADN 1707 520744 Defects in post-translational modification of lysosomal enzymes ND 298 26 141 M 11 ADN 1708 520743 Other Congenital Malformations not classified ND 298 26 142 M 3 ADN 1712 520836 Fragile X ND 304 28 143 M 2 ADN 1731 506843 ASD ND 274 18 144 M 13 ADN 1737 355091 Learning Dificulties ND 289 23 145 M 6 ADN 1739 521269 Fragile X ND 291 24 146 M 12 ADN 1745 521357 Fragile X ND 295 25 147 M 18 ADN 1755 521543 Intelectual disability ND 289 23 148 M 9 ADN 1761 521743 Fragile X ND 262 14 149 M 12 ADN 1762 488203 Intelectual disability ND 313 31 150 M 2 ADN 1764 515488 ASD ND 301 27 151 M 15 ADN 1772 346596 Epilepsy ND 316 32 152 M 14 ADN 1779 388704 ASD ND 295 25 153 M 3 ADN 1804 501276 Behavior Concerns ND 319 33 154 M 3 ADN 1805 512408 ASD ND 286 22 155 M 2 ADN 1811 510733 ASD ND 286 22 156 M 2 ADN 1813 522915 Developmental delay ND 262 14 157 M 3 ADN 1814 494090 Developmental delay ND 259 13 158 M 3 ADN 1819 493599 ASD ND 305 28 159 M 4 ADN 1827 523235 Other Congenital Malformations not classified ND 293 25 160 M 12 ADN 1840 523392 Other Congenital Malformations not classified ND 304 28 161 M 18 ADN 1843 523331 Other Congenital Malformations not classified ND 295 25 162 M 3 ADN 1848 499611 Developmental delay ND 295 25 163 M 6 ADN 1858 523595 ASD ND 307 29 164 M 4 ADN 1862 523701 Fragile X ND 313 31 165 M 2 ADN 1882 517229 Epilepsy ND 325 35 166 M 5 ADN 1889 523909 Fragile X ND 302 27 167 M 6 ADN 1901 460853 Language delay ND 319 33 168 M 2 ADN 1911 523941 ASD ND 328 36 169 M 11 ADN 1914 524243 Intelectual Disability ND 325 35 170 M 15 ADN 1916 524244 Fragile X ND 340 40 171 M 11 ADN 1924 524402 Fragile X ND 307 29 172 M 8 ADN 1927 524434 Intelectual Disability ND 298 26 173 M 4 ADN 1928 524406 Fragile X ND 298 26 174 M 5 ADN 1934 524497 ASD ND 307 29 175 M 6 ADN 1935 476302 ASD ND 313 31 176 M 5 ADN 1939 524590 Intelectual Disability ND 325 35 177 M 4 ADN 1946 524745 ASD ND 337 39 178 M 5 ADN 1951 501283 ASD ND 334 38 179 M 11 ADN 1961 524951 Intelectual Disability ND 331 37 180 M 6 ADN 1963 525017 Intelectual Disability D 181 M 7 ADN 1967 525077 Intelectual Disability ND 331 37 182 M 17 ADN 1968 525078 Intelectual Disability ND 322 34 183 M 13 ADN 1970 SOCIAL ND 316 32 184 M 12 ADN 1982 525269 Fragile X ND 322 34 185 M 5 ADN 1991 525376 Fragile X D 186 M 3 ADN 2002 525184 ASD ND 325 35 187 M 7 ADN 2017 524639 Intelectual Disability ND 298 26 188 M 3 ADN 2025 525737 Fragile X ND 301 27 189 M 2 ADN 2027 524266 Language delay ND 313 31 190 M 3 ADN 2050 502125 Epilepsy ND 277 19 191 M 7 ADN 2052 524355 Epilepsy ND 319 33 192 M 6 ADN 2055 526204 ASD ND 331 37 193 M 14 ADN 2059 526237 Intelectual Disability ND 346 42 194 M 3 ADN 2073 523753 ASD ND 328 36 195 M 6 ADN 2074 526052 Developmental delay ND 340 40 196 M 3 ADN 2085 526387 ASD ND 319 33 197 M 2 ADN 2089 526793 Developmental delay ND 325 35 198 M 15 ADN 2096 379631 Intelectual Disability ND 322 34 199 M 21 ADN 2102 525999 Fragile X ND 313 31 200 M 59 ADN 2020 525639 Myotonic disorders ND 346 42 201 M 12 ADN 2104 399790 ADHD ND 319 33 202 M 6 ADN 2107 526544 Behavior Concerns ND 292 24 203 M 9 ADN 2108 526953 Fragile X ND 334 38 204 M 7 ADN 2112 527079 Fragile X ND 301 27 205 M 3 ADN 2123 510132 Language delay ND 331 37 206 M 3 ADN 2124 526540 Stereotyped movements ND 334 38 207 M 11 ADN 2132 527442 Huntington disease ND 322 34 208 M 5 ADN 2135 527512 Intelectual Disability ND 310 30 209 M 1 ADN 2136 517168 Intelectual Disability ND 313 31 210 M 3 ADN 2139 527569 ASD ND 316 32 211 M 2 ADN 2141 525195 Arteriovenous malformation of the cerebral vessels ND 310 30 212 M 11 ADN 2143 527659 Other Congenital Malformations not classified D 213 M 13 ADN 2154 527915 Developmental delay ND 268 16 214 M 9 ADN 2162 528006 Intelectual Disability ND 298 26 215 M 6 ADN 2174 528266 Fragile X ND 320 34 216 M 14 ADN 2176 528271 Fragile X ND 330 36 217 M 16 ADN 2177 528269 Fragile X ND 347 42 218 M 4 ADN 2185 500261 ASD ND 335 38 219 M ADN 2189 Intelectual disability D 220 M ADN 2194 Intelectual Disability D 221 M 11 ADN 2198 528452 Fragile X D 222 M 4 ADN 2203 528693 Fragile X ND 289 23 223 M 8 ADN 2211 528487 Fragile X ND 301 27 224 M 3 ADN 2212 528883 ASD ND 304 28 225 M 12 ADN 2215 512020 ASD ND 310 30 226 M 12 ADN 2216 528901 Fragile X ND 325 35 227 M 9 ADN 2222 460258 Café au lait spots ND 292 24 228 M 2 ADN 2226 529074 Fragile X ND 310 30 229 M 3 ADN 2227 527760 Language delay ND 307 29 230 M 8 ADN 2238 529539 Other Congenital Malformations not classified ND 301 27 231 M 13 ADN 2259 468312 Intelectual Disability ND 322 34 232 M ADN 2267 SOCIAL Suspicion of Adrenoleukodystrophy ND 304 28 233 M 11 ADN 2301 530096 Developmental delay ND 328 36 234 M 39 ADN 2321 530353 Motor neuron diseases ND 289 23 235 M 3 ADN 2349 530683 Motor delay ND 325 35 236 M 2 ADN 2358 530652 Child brain paralysis ND 292 24 237 M 19 ADN 2368 310775 Learning Dificulties ND 322 34 238 M 2 ADN 2373 526370 ASD ND 322 34 239 M 58 ADN 2380 61529 Parkinson Disease ND 274 18 240 M 5 ADN 2389 500606 Language delay D 241 M 14 ADN 2400 418354 ADHD ND 311 30 242 M 3 ADN 2404 516305 Language delay ND 325 35 2018 2019 2020 2021 2
https://openalex.org/W4255898811	https://periodicos.uffs.edu.br/index.php/RIS/article/download/11261/7464	Portuguese	null	Inter-relação entre metodologias didáticas, motivos e aprendizagem em Bioquímica	Revista Insignare Scientia	2,020	cc-by	7,544	Interrelationship between didactic methodologies, motives and learning in biochemistry Vanessa Aina Person (vaynaperson@hotmail.com) Universidade Federal de Santa Maria (UFSM) Vol. 3, n. 2. Mai./Ago. 2020 João Batista Teixeira da Rocha (jbtrocha@yahoo.com.br) Universidade Federal de Santa Maria (UFSM) Resumo: A Bioquímica é uma disciplina básica de inúmeros cursos de graduação, a qual estuda as relações entre biomoléculas e reações químicas que ocorrem em diferentes organismos vivos nos estados de saúde e de doença. O domínio dos conhecimentos bioquímicos é avaliado como complexo e desafiador principalmente pela necessidade de abstração para compreensão conceitual. O objetivo deste trabalho foi analisar quais são as modalidades didáticas que estão sendo utilizadas para ensinar Bioquímica no Brasil, e, porque estas modalidades poderiam ser consideradas motivadoras de ensino e de aprendizagem, tendo como embasamento teórico a Teoria da Atividade e a psicologia Histórico-cultural. Foram analisados 36 artigos publicados na Revista de Ensino de Bioquímica (REB), entre 2015 e 2019. Emergiram da análise as categorias: motivos relacionados à interação social, motivos relacionados à aprendizagem ativa e motivos relacionados ao desempenho acadêmico. A maioria dos artigos descreveram processos ativos de ensino, apontando para uma tendência focada no aluno como protagonista da aprendizagem. Palavras-chave: Educação em Bioquímica; metodologias; aprendizagem ativa; Teoria da atividade. Abstract: Biochemistry is a basic discipline of numerous undergraduate courses, which studies the relationships between biomolecules and chemical reactions that occur in different living organisms in health and disease states. The mastery of biochemical knowledge is assessed as complex and challenging mainly by the need for abstraction for conceptual understanding, generating great concern for those who teach. The aim of this paper was to analyze what are the didactic modalities that are being used to teach biochemistry in Brazil, and why these modalities are considered motivating of teaching and learning, having as theoretical basis the Activity Theory and the Historical-cultural psychology. We analyzed 36 articles published in the Journal of Biochemistry Education (REB), between 2015 and 2019. The following categories emerged from the analysis: motives related to social interaction, motives related to active learning and motives related to academic performance. Most articles described active teaching processes, pointing to a student-focused tendency as a protagonist of learning. Keywords: Biochemistry Education; methodologies; active learning; activity theory. Recebido em: 16/10/2019 Aceito em: 14/04/2020 101 Vol. 3, n. 2. Mai./Ago. 2020 Vol. 3, n. 2. Mai./Ago. 2020 1. INTRODUÇÃO Um dos grandes desafios do ensino é desenvolver nos alunos a capacidade de refletir/pensar criticamente, dando sentido aos conhecimentos que estão sendo abordados e criando possibilidades de aprendizagem. A Bioquímica enquanto disciplina, é considerada de difícil compreensão, pois exige um alto grau de abstração ao tratar de fenômenos ao nível molecular (isto é, atômico, submicroscópico e microscópico) que ocorrem nos organismos vivos nos estados de saúde e de doença. Além disto, num levantamento recente observamos que as percepções e avaliação sobre a disciplina de Bioquímica por estudantes de graduação, de diversos cursos, foi bastante negativa, mostrando que as aulas de Bioquímica devem ser repensadas e que os conhecimentos básicos devem ser aprimorados (NOGARA, et al, 2018). Esta dificuldade pode estar relacionada com a maneira de ensinar e de aprender, justificando a busca por metodologias de ensino diferenciadas, que atraiam a atenção, colaboração e participação ativa dos alunos. Concordamos com Sforni (2004, p. 13), ao considerar que não existe ensino único capaz de desencadear aprendizagem generalizada, mas “uma variedade de formas de transmissão de saber que podem ser mais ou menos competentes na promoção do desenvolvimento cognitivo”. No entanto, compreendemos que a apropriação do conhecimento não é um processo imediato que ocorre pela simples utilização de uma metodologia, mas deve despertar no aluno um sentimento de necessidade (motivação), ser mediada pelo professor e ter uma intencionalidade. Buscamos analisar as modalidades didáticas utilizadas para ensinar Bioquímica no Brasil e compreender porque elas são ou não consideradas motivadoras de ensino e aprendizagem em Bioquímica, bem como propor uma reflexão sobre o significado dos conceitos motivo/motivação no âmbito educacional levando em consideração a dinâmica do comportamento e desenvolvimento psíquico humano (LEONTIEV, 1978, 1985; VIGOTSKI, 2001, 2002). 2. OS MOTIVOS NAS ATIVIDADES DE ENSINO E APRENDIZAGEM Recebido em: 16/10/2019 Aceito em: 14/04/2020 102 102 Vol. 3, n. 2. Mai./Ago. 2020 A psicologia histórico-cultural do desenvolvimento humano proposta por Vigotski, Leontiev e outros colaboradores, aborda aspectos relacionados aos processos de aprendizagem, linguagem e desenvolvimento (VIGOTSKI; LURIA e LEONTIEV, 2016) sob o referencial teórico-metodológico do materialismo histórico-dialético (CALVE; ROSSLER; SILVA, 2015). Assim, a base do desenvolvimento psíquico humano é social, o homem é considerado produto da apropriação da cultura e da sociedade, o qual se desenvolve principalmente pelo uso da linguagem, instrumentos e de seus significados. Neste contexto, as atividades ocorrem num sistema de relações sociais que estão associadas a processos de cooperação, organizadas coletivamente e reguladas pela linguagem – o agir comunicativo (BONOTTO, SCHELLER, 2018). Nesta perspectiva, as atividades de ensino e aprendizagem são atividades sociais de produção de conhecimento científico bem como de desenvolvimento psíquico, sendo fundamental mobilizar o pensamento dos alunos para transformá-lo em ação mental. A motivação nesse contexto é considerada um estímulo para os estudantes participarem das atividades propostas pelo professor. Conforme Marino Filho (2011), [...] não basta que haja à disposição dos alunos os objetos, ou conteúdos disciplinares, para que eles se envolvam cognitivamente de forma produtiva e desenvolvedora. Há que se produzir uma atividade que crie a necessidade de envolvimento do aluno e que ela faça sentido para ele, no conjunto das suas ações, e que este sentido possa reconhecer-se como vital para o seu desenvolvimento (MARINO, FILHO, p.59, 2011). Leontiev (1978) propõe que a atividade é em termos mais gerais, uma forma de agir mediada por uma ferramenta (material ou intelectual) e conduzida por uma necessidade. No entanto, em estudos posteriores, Leontiev (1985, p. 83 [trad. nossa]) destacou que “na realidade sempre estamos na presença de atividades específicas, cada uma das quais responde a uma determinada necessidade do sujeito”. O autor ressalta que o mais importante que distingue uma atividade de outra é o objeto da atividade, o seu motivo, que pode ser externo ou ideal. Nesta perspectiva, distintos motivos podem emergir das atividades de ensino e aprendizagem, por exemplo, podem ser motivos eficazes ou apenas compreensíveis. De acordo com Leontiev (1978, 1985), os motivos apenas compreensíveis são aqueles que não geram significado real para quem realiza uma atividade, ou seja, mesmo sabendo que existe uma necessidade por detrás da atividade proposta, não há sentido pessoal para realizá-la. 2. OS MOTIVOS NAS ATIVIDADES DE ENSINO E APRENDIZAGEM Já nos motivos realmente eficazes, as atividades são geradoras de sentido pessoal, o autor aponta que “ao satisfazer a sua necessidade de conhecimento, o Recebido em: 16/10/2019 Aceito em: 14/04/2020 103 Recebido em: 16/10/2019 Aceito em: 14/04/2020 Recebido em: 16/10/2019 Aceito em: 14/04/2020 103 Vol. 3, n. 2. Mai./Ago. 2020 Vol. 3, n. 2. Mai./Ago. 2020 homem pode fazer de um conceito o seu conceito, isto é, apropriar-se de sua significação” (LEONTIEV, 1978, p. 168[grifo do autor]). Por exemplo, estudar Bioquímica pode ser apenas um meio de não reprovar nesta disciplina, sendo, portanto, um motivo apenas compreendido. Porém, se o aluno estuda para compreender o que ocorre nos níveis moleculares, relacionando com a sua própria vida e futura profissão, o conteúdo estudado fará outro sentido para ele. Consequentemente as metodologias didáticas seguem uma lógica parecida, elas podem ser realizadas mecanicamente pelo estudante apenas para realizar o que lhe foi proposto, ou elas podem auxiliar efetivamente na aprendizagem. Assim é importante conhecer os motivos que conduzem a realização das atividades propostas. Dependendo da intencionalidade do professor e da necessidade despertada no aluno, o uso de metodologias didáticas diferenciadas pode ou não desencadear o aprendizado. 1. METODOLOGIA A metodologia utilizada neste trabalho é de caráter qualitativo bibliográfico, que se caracteriza por ser aquela realizada a partir de registros disponíveis, decorrentes de pesquisas anteriores. Conforme Severino (2007) neste processo utiliza-se [...] dados ou de categorias teóricas já trabalhadas por outros pesquisadores e devidamente registrados. Os textos tornam-se fontes dos temas a serem pesquisados. O pesquisador trabalha a partir das contribuições dos autores dos estudos analíticos constantes dos textos (SEVERINO, 2007, p. 122). Para realizar nossa análise, selecionamos Revista de Ensino de Bioquímica (REB), que está vinculada a Sociedade Brasileira de Bioquímica e Biologia Molecular (SBBq). Conforme o website da REB, a revista é um meio de divulgar pesquisas em ensino de Bioquímica, Biologia Molecular e áreas afins, contribuindo para os avanços científicos, tecnológicos e pedagógicos. Utilizamos como critério de inclusão apenas os artigos publicados nos últimos 5 anos (2015-2019), excluindo as edições especiais (resumos publicados das reuniões anuais SBBq) deste período. Neste intervalo de tempo foram publicados 49 artigos alocados nas seções “Inovações Educacionais”, “Pesquisa em Ensino”, “REB Na Escola”, “Imagem Pública e Divulgação Científica”. Recebido em: 16/10/2019 Aceito em: 14/04/2020 104 Recebido em: 16/10/2019 Aceito em: 14/04/2020 Recebido em: 16/10/2019 Aceito em: 14/04/2020 104 Vol. 3, n. 2. Mai./Ago. 2020 Nosso objetivo inicial foi verificar quais são as metodologias didáticas utilizadas atualmente para ensinar Bioquímica no Brasil. Após a primeira leitura, percebemos que muitas das pesquisas publicadas faziam alguma correlação entre a metodologia didática utilizada e a motivação, emergindo assim, outro questionamento: por que os pesquisadores consideram as metodologias didáticas utilizadas como motivadoras de ensino e aprendizagem em Bioquímica? Para a segunda etapa da pesquisa utilizamos como ferramenta analítica a Análise Textual Discursiva (ATD), que consiste em três estágios: Para a segunda etapa da pesquisa utilizamos como ferramenta analítica a Análise Textual Discursiva (ATD), que consiste em três estágios: 1. Desmontagem dos textos: também denominado de processo de unitarização, implica examinar os materiais em seus detalhes, fragmentando-os no sentido de atingir unidades constituintes [...] 2. Estabelecimento de relações: processo denominado de categorização, implicando construir relações entre as unidades de base, combinando-as e classificando-as no sentido de compreender como esses elementos unitários podem ser reunidos na formação de conjuntos mais complexos, as categorias. 3. Captando o novo emergente: a intensa impregnação nos materiais da análise desencadeada pelos dois estágios anteriores possibilita a emergência de uma compreensão renovada do todo. [...] O metatexto resultante desse processo representa um esforço em explicitar a compreensão que se apresenta como produto de uma nova combinação dos elementos construídos ao longo dos passos anteriores (MORAES, 2003, p. 192 [grifos do autor]). Por meio da utilização da ATD e tendo como embasamento teórico a psicologia histórico-cultural do desenvolvimento humano emergiram as categorias: motivos relacionados à interação social; motivos relacionados à aprendizagem ativa e motivos relacionados ao desempenho acadêmico que serão descritas e analisadas na sequência. 1. Desmontagem dos textos: também denominado de processo de unitarização, implica examinar os materiais em seus detalhes, fragmentando-os no sentido de atingir unidades constituintes [...] 2. Estabelecimento de relações: processo denominado de categorização, implicando construir relações entre as unidades de base, combinando-as e classificando-as no sentido de compreender como esses elementos unitários podem ser reunidos na formação de conjuntos mais complexos, as categorias. g . Captando o novo emergente: a intensa impregnação nos materiais da análise desencadeada pelos dois estágios anteriores possibilita a emergência de uma compreensão renovada do todo. [...] O metatexto resultante desse processo representa um esforço em explicitar a compreensão que se apresenta como produto de uma nova combinação dos elementos construídos ao longo dos passos anteriores (MORAES, 2003, p. 192 [grifos do autor]). Por meio da utilização da ATD e tendo como embasamento teórico a psicologia histórico-cultural do desenvolvimento humano emergiram as categorias: motivos relacionados à interação social; motivos relacionados à aprendizagem ativa e motivos relacionados ao desempenho acadêmico que serão descritas e analisadas na sequência. 2. RESULTADOS E DISCUSSÃO No período de 2015 a 2019 foram publicados na REB 49 artigos, sendo que 36 deles continham em algum momento as palavras “motivo”, “motivação”, “motivador” e/ou “motivados” (Quadro 1). Recebido em: 16/10/2019 Aceito em: 14/04/2020 105 Quadro 1- Total de artigos publicados na REB entre 2015 a 2019. Ano Total de artigos Motivo/motivação/motivador/motivados 2015 10 7 2016 12 6 2017 11 10 2018 10 8 Quadro 1- Total de artigos publicados na REB entre 2015 a 2019. Quadro 1- Total de artigos publicados na REB entre 2015 a 2019. Quadro 1- Total de artigos publicados na REB entre 2015 a 2019. Ano Total de artigos Motivo/motivação/motivador/motivados 2015 10 7 2016 12 6 2017 11 10 2018 10 8 105 Vol. 3, n. 2. Mai./Ago. 2020 2019 6 5 TOTAL 49 36 Fonte: os autores, 2019. Vol. 3, n. 2. Mai./Ago. 2020 2019 6 5 TOTAL 49 36 Fonte: os autores, 2019. Vol. 3, n. 2. Mai./Ago. 2020 No quadro 2 destacamos as metodologias encontradas, sendo possível observar que as atividades lúdicas, o uso de softwares e a experimentação foram as mais citadas e a educação continuada, fóruns de discussão, apresentação de seminários, mapas conceituais e sequências didáticas as metodologias menos citadas. Quadro 2- Metodologias didáticas Metodologias Total 1. Atividades lúdicas (incluem jogos, teatro e música) 8 2. Uso de Softwares 5 3. Experimentação 4 4. Avaliação da qualidade do ensino / disciplina / metodologia 4 5. Extensão universitária 3 6. Estudos de Caso/Situações problema 3 7. Modelos tridimensionais 2 8. Sala de aula invertida 2 9. Educação continuada 1 10. Fóruns de discussão via Moodle 1 11. Apresentação de seminários 1 12. Mapas conceituais 1 13. Sequências didáticas 1 Fonte: os autores, 2019. Quadro 2- Metodologias didáticas Quadro 2- Metodologias didáticas No quadro 3 estão representados os artigos que selecionamos para a análise, respeitando os critérios apontados anteriormente. Estão dispostos conforme o título, o ano, os autores, e a seção da revista. Recebido em: 16/10/2019 Aceito em: 14/04/2020 106 Quadro 3- Artigos que contêm as palavras motivo, motivação, motivar e/motivados publicados na REB entre 2015 a 2019. Título Ano Autores Seção 1-Ensino de Imunologia pela incorporação do conhecimento pelo teatro e a música 2015 ALMEIDA, M. E. F.; SANTOS, V. S. Pesquisa em Ensino 2-Seminários: proposta didática para o ensino de Genética 2015 GOUVÊA, C. M. C. P. Pesquisa em Ensino 3-Elaboração e utilização de um aplicativo como ferramenta no ensino de Bioquímica: carboidratos, 2015 ALCÂNTARA, N. R.; MORAES FILHO, Inovações Educacionais Quadro 3- Artigos que contêm as palavras motivo, motivação, motiva e/motivados publicados na REB entre 2015 a 2019. 106 106 Vol. 3, n. 2. Mai./Ago. 2020 Vol. 3, n. 2. Mai./Ago. 2020 lipídios, proteínas e ácidos nucleicos A.V. 4-Liga acadêmica de bioquímica clínica: experiência de implantação e participação na primeira liga acadêmica numa instituição de ensino superior de Juazeiro do Norte – Ceará 2015 HOLANDA, V. N.; et al. Imagem Pública e Divulgação Científica da Bioquímica 5-Análise da percepção de estudantes de graduação da área da saúde sobre o tema Biologia Celular 2015 MONERAT, C. A. A.; ROCHA, M. B. Pesquisa em ensino 6-Um jogo de construção para o aprendizado colaborativo de Glicólise e Gliconeogênese 2015 OLIVEIRA, O. S. Inovações educacionais 7-Twister Proteico: uma ferramenta lúdica envolvendo a síntese de proteínas 2015 WEYH, A. CARVALHO, I. G. B.; GARNERO, A. V. Inovações educacionais 8- Produção científica sobre estratégias didáticas utilizadas no ensino de Bioquímica: uma revisão sistemática 2016 SILVEIRA, J. T.; ROCHA, J. B. T. Pesquisa em ensino 9-Unidade Metabólica Baseada em Casos (CBMU): Um modelo para melhor compreensão do metabolismo no segundo ano do programa modular extendido de estudantes de medicina 2016 EISSA, S. SABBOUR, A. Inovações educacionais 10- Jogo Bozó Genético: uma proposta didática como alternativa para o ensino da replicação do DNA no ensino médio 2016 VILHENA, L. et al. REB na escola 11- Apresentação de resultados de pesquisa científica como estratégia para aumentar o interesse dos alunos em fisiologia 2016 ALTERMANN, C. D.; GARCIA, A.; MELLO, P. C. Inovações educacionais 12- Observação do efeito redutor da N-acetilcisteína no metabolismo da levedura 2016 CHAPELA, S. et al. Inovações educacionais 13- ProtVirt: simulação da dosagem de proteínas por espectrometria auxiliando as aulas práticas de Bioquímica 2016 HORNINK, G. G. Inovações educacionais 14- Impacto do ensino do Ciclo da Ureia por meio do “vídeo animado” versos o método de retroprojetor: percepção dos estudantes do primeiro ano de Bacharelado em odontologia 2017 AZIL, A. S.; SURYAKAR, A.; DIKSHIT, M. Pesquisa em ensino 15- Estudos dirigidos inovadores para a aprendizagem significativa de bioquímica no curso de biologia: uma pesquisa baseada em design 2017 COSTA. C.; GALEMBECK, E. Pesquisa em ensino 16- O hormônio insulina como um modelo para ensinar a estrutura tridimensional das proteínas 2017 OLIVEIRA, C. S. et al. Inovações educacionais lipídios, proteínas e ácidos nucleicos 107 Vol. 3, n. 2. Mai./Ago. 2020 Vol. 3, n. 2. Mai./Ago. Vol. 3, n. 2. Mai./Ago. 2020 Vol. 3, n. 2. Mai./Ago. 2020 32- Liga Acadêmica de Fisiologia Médica: um olhar dos discentes sobre metodologias ativas de ensino- aprendizagem 2019 FRIZZO, R. A.; et al. Pesquisa em ensino 33- Análise do conhecimento dos acadêmicos das áreas biológicas e saúde sobre o dogma "DNA – RNA – Proteína" 2019 ANDRADE, V. R. M.; et al. Pesquisa em ensino 34- As aventuras de Kreber: jogo digital sobre o metabolismo energético 2019 BAÊTA, F. J. M.; HORNINK, G. G. Inovações educacionais 35- Ferramentas de bioinformática aplicadas ao ensino da biotecnologia 2019 NASCIMENTO, Y. A. P.; SARAIVA, F. M. REB na escola 36- Mês do cérebro: uma ação visando a popularização da neurociência com alunos de ensino médio, em uma escola do DF, Brasil 2019 CARVALHO, K. M.; MENEZES, J. P. C. Imagem pública e divulgação científica. 2019 Fonte: os autores, 2019. 4.1 Motivos relacionados à interação social Na perspectiva da psicologia histórico-cultural a atividade de estudo antes de ser individual é social, as interações sociais desencadeiam a aquisição do conhecimento e o desenvolvimento humano, especialmente através da comunicação entre as pessoas e pelo uso de instrumentos (LEONTIEV, 1978). Esses elementos criam a possibilidade de reorganizar as ideias que os estudantes já possuem e favorece a construção de novos conhecimentos. A interação social nesta perspectiva, “não tem sentido de adaptação ao meio, mas de diálogo, de participação consciente, de possibilidade de intervenção” (SFORNI, 2004, p. 11), tem função formadora. Esses elementos criam a possibilidade de reorganizar as ideias que os estudantes já possuem e favorece a construção de novos conhecimentos. A interação social nesta perspectiva, “não tem sentido de adaptação ao meio, mas de diálogo, de participação consciente, de possibilidade de intervenção” (SFORNI, 2004, p. 11), tem função formadora. Os artigos que contemplam esta categoria de motivos envolvem as seguintes metodologias: o teatro e a música (Artigo 1), extensão universitária (Artigos 4, 11, 32), estudos de caso (Artigos 9, 15, 20), formação continuada (Artigo 18), fóruns de discussão (artigo 19), atividades lúdicas/jogos (Artigos 6, 7, 10, 23, 24, 34 e 36). Estas metodologias criam conexões entre as pessoas e realidade das futuras práticas profissionais, estimulando a necessidade de aprendizado de Bioquímica e áreas afins. O teatro e a música são formas lúdicas de ensinar e aprender. 3, n. 2. Mai./Ago. 2020 32- Liga Acadêmica de Fisiologia Médica: um olhar dos discentes sobre metodologias ativas de ensino- aprendizagem 2019 FRIZZO, R. A.; et al. Pesquisa em ensino 33- Análise do conhecimento dos acadêmicos das áreas biológicas e saúde sobre o dogma "DNA – RNA – Proteína" 2019 ANDRADE, V. R. M.; et al. Pesquisa em ensino 34- As aventuras de Kreber: jogo digital sobre o metabolismo energético 2019 BAÊTA, F. J. M.; HORNINK, G. G. Inovações educacionais 35- Ferramentas de bioinformática aplicadas ao ensino da biotecnologia 2019 NASCIMENTO, Y. A. P.; SARAIVA, F. M. REB na escola 36- Mês do cérebro: uma ação visando a popularização da neurociência com alunos de ensino médio, em uma escola do DF, Brasil 2019 CARVALHO, K. M.; MENEZES, J. P. C. Imagem pública e divulgação científica. Fonte: os autores, 2019. 4.1 Motivos relacionados à interação social Na perspectiva da psicologia histórico-cultural a atividade de estudo antes de ser individual é social, as interações sociais desencadeiam a aquisição do conhecimento e o desenvolvimento humano, especialmente através da comunicação entre as pessoas e pelo uso de instrumentos (LEONTIEV, 1978). Esses elementos criam a possibilidade de reorganizar as ideias que os estudantes já possuem e favorece a construção de novos conhecimentos. A interação social nesta perspectiva, “não tem sentido de adaptação ao meio, mas de diálogo, de participação consciente, de possibilidade de intervenção” (SFORNI, 2004, p. 11), tem função formadora. Os artigos que contemplam esta categoria de motivos envolvem as seguintes metodologias: o teatro e a música (Artigo 1) extensão universitária (Artigos 4 11 32) 2020 b d 17- Construção e aplicação de um modelo tridimensional como recurso didático para o ensino de síntese proteica 2017 LAZZARONI, A. A.; TEIXEIRA, G. A. P. B. REB na escola 18- Estratégias didático-pedagógicas como alternativas para o ensino de Biologia Celular: curso aos professores de escolas públicas de Ensino Médio de Curitibanos-SC 2017 GLASER, V.; PIERRE, P. M. O.; FIOREZE, A. C. C. L. REB na escola 19- O uso de fóruns de discussão sobre tópicos de interesse geral como estratégia para melhorar o interesse dos estudantes em Fisiologia 2017 GONÇALVES, R. et al. Pesquisa em ensino 20- Mapeando tendências da pesquisa na área de Educação em Bioquímica da SBBq de 2006 a 2016 2017 FERREIRA, C. R. C.; GONÇALVES, J. L. Pesquisa em ensino 21-Avaliação das dificuldades de aprendizado em Bioquímica dos discentes da Universidade Federal do Piauí 2017 ANDRADE, R. S. B.; SILVA, A. F. S.; ZIERER, M. S. Pesquisa em ensino 22- Atividades experimentais nos anos iniciais do ensino fundamental: ferramenta metodológica para a construção do processo de ensino aprendizagem 2017 LIMA, A. S. et al. REB na escola 23- Uso do Role-Playing Game (RPG) como complemento didático no ensino de Imunologia 2017 LAUREANO, M. O. L. et al. Inovações educacionais 24- Na trilha dos genes: uma proposta de jogo didático para o ensino de Genética 2018 LOVATO, F. L. Inovações educacionais 25- Ensino híbrido e gamificação aplicado no ensino de Bioquímica 2018 JUNIOR SOUZA, A. A.; SOUZA, G. P. V. A.; SANTOS, E. A. Inovações educacionais 26- Resolução de problemas no ensino de Ciências: utilização de Artemia salina como modelo experimental para o estudo de plantas medicinais na escola básica 2018 SALGUEIRO, A. C. F.; et al. REB na escola 27- Ciclo celular: construção e validação de uma sequência didática pela metodologia da engenharia didática 2018 KIELING, K. M. C.; GOULART, A. S.; ROEHRS, R. REB na escola 28- Percepção de estudantes de graduação de uma Universidade brasileira sobre a disciplina de Bioquímica 2018 NOGARA, P. A.; et al. Pesquisa em ensino 29- Avaliação da qualidade de ensino de Bioquímica em cursos de Nutrição 2018 SILVEIRA, J. T.; TEIXEIRA, J. B. Pesquisa em ensino 30- Realidade virtual no ensino de vias metabólicas 2018 GARZÓN, J. C. V.; MAGRINI, M. L.; GALEMBECK, E. Inovações educacionais 31- Estratégia de ensino: Aprenda em sala de aula 2018 SANTIAGO, S. A.; CARVALHO, H. F. Inovações educacionais 108 Vol. O artigo 1 (ALMEIDA; SANTOS, 2015) demonstrou que este método implica na responsabilidade Recebido em: 16/10/2019 Aceito em: 14/04/2020 Recebido em: 16/10/2019 Aceito em: 14/04/2020 109 Vol. 3, n. 2. Mai./Ago. 2020 dos “atores” e “telespectadores” contribuindo para a construção de um pensamento crítico, bem como para a formação social e cultural dos alunos que têm a oportunidade de prestigiá-lo. Através das narrativas que são desencadeadas nesta metodologia tem-se a oportunidade de colocar o pensamento em palavras tornando a universidade em um espaço de socialização do conhecimento (BOLZAN, 2009). No artigo 4 (2015) a extensão universitária é uma metodologia que aproxima os estudantes das suas profissões. Conforme Holanda et. al (2015, p. 94), uma das principais motivações apontadas pelos participantes foi “o desejo de atuar de forma mais direta na comunidade, através de ações sociais”. Os autores concluem que a criação da Liga acadêmica foi extremamente positiva, possibilitando “uma extensa contribuição sócio-científica aos participantes considerando a vivência com o social, os novos conhecimentos científicos e clínicos” (Ibidem, p. 98). O artigo 18 (GLASER; PIERRE; FIOREZE, 2017) refere-se à formação continuada de professores, a qual permitiu a troca de experiências através da elaboração de modelos didáticos para ensinar Biologia celular. A interação social foi um dos fatores que motivou os professores a buscarem por estratégias didáticas mais atrativas, pois propiciou um ambiente de discussão de práticas pedagógicas. No artigo 19 (GONÇALVES et al; 2017) também é possível observar, que a metodologia de fóruns de discussão é utilizada como estratégia para melhorar o interesse dos estudantes e estimular o pensamento crítico. O artigo 18 (GLASER; PIERRE; FIOREZE, 2017) refere-se à formação continuada de professores, a qual permitiu a troca de experiências através da elaboração de modelos didáticos para ensinar Biologia celular. A interação social foi um dos fatores que motivou os professores a buscarem por estratégias didáticas mais atrativas, pois propiciou um ambiente de discussão de práticas pedagógicas. No artigo 19 (GONÇALVES et al; 2017) também é possível observar, que a metodologia de fóruns de discussão é utilizada como estratégia para melhorar o interesse dos estudantes e estimular o pensamento crítico. As metodologias de estudo de casos clínicos e apresentação de trabalhos científicos como as descritas nos artigos 9 (EISSA; SABBOUR, 2016) e 11 (ALTERMANN; GARCIA; MELLO, 2016) respectivamente, aproximam os alunos da realidade profissional e são utilizadas para melhorar as capacidades intelectuais, possibilitando desenvolver as habilidades de pensamento crítico nos estudantes, pois geram discussão dos dados analisados. Podemos inferir que esta categoria de motivos é caracterizada pela atividade compartilhada, que estimula as capacidades de atenção, memória, abstração e generalização que são desencadeadas no meio social, cultural, através da comunicação entre as pessoas. A apropriação do conhecimento, ou seja, o desenvolvimento cognitivo Recebido em: 16/10/2019 Aceito em: 14/04/2020 110 Recebido em: 16/10/2019 Aceito em: 14/04/2020 110 Vol. 3, n. 2. Mai./Ago. 2020 vai da dimensão social para a individual e vice-versa (SFORNI, 2004), destacando a importância da interação social nos processos de ensino e aprendizagem. Vol. 3, n. 2. Mai./Ago. 2020 vai da dimensão social para a individual e vice-versa (SFORNI, 2004), destacando a importância da interação social nos processos de ensino e aprendizagem. vai da dimensão social para a individual e vice-versa (SFORNI, 2004), destacando a importância da interação social nos processos de ensino e aprendizagem. 4.2 Aprendizagem ativa Leontiev estudou o aparecimento da consciência humana em comparação ao aparecimento do trabalho. Em sua obra “O desenvolvimento do psiquismo” (LEONTIEV, 1978), o autor assinala que o trabalho se efetua em condições de atividade coletiva, orientados por uma necessidade e efetivados pelo uso de instrumentos. Assim como o trabalho, a atividade estudo passou a ser considerada uma atividade social que exige organização do processo didático e educativo e o uso de instrumentos a fim de satisfazer uma necessidade de aprendizagem, transformando o conteúdo externo em mental e social em individual. Diferente do ensino tradicional, metodologias ativas de ensino tem o aluno como principal agente responsável por sua aprendizagem e o professor um facilitador deste processo, elas favorecem a autonomia dos estudantes e apresentam oportunidades de problematização (BERBEL, 2011). Os motivos relacionados à aprendizagem ativa constituem ações e operações que possibilitam desencadear nos alunos o desenvolvimento do pensamento, por meio da reflexão e análise coletiva. Nesta categoria de motivos destacamos metodologias didáticas como a aprendizagem baseada em problemas (Artigos 9,15 e 20), construção de modelos didáticos (Artigos 16 e 17), experimentação (Artigos 5, 12, 22 e 26), jogos (Artigos 6, 7, 10, 23,24, 34 e 36), sala de aula invertida (Artigos 25 e 31), uso de softwares (Artigos 3, 13, 14, 30 e 35), mapas conceituais (Artigo 33) e sequências didáticas (Artigo 27). Conforme o artigo 15 (COSTA; GALEMBECK, 2017), a metodologia de resolução problemas permite despertar a curiosidade dos alunos na busca por solucioná-las, os alunos elaboram hipóteses que são discutidas em grupos com a ajuda do professor. Os autores destacam que para ensinar Bioquímica, “é indispensável o uso de estratégias motivacionais voltadas para cada público-alvo, com o objetivo de contextualizar o assunto para despertar interesse, estudo rápido e promover o aprendizado” (COSTA; GALEMBECK, 2017, p. 76). O principal propósito da aprendizagem baseada em problemas é criar hábitos de estudo e de pensamento pela experiência reflexiva, principalmente a autonomia de Recebido em: 16/10/2019 Aceito em: 14/04/2020 111 111 2020 aprendizado, ao tentar solucionar os problemas apresentados pelo confronto direto com a realidade” (SALGUEIRO, et al, 2018, p. 45). O Artigo 10 (VILHENA, et al, 2016) trata os jogos didáticos como instrumentos úteis para instigar a curiosidade e a criatividade dos alunos. A metodologia de sala de aula invertida provoca a inversão da postura do professor e do aluno. Conforme descrito no artigo 25 (JUNIOR SOUZA; SOUZA; SANTOS, 2018), o professor assume uma postura de orientador e facilitador e o aluno sai da postura de expectador para um perfil mais ativo e colaborativo, contribuindo para a aprendizagem. O uso de softwares como o descrito no artigo 35 (NASCIMENTO; SARAIVA, 2019) é considerado pelos autores um material didático dinâmico. Eles destacam que a simples utilização de um software (bioinformática) já desperta a curiosidade e produz melhores resultados na aprendizagem. Ainda mapas conceituais e sequências didáticas representados no artigo 33 (ANDRADE, et al, 2019) e no artigo 27 (KIELING; GOULART; ROEHRS, 2018), respectivamente, são ferramentas de ensino que colocam os alunos numa condição ativa de aprendizagem, pois diferente das aulas tradicionais, são os alunos que desenvolvem a aula. Cada metodologia inclusa nesta categoria de motivos tem sua peculiaridade, mas percebemos que apesar das diferenças, a grande maioria busca colocar o aluno no centro da aprendizagem através da resolução de problemas e desafios que mobilizem o potencial intelectual. Assim é possível que ocorra o desenvolvimento do pensamento científico e reflexivo, contribuindo para a autonomia dos estudantes (BERBEL, 2011). Vol. 3, n. 2. Mai./Ago. 2020 aprendizagem e trabalho em equipe (FREITAS, 2012). Esta metodologia é considerada ativa, interativa e colaborativa visando criar possibilidades de desenvolvimento argumentativo. Vol. 3, n. 2. Mai./Ago. 2020 aprendizagem e trabalho em equipe (FREITAS, 2012). Esta metodologia é considerada ativa, interativa e colaborativa visando criar possibilidades de desenvolvimento argumentativo. A construção de modelos didáticos tridimensionais promove a aprendizagem ativa como demonstrado no artigo 16. Oliveira et al (2017) consideram que elaboração de um modelo tridimensional para ensinar as estruturas de proteínas promove o aprendizado, pois os alunos interagem com modelos concretos sobre assuntos abstratos e precisam sair da sua zona de conforto, ou seja, tornam-se ativos na elaboração do modelo e assim, consequentemente tornam-se mais motivados. O artigo 16 (OLIVEIRA et al, 2017) conclui que a construção de modelos tridimensionais gera resultados significativos e as dificuldades de aprendizagem de conceitos abstratos são superadas por meio da atividade que é realizada em colaboração. A característica lúdica dos jogos didáticos, como no exemplo do artigo 34 (BAÊTA; HORNINK, 2019), aproxima os alunos e o professor, de maneira semelhante com a metodologia de construção de modelos tridimensionais que implica a participação ativa. Os autores concluem que a partir do uso do jogo, “observou-se que os usuários puderam, de forma lúdica, interagir ativamente com os conteúdos abordados e, por meio das dificuldades apresentadas no jogo, tiveram a oportunidades de ampliar e rever seus conhecimentos” (BAÊTA; HORNINK, 2019, p. 33). A experimentação também está relacionada à aprendizagem ativa, como destacamos nos artigos 22 (2017) e 26 (2018). No artigo 22, Lima et al (2017) consideram a experimentação um fator que motiva e auxilia fortemente a aprendizagem, a memorização e mudança conceitual. Para os autores a participação ativa dos alunos pode acionar uma série de estruturas cerebrais associadas com a cognição e evidenciam que “motivação, envolvimento e experimentação são elementos de um mesmo viés educacional e se apresentam como indispensáveis na ação pedagógica” (Ibidem, 2017, p. 55). Os autores do artigo 26 (2018) relatam que os alunos consideram o ensino por experimentação mais atraente e produtivo e justificam dizendo que “isso ocorre especialmente pelo fato de o estudante participar ativamente de seu processo de Recebido em: 16/10/2019 Aceito em: 14/04/2020 112 Recebido em: 16/10/2019 Aceito em: 14/04/2020 Recebido em: 16/10/2019 Aceito em: 14/04/2020 112 Vol. 3, n. 2. Mai./Ago. 2020 Vol. 3, n. 2. Mai./Ago. 4.3 Desempenho acadêmico Destacamos nesta categoria de motivos relacionados ao desempenho acadêmico os artigos (2, 8, 21, 28 e 29) que avaliaram a qualidade do ensino, as disciplinas e as metodologias didáticas utilizadas para ensinar bioquímica e áreas afins. A avaliação faz parte do cotidiano dos alunos e professores em qualquer sistema de ensino e, portanto, o desempenho acadêmico é um dos motivos melhor observado 0/2019 2020 113 Recebido em: 16/10/2019 Aceito em: 14/04/2020 Recebido em: 16/10/2019 Aceito em: 14/04/2020 113 113 Vol. 3, n. 2. Mai./Ago. 2020 Vol. 3, n. 2. Mai./Ago. 2020 quando nos referimos à educação e pode estar relacionado a um sistema de recompensas, normalmente a busca da aprovação. quando nos referimos à educação e pode estar relacionado a um sistema de recompensas, normalmente a busca da aprovação. Leontiev (1978) descreve o exemplo de um aluno fazendo a leitura de uma obra científica em preparação para uma avaliação: Imaginemos um aluno lendo uma obra científica que lhe foi recomendada. Eis um processo consciente que visa um objetivo preciso. O seu fim consciente é assimilar o conteúdo da obra. Mas qual é o sentido particular que toma para o aluno este fim e por consequência a ação que lhe corresponde? Isso depende do motivo que estimula a atividade realizada na ação da leitura. Se o motivo consiste em preparar o leitor para a sua futura profissão, a leitura terá um sentido. Se, em contrapartida, se trata para o leitor de passar nos exames, que não passam de uma simples formalidade, o sentido da sua leitura será outro, ele lerá a obra com outros olhos; assimilá-la-á de maneira diferente (LEONTIEV, 1978, p. 97). O autor continua narrando que outro aluno comenta ser desnecessária a leitura da obra, pois não fará parte da avaliação. Temos, portanto, algumas situações: este aluno pode simplesmente parar a leitura, pode continuar porque estava achando interessante ou ainda, ler porque será útil para sua profissão futura. Esses motivos nos fazem refletir sobre a intencionalidade dos professores ao propor uma atividade, exercício, experimento e avaliação. Além do sistema de recompensas, o desempenho do indivíduo pode mudar à medida que ele atua com outros sujeitos no processo de ensino e de aprendizagem. Bolzan (2009, p. 41) enfatiza que precisamos considerar as experiências vividas pelo individuo, portanto, “a bagagem sociocultural de cada sujeito é um fator que distingue suas condições para aprender durante as situações de ensino”. 4.3 Desempenho acadêmico O autor continua narrando que outro aluno comenta ser desnecessária a leitura da obra, pois não fará parte da avaliação. Temos, portanto, algumas situações: este aluno pode simplesmente parar a leitura, pode continuar porque estava achando interessante ou ainda, ler porque será útil para sua profissão futura. Esses motivos nos fazem refletir sobre a intencionalidade dos professores ao propor uma atividade, exercício, experimento e avaliação. Além do sistema de recompensas, o desempenho do indivíduo pode mudar à medida que ele atua com outros sujeitos no processo de ensino e de aprendizagem. Bolzan (2009, p. 41) enfatiza que precisamos considerar as experiências vividas pelo individuo, portanto, “a bagagem sociocultural de cada sujeito é um fator que distingue suas condições para aprender durante as situações de ensino”. O artigo 2 (GOLVÊA, 2015) se destaca ao demonstrar claramente essa categoria de motivos. Na metodologia descrita, foi proposta aos alunos a apresentação de seminários sobre um determinado assunto, sendo aberto para comunidade acadêmica. Conforme a autora, a execução dos seminários não proporcionou ganho de conhecimento para todas as turmas analisadas e um dos motivos poderia estar relacionado com a avalição. Na proposta Golvêa (2015), os alunos tinham a liberdade de escolher se os seminários iriam fazer parte do sistema de notas da disciplina ou não. Os resultados indicaram que os grupos que optaram por não fazer parte da nota demonstraram desinteresse pela proposta, ao contrário dos grupos em que esta atividade integrou a avaliação. Recebido em: 16/10/2019 Aceito em: 14/04/2020 114 Recebido em: 16/10/2019 Aceito em: 14/04/2020 Recebido em: 16/10/2019 Aceito em: 14/04/2020 114 114 Vol. 3, n. 2. Mai./Ago. 2020 No artigo 21 (ANDRADE; SILVA; ZIERER, 2017) foram analisadas múltiplas causas para dificuldades de aprendizagem em Bioquímica, mas enfatizou-se como mais importante à deficiência na educação básica. Os autores também destacaram que a rotina de estudos inadequada contribui para o baixo desenvolvimento acadêmico. Além disso, sugerem que diferentes atividades podem facilitar o ensino e aprendizado, tornando-se mais atrativas aos alunos. Os artigos 8, 28 e 29 apontam que disciplinas como a Bioquímica precisam se adequar com as características dos cursos, dando ênfase as profissões. No artigo 28 (NOGARA, et al, 2018) analisou-se diferentes metodologias utilizadas para ensinar Bioquímica, no entanto, os autores concluem que apesar de existirem diversos trabalhos que versam sobre metodologias didáticas diferenciadas há poucos estudos que analisam a efetividade do aprendizado propriamente dito. Assim, essa categoria demonstrou que existem grandes esforços para elevar os níveis de aprendizagem dos alunos, mas que o desempenho acadêmico ainda é mensurado apenas por notas, sendo mais difícil evitar as avaliações tradicionais e consequentemente validar outras metodologias para mensurar os níveis de aprendizagem dos alunos. 3. CONCLUSÃO 3. CONCLUSÃO Neste estudo, verificamos que a escolha por metodologias diferenciadas no ensino de Bioquímica e de áreas afins, se justifica pelas dificuldades de aprendizagem apresentadas, principalmente pela necessidade de compreensão dos conteúdos abstratos em um curto espaço de tempo. Percebemos que apesar de existirem inúmeros motivos que versam sobre os processos de ensino e de aprendizagem, muitas vezes, não compreendemos o significado conceitual dos motivos elencados. Embora não estejam explícitos nos textos analisados quais são os significados destes motivos, acreditamos que a justificativa possa estar relacionada com a perspectiva da psicologia histórico-cultural do desenvolvimento humano. Por esse viés destacamos os motivos relacionados à interação social, a aprendizagem ativa e ao desempenho acadêmico. Recebido em: 16/10/2019 Aceito em: 14/04/2020 Recebido em: 16/10/2019 Aceito em: 14/04/2020 115 Vol. 3, n. 2. Mai./Ago. 2020 Portanto, levando em consideração os aspectos apontados ao longo do texto, enfatizamos a complexidade que é ensinar e aprender Bioquímica e outras áreas, e, ressaltamos que nenhum dos motivos apontados deve ser pensado isoladamente. Os profissionais responsáveis pelo ensino necessitam prestar atenção nas escolhas metodológicas e em como essas escolhas vão refletir positivamente ou negativamente no aprendizado dos alunos. Nesse sentido, concluímos que embora haja a intenção de interrelacionar as metodologias didáticas com a eficácia do aprendizado, ainda não conseguimos aferir se houve um aprendizado real de médio e longo prazo com as metodologias utilizadas nos artigos citados aqui. Sugerimos que a forma de avaliação decorrente destes instrumentos seja mais eficiente, que a motivação seja pela necessidade de aprender, e sim, que esteja relacionada com a interação social, a aprendizagem ativa e o desempenho acadêmico. 4. REFERÊNCIAS BIBLIOGRÁFICAS ALMEIDA, M. E. F.; SANTOS, V. S. Ensino de Imunologia pela incorporação do conhecimento pelo teatro e a música. Revista de ensino de Bioquímica, v. 13, n. 3, 2015, pp. 06-20. ALTERMANN, C. D.; GARCIA, A.; MELLO, P. C. Apresentação de resultados de pesquisa científica como estratégia para aumentar o interesse dos alunos em fisiologia. Revista de ensino de Bioquímica, v. 14, n. 1, 2016, pp. 90-98. pesquisa científica como estratégia para aumentar o interesse dos alunos em fisiologia Revista de ensino de Bioquímica, v. 14, n. 1, 2016, pp. 90-98. ANDRADE, V. R. M.; et al. Análise do conhecimento dos acadêmicos das áreas biológicas e saúde sobre o dogma "DNA – RNA – Proteína". Revista de ensino de Bioquímica, v. 17, n. 1, 2019, pp. 1-15. ANDRADE, R. S. B.; SILVA, A. F. S.; ZIERER, M. S. Avaliação das dificuldades de aprendizado em Bioquímica dos discentes da Universidade Federal do Piauí. Revista de ensino de Bioquímica, v. 15, n. 1, 2017, pp. 24-39. BAÊTA, F. J. M; HORNINK, G. G. As aventuras de Kreber: jogo digital sobre o metabolismo energético. Revista de ensino de Bioquímica, v. 17, n. 1, 2019, pp. 16- 36. BERBEL, N. As metodologias ativas e a promoção da autonomia de estudantes. Semina: Ciências Sociais e Humanas, Londrina, v. 32, n. 1, p. 25-40, jan./jun. 2011. BOLZAN, D. Formação de professores: Compartilhando e reconstruindo conhecimentos. Porto Alegre: Mediação, 2002. BONOTTO, D.; SCHELLER, M. O agir modelagem. Revista Insignare Scientia - RIS, v. 1, n. 2, 20 jun. 2018. CALVE, T. M.; ROSSLER, J. H.; SILVA, G. L. R. A aprendizagem escolar e o sentido pessoal na Psicologia de A. N. Leontiev. Revista Quadrimestral da Associação Recebido em: 16/10/2019 Aceito em: 14/04/2020 Recebido em: 16/10/2019 Aceito em: 14/04/2020 116 Vol. 3, n. 2. Mai./Ago. 2020 Vol. 3, n. 2. Mai./Ago. 2020 Brasileira de Psicologia Escolar e Educacional. v.19, n. 3, Set/Dez 2015, pp. 435- 444. EISSA, S.; SABBOUR, A. Unidade Metabólica Baseada em Casos (CBMU): Um modelo para melhor compreensão do metabolismo no segundo ano do programa modular extendido de estudantes de medicina. Revista de ensino de Bioquímica, v. 14, n. 3, 2016, pp. 22-33. FREITAS, R. A. M. M. Ensino por problemas: uma abordagem para o desenvolvimento do aluno. Educação e Pesquisa, São Paulo, v. 38, n. 2, p. 403-418, abr./jun. 2012. Acesso em 17 jul. 2019. Disponível em: http://www.scielo.br/pdf/ep/v38n2/aop478.pdf GLASER, V.; PIERRE, P. M. O.; FIOREZE, A. C. C. L. Estratégias didático- pedagógicas como alternativas para o ensino de Biologia Celular: curso aos professores de escolas públicas de Ensino Médio de Curitibanos-SC. Revista de ensino de Bioquímica, v. 15, n. 2, 2017, pp. 49-74. GOLVÊA, C. M. C. P. Seminários: proposta didática para o ensino de Genética. Revista de ensino de Bioquímica, v. 13, n. 3, 2015, pp. 22-35. GONÇALVES, R. et al. O uso de fóruns de discussão sobre tópicos de interesse geral como estratégia para melhorar o interesse dos estudantes em Fisiologia. Revista de ensino de Bioquímica, v. 15, n. 1, 2017, pp. 06-14. HOLANDA, V. N. et al. Liga acadêmica de bioquímica clínica: experiência de implantação e participação na primeira liga acadêmica numa instituição de ensino superior de Juazeiro do Norte –Ceará. Revista de ensino de Bioquímica, v. 13, n. 3, 2015, pp. 87-99. JUNIOR SOUZA, A. A.; SOUZA, G. P. V. A.; SANTOS, E. A. Ensino híbrido e gamificação aplicado no ensino de Bioquímica. Revista de ensino de Bioquímica, v. 16, n. 2, 2018, pp. 87-103. KIELING, K. M. C; GOULART, A. S.; ROEHRS, R. Ciclo celular: construção e validação de uma sequência didática pela metodologia da engenharia didática. Revista de ensino de Bioquímica, v. 16, n. 2, 2018, pp. 49-70. LEONTIEV, A. O desenvolvimento do psiquismo humano. Lisboa: Horizonte universitário. 1978. LEONTIEV, A. O desenvolvimento do psiquismo humano. Lisboa: Horizonte universitário. 1978. ______. Actividade, conciencia, personalidad. 2ª reimpresión. Ciudad de La Habana: Editorial Pueblo e Educación, 1985. LIMA, A. S. et al. Atividades experimentais nos anos iniciais do ensino fundamental: ferramenta metodológica para a construção do processo de ensino aprendizagem. Revista de ensino de Bioquímica, v. 15, n. 2, 2017, pp. 40-63. LOMPSCHER, J. Motivation and activity. European Journal of Psychology of Education, 1999, Vol.XIV, n° 1, pp.11-22. MARINO FILHO, Armando. A atividade de estudo no ensino fundamental: necessidade e motivação. Marília, 2011, 236 f. Tese (Doutorado em Educação) – Faculdade de Filosofia e Ciências, Universidade Estadual Paulista, 2011. Recebido em: 16/10/2019 Aceito em: 14/04/2020 Recebido em: 16/10/2019 Aceito em: 14/04/2020 117 Vol. 3, n. 2. Mai./Ago. 2020 Vol. 3, n. 2. Mai./Ago. 2020 Vol. 3, n. 2. Mai./Ago. 2020 MORAES, R. Uma tempestade de luz: a compreensão possibilitada pela análise textual discursiva. Ciência & Educação, v. 9, n. 2, p. 191-211, 2003. Acesso em 03 out. 2019. Disponível em http://dx.doi.org/10.1590/S1516-73132003000200004. MORAES, R. Uma tempestade de luz: a compreensão possibilitada pela análise textual discursiva. Ciência & Educação, v. 9, n. 2, p. 191-211, 2003. Acesso em 03 out. 2019. Disponível em http://dx.doi.org/10.1590/S1516-73132003000200004. NASCIMENTO, Y. A. P.; SARAIVA, L. F. M. Ferramentas de bioinformática aplicadas ao ensino da biotecnologia. Revista de ensino de Bioquímica, v. 15, n. 2, 2019, pp. 77-90. NOGARA, P. A.; et al. Percepção de estudantes de graduação de uma Universidade brasileira sobre a disciplina de Bioquímica. Revista de ensino de Bioquímica, v. 16, n. 1, 2018, pp. 5-24. OLIVEIRA, C. S. et al. O hormônio insulina como um modelo para ensinar a estrutura tridimensional das proteínas. Revista de ensino de Bioquímica, v. 17, n. 1, 2019, pp. 114-130. REVISTA DE ENSINO DE BIOQUÍMICA. ISSN: 2318-8790, 2015-2019. REVISTA DE ENSINO DE BIOQUÍMICA. ISSN: 2318-8790, 2015-2019. SALGUEIRO, A. C. F. et. al. Resolução de problemas no ensino de Ciências: utilização de Artemia salina como modelo experimental para o estudo de plantas medicinais na escola básica. Revista de ensino de Bioquímica, v. 16, n. 2, 2018, pp. 31-47. SEVERINO, A. J. Metodologia do trabalho científico. 23ª edição. São Paulo: Cortez Editora, 2007. SFORNI, M. S. F. Aprendizagem conceitual e organização do ensino: contribuições da teoria da atividade. Araraquara: JM Editora, 2004, p.200. SILVEIRA, J. T; ROCHA, J. B. T. Produção científica sobre estratégias didáticas utilizadas no ensino de Bioquímica: uma revisão sistemática. Revista de ensino de Bioquímica, v. 14, n.1, 2016, pp. 07-21. VIGOTSKI, L. S. A construção do pensamento e da linguagem. Tradução Paulo Bezerra. São Paulo: Martins Fontes, 2001. p.496. ______. A formação social da mente. Tradução José Cipolla Neto, Luís Silveira Menna Barreto, Solange Castro Afeche. 6. ed. São Paulo: Martins Fontes, 2002. ______; LURIA, A; R. LEONTIEV, A. N. Linguagem, desenvolvimento e aprendizagem. Tradução: Maria da Pena Villalobos. 14ª ed. São Paulo: Ícone, 2016. VILHENA, L. et al. Jogo Bozó Genético: uma proposta didática como alternativa para o ensino da replicação do DNA no ensino médio. Revista de ensino de Bioquímica, v. 14, n. 3, 2016, pp. 57-67. Recebido em: 16/10/2019 Aceito em: 14/04/2020 Recebido em: 16/10/2019 Aceito em: 14/04/2020 118
https://openalex.org/W1631983050	http://masujournal.org/store_file/archive/99-4-6-390-393.pdf	English	null	Influence of Artificial Diet on Larvae of Eri Silkworm, Samia cynthia ricini Boisduval	Madras Agricultural Journal	2,012	cc-by	3,230	Influence of Artificial Diet on Larvae of Eri Silkworm, Samia cynthia ricini Boisduval P. Mangammal* and G. Sri Devi1 Department of Sericulture, 1Department of Soil Science and Agricultural Chemistry, University of Agricultural Sciences, GKVK, Bangalore - 560 065. An Artificial Diet composed of castor leaf powder (11.5 g), parched soybean powder (1.0 g), sucrose (1.0 g), agar-agar (1.5 g), inhibitor solution (5 ml) and water (20 ml) was evaluated against eri silkworm larvae. Castor fed eri silkworms were maintained as control for comparison. Larval weight was significantly maximum in eri silkworms fed with artificial diet upto first instar which was followed by feeding of eri silkworms on artificial diet upto second instar + castor leaves from upto fifth instar and feeding of eri silkworm on artificial diet upto third instar + castor during remaining instars. Whereas, the larval mortality was minimum in eri silkworms reared on artificial diet upto first instar followed by artificial diet upto second and third instars + castor during remaining instars, whereas, they were higher in eri silkworms fed on artificial diet upto fourth and fifth instars + castor during remaining instars. Key words: Artificial diet, eri silkworm, larval parameters. Rearing, B. mori L. entirely on an artificial diet was also achieved (Fukuda et al., 1960b; Ito and Tanaka, 1960; Yoshida et al., 1960). The composition of diet used by Fukuda et al. (1960b) is entirely the same as that devised by Yoshida et al. (1960) but that employed by Ito and Tanaka (1960) is slightly modified. Main constituents of these diets are starch, sucrose, soybean powder and mulberry leaf powder. Growth was suboptimal on all these diets. Sericulture is broadly classified into two distinct sectors as Mulberry and Non-mulberry. Mulberry silk is produced on large scale around the World. India ranks second in the World with respect to raw silk production and has unique distinction of producing all the four popular kinds on natural silks viz., Mulberry, Tasar, Eri and Muga. Among the commercially exploited non- mulberry silkworms, the eri silkworm, Samia cynthia ricini Boisduval is the only species domesticated completely and adapted to indoor rearing all through the year (Reddy, 2000). In this context, the influence of an artificial diet on larval parameters was studied in comparison with natural diet in S.c. ricini. Rearing silkworms on leaves require more labour. Influence of Artificial Diet on Larvae of Eri Silkworm, Samia cynthia ricini Boisduval The leaves used must be fresh enough to meet the preferences of silkworm; therefore, it has to be given to the silkworm three to four times a day. The year- round rearing of eri silkworm on fresh leaves is not convenient, because the food plants, Ricinus communis and Ailanthus glandulosa shed their leaves. The low quality of the leaf fed to early instars of silkworms and the risk of infection in chawki are the major constraints. A system of rearing with artificial diet may solve the above problems. Rearing on artificial diet allows complete prevention of various pathogenic microorganisms infecting through food source. Farmers who rear silkworms can buy the diet instead of cultivating and need not have a field for themselves. Materials and Methods Studies on economic parameters and consumption indices of eri silkworm, Samia cynthia ricini Boisduval on artificial diet were carried out in the Department of Sericulture, University of Agricultural Sciences, Gandhi Krishi Vignana Kendra, Bangalore. Three consecutive rearings were conducted. Corresponding author email: mangammalseri@gmail.com Madras Agric. J., 99 (4-6): 390-393 June 2012 https://doi.org/10.29321/MAJ.10.100095 Madras Agric. J., 99 (4-6): 390-393 June 2012 https://doi.org/10.29321/MAJ.10.100095 Treatment details T1 : Feeding eri silkworms on artificial diet upto first instar + further feeding on castor leaves from second to fifth instar. T2 : Feeding eri silkworms on artificial diet upto second instar + further feeding on castor leaves from third to fifth instar. T3 : Feeding eri silkworms on artificial diet upto third instar + further feeding on castor leaves from fourth to fifth instar. T4 : Feeding eri silkworms on artificial diet up to fourth instar + castor during fifth instar. T 5 : Feeding eri silkworms on artificial diet up to spinning. T6 (Control): Feeding eri silkworms on castor leaves throughout the larval period. T1 : Feeding eri silkworms on artificial diet upto first instar + further feeding on castor leaves from second to fifth instar. : Feeding eri silkworms on artificial diet upto second instar + further feeding on castor leaves from third to fifth instar. : Feeding eri silkworms on artificial diet upto third instar + further feeding on castor leaves from fourth to fifth instar. Preparation of Rearing Room and Equipment Prior to commencement of rearing, the rearing room with rearing stands were cleaned, washed thoroughly and properly disinfected with 4 per cent formalin solution using a foot pump as adopted by Dandin et al. (2003). After spraying, the room was kept closed for 48 hours for effective disinfection. After 24 hours of disinfection, the doors and windows were kept open to allow the fresh air into the rearing room. Raising of insects on artificial food has been successful in a few cases, but raising of insects, on artificial food is not so easy and there are very few successful cases. 391 Results and Discussion The eggs of plain white eri silkworm breeds were prepared as per the procedure outlined by Reddy et al. (2000). The eggs laid in a span of 24 hours were collected, surface disinfected with 2 per cent formalin solution for 5 minutes, washed with tap water, shade dried and incubated at ambient room temperature. Larval weight The larval weight was 370.97, 369.97 and 360.86 g / 50 larvae when larvae were fed with artificial diet for first, second and third instars + castor for remaining instars, respectively. Whereas, larval weight was 375.75 when silkworms were fed with castor throughout the larval duration (Table 2). The Table 1. Composition of Artificial Diet Table 1. Composition of Artificial Diet Composition Weight / Volume Castor leaf powder 11.5g Powder of parched soybean 1.0g Sucrose 1.0g Agar-agar 1.5g Inhibitor solution 5ml Water 20ml variation in larval weight may be due to the difference in nutritional composition of the artificial diet and leaf. There was a gradual increase in larval weight from first to fourth instar and steep increase during fifth instar. This may be due to maximum food consumption and growth rate during fifth instar. The present results are comparable to the findings of Fukuda et al. (1960a), who opined that the growth of eri silkworms reared on artificial food were almost same as those in case of the eri silkworms reared on fresh leaves of castor. Chen et al. (1992) reported that larval growth of silkworms reared on artificial diet was satisfactory. Sumida et al. (1995) reported that the weight of silkworm reared on low-cost artificial diet containing a residue byproduct of soybean crude as principal ingredients was satisfactory. However, Choudhuri (2000) reported that higher larval weight with artificial diet fed eri silkworms had lesser disease incidence compared to castor leaves fed batch. variation in larval weight may be due to the difference in nutritional composition of the artificial diet and leaf. With this artificial diet, castor leaves were supplemented in different larval instars as below and larval growth parameters were recorded. Table 2. Larval weight of S. c. ricini in different instars as influenced by feeding on artificial diet Table 2. Larval weight of S. c. ricini in different instars as influenced by feeding on artificial diet Leaves picked from castor plant were dried (not higher than 40°C) under air-blast and then powdered to pass through 80-mesh sieve. The inhibitor solution was prepared by dissolving 0.1g of Vitamin K3, 0.2g of sodium dehydroacetate and 0.2g sodium sorbate in 100ml of distilled water by heat. To the hot mixture containing agar-agar, sucrose, inhibitor solution and water, powdered leaves and parched soybean were added. The mixture was stirred well and was hardened at room temperature. The composition of artificial diet is given in Table 1. Treatment Mature Larval Total larval weight (g/ 50 duration larvae) T1 370.97 21.76 T2 369.97 21.34 T3 360.86 22.51 T4 - 10.67 T5 - 11.01 T6 (Control) 375.75 20.78 F- Test * * S.Em ± 0.102 0.006 CD at 5% 0.302 0.060 Significant at 5% Preparation of artificial diet Table 2. Larval weight of S. c. ricini in different instars as influenced by feeding on artificial diet Larval mortality The larval mortality was significantly low (2.56 and 1.88 per cent during first and second instar, respectively) in eri silkworms fed on artificial diet upto first instar + castor from second to fifth instar followed by eri silkworms fed on artificial diet upto second instar + castor from third to fifth instar (2.50, 1.93 and 1.38 per cent during first, second and third instar, respectively). The eri silkworms fed on artificial diet upto third instar + castor upto fifth instar had significantly higher mortality (2.43, 2.81, 4.88 and 1.55 per cent during first, second, third and fourth instar, respectively) (Table 4). However, there was no mortality in castor fed eri silkworms during all the instars. There was maximum mortality when eri silkworms were provided with artificial diet and when they were transferred to castor leaves the mortality was reduced and there was no mortality during subsequent instars. But the mortality was maximum when eri silkworms were fed on artificial diet upto fourth instar. There was severe mortality during fourth instar i.e., immediately after third moult. So the worms in the treatments provided with artificial diet upto fourth instar + castor during fifth instar and artificial diet throughout the larval period were unable to reach fifth instar and spin cocoons. These results are in conformity with Hamamura (2001), who reported that when silkworms reared on artificial diet, mortality was observed twice during the larval period, once during the early instar or during the first moulting and again at the end of fifth instar. Yanagawa et al. (1991) also reported that the rate of survival was slightly lower when larvae were reared on artificial diet. The reason for the mortality of the larvae during first instar (when reared on artificial diet) is attributed to the composition of the diet. In most of the cases it is due to the factor preventing the feeding or the presence of growth inhibitor in the diet. + castor during remaining larval period. However, the fourth moult duration of castor fed eri silkworms was 24.19 hours (Table 3). In general, moulting duration was found to be higher when eri silkworms were reared on artificial diet and transferred to castor leaves. This may be due to sudden change in feeding habit, which may take some time for acclimatization for the larvae when they were transferred from artificial diet to leaves. Larval duration : Feeding eri silkworms on artificial diet up to fourth instar + castor during fifth instar. The total larval duration was significantly shorter (20.78 days) when eri silkworms were reared on castor leaves during the entire larval period. The total larval duration was longer in case of artificial diet fed eri silkworms upto third instar + castor from fourth to fifth instar (22.51 days) followed by the larval duration of 21.76 and 21.34 days in eri silkworms provided with artificial diet during first and second instars, respectively + castor during remaining instars. There was no significant difference between the treatments and control with respect to larval duration during first three instars, but significant : Feeding eri silkworms on artificial diet up to spinning. T6 (Control): Feeding eri silkworms on castor leaves throughout the larval period. T6 (Control): Feeding eri silkworms on castor leaves throughout the larval period. The observations on larval weight, larval duration, moulting duration and larval mortality were recorded and analyzed using completely randomized design (Cochran and Cox, 2000). 392 Table 3. Larval duration of different moults in S. c. ricini as influenced by feeding on artificial diet Table 3. Larval duration of different moults in S. c. ricini as influenced by feeding on artificial diet Treatment Moulting duration (hours) I Moult II Moult III Moult IV Moult T1 48.69 120.69 24.31 24.38 T2 48.63 96.63 72.38 24.19 T3 48.31 96.63 120.31 24.63 T4 48.25 96.81 48.75 - T5 48.50 96.63 48.38 - T6 (Control) 48.25 24.25 24.25 24.19 F- Test NS * * S.Em ± - 0.157 0.149 0.116 CD at 5% - 0.467 0.442 0.345 : Significant at 5% NS: Non-Significant drops of ecdysone to the diet. Choudhuri (2000) also reported that eri silkworms reared on artificial diet containing castor leaf powder recorded longer larval duration over eri silkworms reared on natural food plant, where the duration was slightly less. Table 3. Larval duration of different moults in S. c. ricini as influenced by feeding on artificial diet differences were noticed during latter two instars (Table 2). These results are in close agreement with the findings of Choudhuri (2000), who reported that the eri silkworms reared on artificial diet containing castor leaf powder recorded longer larval duration over natural food plant, where the duration was slightly less. Moulting duration There was no significant difference between the treatments and control in respect of first moult duration. It was minimum in eri silkworms which received artificial diet upto third instar + castor from fourth to fifth instar (48.31hours) followed by the moulting duration of 48.63 and 48.69 hours in eri silkworms reared on artificial diet upto second and first instars, respectively + castor during remaining instars. The first moult duration in eri silkworms fed with castor was 48.25 hours. The second moult duration was significantly minimum (24.25 hours) in individuals reared on castor upto fifth instar, whereas, it was significantly maximum (120.69 hours) in eri silkworms reared on artificial diet upto first instar + castor upto fifth instar followed by the moulting duration of 96.63 in eri silkworms which received artificial diet upto second and third instars, respectively + castor during remaining instars. The third moult duration was significantly minimum (24.31 hours) in eri silkworms reared on artificial diet upto first instar + castor from second to fifth instar, while it was significantly maximum (120.31 hours) in eri silkworms reared on artificial diet upto third instar + castor from fourth to fifth instar followed by the moulting duration of eri silkworms fed on artificial diet upto second instar + castor upto fifth instar (72.38 hours). The third moult duration of eri silkworms fed with castor throughout the larval period was 24.25 hours, whereas, fourth moult duration was found to be significantly minimum (24.19 hours) in eri silkworms reared on artificial diet upto second instar + castor leaves upto fifth instar followed by the moulting duration of 24.38 and 24.63 hours in eri silkworms provided with artificial diet upto first and third instars, respectively drops of ecdysone to the diet. Choudhuri (2000) also reported that eri silkworms reared on artificial diet containing castor leaf powder recorded longer larval duration over eri silkworms reared on natural food plant, where the duration was slightly less. drops of ecdysone to the diet. Choudhuri (2000) also reported that eri silkworms reared on artificial diet containing castor leaf powder recorded longer larval duration over eri silkworms reared on natural food plant, where the duration was slightly less. Larval mortality The larval duration was maximum in artificial diet fed eri silkworms compared to those fed on castor leaves because of increased moulting duration, so they were unable to moult simultaneously and were lagging behind. These results conform to the results of Okauchi (1969), who reported that the larvae reared on artificial diet were lagging behind in growth, which can be made uniform by adding few 393 Table 4. Larval mortality in S. c. ricini in different instars as influenced by feeding on artificial diet Table 4. Larval mortality in S. c. ricini in different instars as influenced by feeding on artificial diet Table 4. Larval mortality in S. c. ricini in different instars as influenced by feeding on artificial diet using an aseptic rearing system of silkworms on an artificial diet: Thirty rearing per annum. J. Seric. Sci. Jpn., 61: 172-179. Treatment Larval mortality (%) I Instar II Instar III Instar IV Instar V Instar T1 2.56 1.18 - - - T2 2.50 1.93 1.38 - - T3 2.43 2.81 4.88 1.55 - T4 2.63 2.38 4.88 89.76 - T5 2.49 2.69 4.48 90.00 - T6 (Control) - - - - - F- Test * * * - S.Em ± 0.187 0.235 0.176 0.159 - CD at 5% 0.556 0.697 0.522 0.474 *: Significant at 5% NS: Non-Significant Choudhuri, C.C. 2000. Rearing of eri silkworm, Philosamia ricini and Philosamia cynthia ricini on artificial diet. Natl. Conf. Strat. Seric. Res. Dev., CSR&TI, Mysore, p. 94-95. Cochran and Cox. 2000. Experimental Design Procedures for the Behavioural Sciences, Cole Publishing Company, 319-380pp. Dandin, S.B., Jayaswal, J. and Giridhar, K. 2003. Handbook of Sericultural Technologies, CSB publications, 103-132pp. Fukuda, T., Higuchi, Y. and Matsuda, M. 1960a. Artificial food for eri silkworm. Indian J. Seric., 1: 12-16. Fukuda, T., Suto, M. and Higuchi, Y. 1960b. Silkworm rearing on the artificial food. J. Seric. Sci. Jpn., 29: 1-3. As reported by Sengupta (1990), artificial diet for silkworm is restricted to the first and second instars only, covering chawki rearing. Limitation for their bulk use during the later stages is mainly due to difficulty in preservation and use i.e., maintenance of aseptic condition, besides the correct temperature and humidity to prevent the physical and chemical degradation of the diet. Hamamura, Y. 2001. Silkworm Rearing on Artificial Diet (Translated from Japanese). Oxford & IBH Publishing Co. Pvt. Ltd., New Delhi, 79-103pp. Ito, T. and Tanaka, M. 1960. Larval mortality Rearing of the silkworm on an artificial diet and the segregation of pentamolters. J. Seri. Sci. Tokyo, 29: 191-196pp. Okuachi, T. 1969. Recent study of phytoecdysone. Insect Repellents, 38: 140-156pp. Reddy, D.N.R. 2000. On the nomenculature of eri silkworm. Sericologia, 40: 665-667pp. In general, eri silkworms fed on castor during first instar + castor from second to fifth instar were able to consume and digest more food which in turn led to increased larval growth. Eri silkworms provided with artificial diet upto fourth and fifth instars failed to complete their larval life, which might be attributed to the diet composition. The nutrition plays a vital role during last two instars, as the silkworms consume about 90 per cent of food during fourth and fifth instars to increase their body size and to synthesize silk. Reddy, D.N.R., Barauah, A.M. and Reddy, R. N. 2000. Effective utilization of eri silkworm wastes. Int. J. Wild Silkmoth & Silk, 28: 109-110pp. Sengupta, K. 1990. Artificial diet for silkworm: Are we nearer the goal? Indian Silk, 29: 16-18 Yanagawa, H., Watanabe, K. and Nakamura, M. 1991. Application of feed ingredients for livestock diet by using polyphagous strains of the silkworm. J. Seric. Sci. Jpn., 58: 401-406. Yoshida, T., Matuoka, M. and Kimura, K. 1960. On the rearing of silkworm larvae with an artificial diet containing dried mulberry leaf powder as its basic material. Bull. Seri. Exp. Stn. Japan, 15: 543-586. Chen, R.Y., Mori, H., Sumida, M., Yuan, X.L., Kitamaru, I. and Matsubara, F. 1992. All year round sericulture by Received: January 9, 2012; Accepted: May 8, 2012 References Chen, R.Y., Mori, H., Sumida, M., Yuan, X.L., Kitamaru, I. and Matsubara, F. 1992. All year round sericulture by Received: January 9, 2012; Accepted: May 8, 2012
https://openalex.org/W3119986206	https://www.researchsquare.com/article/rs-39438/v2.pdf?c=1605124043000	English	null	The dual amylin and calcitonin receptor agonist KBP-089 and the GLP-1 receptor agonist liraglutide act complimentarily on body weight reduction and metabolic profile	BMC endocrine disorders	2,021	cc-by	6,829	The Dual Amylin and Calcitonin Receptor Agonist KBP-089 and the GLP-1 receptor agonist liraglutide act complimentarily on body weight reduction and metabolic profile Anna Thorsø Larsen Nordic Bioscience Sofie Gydesen Nordic Bioscience Nina Sonne Nordic Bioscience Morten Asser Karsdal Nordic Bioscience Kim Henriksen (  kh@nordicbio.com ) Nordic Bioscience Research article Keywords: weight loss therapy, amylin receptor agonists, GLP-1 receptor agonists, dual use Posted Date: November 11th, 2020 DOI: https://doi.org/10.21203/rs.3.rs-39438/v2 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 January 7th, 2021. See the published version at https://doi.org/10.1186/s12902-020-00678-2. Page 1/18 Abstract Background: Weight loss therapy is becoming more and more important, and two classes of molecules, namely amylin receptor and GLP-1 receptor agonists, have shown promise in this regard. Interestingly, these molecules have several overlapping pharmacological effects, such as suppression of gastric emptying, reduction of glucagon secretion and weight loss in common; however, they also have distinct effects on prandial insulin secretion. Hence, a combination of these two mechanisms is of significant interest. Methods: In this study, we investigated the add-on potential of the dual amylin and calcitonin receptor agonist (DACRA) KBP-089 in combination with the GLP-1 receptor agonist liraglutide as obesity treatment in high-fat diet (HFD) fed rats. Results: Increasing doses of KBP-089 and liraglutide alone and in combination were studied with respect to their effects on body weight, food intake and glucose metabolism during a 9-week intervention study conducted in HFD rats. Further, the gastric emptying rate during an oral glucose tolerance was assessed. Treatment with KBP-089 and liraglutide dose-dependently lowered body weight 15% (at 2.5 µg/kg/day) and 7% (at 400 µg/kg/day) in HFD rats, respectively, while the combination resulted in a 21% body weight reduction, which was mirrored by reduction in fat depot sizes. Gastric emptying and glucose metabolism were improved, primarily by KBP-089, although liraglutide led to a reduction in fasting plasma glucagon. Conclusion: DACRAs complement GLP-1 on food intake, body weight, and glucose tolerance indicating the potential for an add-on therapy. Background Obesity is the result of modern lifestyle and excessive caloric intake, and the number of obese people is increasing. Multiple complications such as insulin resistance, type 2 diabetes, cardiovascular disease, cancer and non-alcoholic fatty liver disease, among others, are frequently associated with obesity (Haslam and James, 2005; Kahn et al., 2006; Guh et al., 2009). A sustained weight loss is key in treatment of obesity; however, treatments beside lifestyle intervention are still few. Bariatric surgery is effective, but is only used in severe obesity due to risk of surgical complications (Pories, 2008), therefore alternative therapies with improved efficacy and low risk of side effects are of great interest. Furthermore, a significant weight loss is important in treatment of non-alcoholic fatty liver disease (NAFLD), non- alcoholic steatohepatitis (NASH), and other obesity related morbidities (Cusi, 2009; Milić et al., 2014). Multiple GLP-1 agonists are approved for treatment of type 2 diabetes and recently high dose liraglutide was also approved for treatment of obesity, as it promotes sustained weight loss via effects on satiety and appetite (Van Can et al., 2014; Crane and McGowan, 2016). Additionally, liraglutide improves postprandial blood glucose concentration, although still with limitations in terms of tolerability challenges, in particular nausea (Astrup et al., 2012; Kanoski et al., 2012; Lean et al., 2014; Davies et al., 2015). There is an increasing focus on combining GLP-1 receptor agonists with additional therapy to Page 2/18 Page 2/18 obtain greater therapeutic efficacy. In this aspect, amylin receptor agonists (davalintide and pramlintide) have shown potential for significant body weight reduction and improved appetite control in both preclinical and clinical studies (Aronne et al., 2007; Smith et al., 2008; Mack et al., 2010). Additionally, pramlintide has been approved as adjunct to insulin therapy for treatment of type 1 and type 2 diabetes due to its ability to regulate post-prandial glucose levels, reduce body weight, and HbA1c (Weyer et al., 2001; Ratner et al., 2004; Ryan et al., 2009). Dual Amylin and Calcitonin Receptor Agonists (DACRAs) activate both the amylin receptor and the calcitonin receptor for an extended period of time and elicit superior activity in terms of typical amylin-induced responses (Mack et al., 2010; Andreassen et al., 2014; Gydesen et al., 2016; Hjuler et al., 2016). Background Importantly, GLP-1 and amylin analogues have several overlapping pharmacological effects including marked reductions in food intake, delay of gastric emptying and inhibition of glucagon secretion, although they act through different sites and mechanisms of action (Roth et al., 2012). Previously, combinations of sub-optimal doses of the DACRA KBP-089 and the GLP-1 agonist liraglutide were shown to act complementarily on body weight, food intake and glucose tolerance (Gydesen, Andreassen, et al., 2017), indicating the add-on potential of KBP-089 to liraglutide in obesity treatment. As only sub-optimal doses of KBP-089 and liraglutide have been examined chronically, we here evaluate combination-effects of doses that elicits full response separately (Gydesen, Hjuler, et al., 2017). In this study, we investigated the weight reducing potential of KBP-089 as monotherapy and in combination with the GLP-1 analogue liraglutide in obese high-fat diet fed (HFD) rats. Peptide therapy Synthetic KBP-089 (American Peptide Company, CA, USA) and liraglutide (SynPeptide, Shanghai, China) were dissolved in saline for subcutaneous delivery. The doses chosen for KBP-089 administration in the current in vivo investigations were based on previous comparable studies in animal models of obesity using KBP-089 (Gydesen, Andreassen, et al., 2017; Gydesen, Hjuler, et al., 2017) and previous studies with liraglutide (Knudsen, 2010; Gydesen, Andreassen, et al., 2017). Glucose tolerance tests OGTTs were performed at 4 and 8 weeks of treatment in animals fasted for 12 hours. Animals were administered with either vehicle or drug at t=-30 and received glucose p.o. gavage (2 g/kg) at t=0. EDTA blood samples were collected from the tail vein before drug administration and glucose challenge (0 min) and then the following 15, 30, 60 and 120 min post glucose challenge. Biochemical analysis Blood samples were collected in EDTA tubes and centrifuged at 5000 rpm for 10 min at 4 °C and the plasma was kept at -20 °C until further analysis. Blood glucose was monitored by Accu-Check® Avia monitoring system (Roche Diagnostics, Rotkreuz, Switzerland). Plasma levels of insulin (Mercodia Rat Insulin ELISA, Mercodia AB, Uppsala, Sweden), glucagon (Mercodia glucagon ELISA, Mercodia AB, Uppsala, Sweden) and acetaminophen (Acetaminophen Direct ELISA Kit, Immuneanalysis, Pomona, CA) were analyzed according to manufacturer's instruction. Animal experiments All animal procedures were performed in accordance with guidelines from the Animal Welfare Division of the Danish Ministry of Justice under the institutional license issued to Nordic Bioscience (2016-15-0201- 00910). 108 male Sprague Dawley (SD) rats (Envigo, Horst, The Netherlands) were obtained at 6 weeks of age and housed pair-wise in standard Type IV cages at the Nordic Bioscience animal facility (21-23 °C, 55-65% relative humidity, 12-h light/dark cycle) with ad libitum access to food and water. Page 3/18 From arrival and throughout the study period, the age-matched male SD rats were fed a 60 kcal% fat diet (#58Y1, TestDiet, London, UK). After 10 weeks on high fat diet (HFD), the rats were allocated into treatment groups according to body weight (n = 8-10 rats/treatment group – 8 rats in monotherapy groups and 10 rats in combination therapy groups). The rats received doses of KBP-089 (KBP) (0.625, 1.25 and 2.5 µg/kg sc), liraglutide (L) (200 and 400 µg/kg sc) and the combinations (KBP 0.625 + L 200 µg/kg, KBP 0.625 + L 400 µg/kg, KBP 1.25 + L 200 µg/kg, KBP 1.25 + L 400 µg/kg and KBP 2.5 + L 200 µg/kg, KBP 2.5 + L 400 µg/kg) and vehicle (saline) for 9 weeks. Body weight and food intake were monitored daily in the initial three weeks, then once weekly. Following 4 and 8 weeks of treatment, oral glucose tolerance tests (OGTT) were performed. To assess the treatment effect on gastric emptying, rats received acetaminophen (40 mg/kg) p.o. gavage (4 mL/kg) together with the glucose bolus during OGTT and the appearance of acetaminophen in plasma was measured after 30 min. At study end, animals were euthanized by exsanguination (under isoflurane anaesthesia) followed by dissection. Epididymal, perirenal and subcutaneous inguinal fat depots were surgically removed and weighed. KBP-089 acts complementary with GLP-1 on food intake and body weight loss To assess whether KBP-089 acts complimentary with GLP-1. KBP-089 and the GLP-1 analogue liraglutide were administered alone or in combination for 9 weeks in HFD rats. 9 weeks of treatment with KBP-089 (0.625, 1.25 and 2.5 µg/kg) resulted in a dose dependent weight loss (Figure 1C and supplementary figure 1A). Chronic treatment with high concentrations of liraglutide (200 and 400 µg/kg) and KBP-089 (2.5 µg/kg) resulted in a 7% and 15% vehicle-corrected body weight loss, respectively, while the combinations (L 200 µg/kg + KBP 2.5 µg/kg and L 400 µg/kg + KBP 2.5 µg/kg) resulted in a 17% and 21% weight reduction, respectively (Figure 1C and supplementary figure 1A). All treatments significantly reduced food intake in the initial phase of the study (Figure 1A and supplementary figure 1B-C), while only high dose KBP-089 and combination therapy reduced food intake during the entire study (Figure 1B). Based on food intake and body weight change, food efficiency was calculated. Treatment with the two highest KBP-089 doses (1.25 and 2.5 µg/kg) as well as their combinations with liraglutide resulted in a significant reduction in food efficiency compared with vehicle. Additionally, combination treatment was superior to treatment with liraglutide and showed a trend to towards superiority to KBP-089 alone (Figure 1D). Statistical analysis The endpoints were: change in bodyweight, food intake, glucose tolerance and insulin levels. All data are presented as mean ± standard error of the mean (SEM). The statistical analysis of group differences were assessed using one-way ANOVA followed by Tukey’s post-hoc test for multiple comparison. Statistical analyses of non-parametric data were conducted using Kruskal Wallis test followed by Dunn’s post-hoc test for multiple comparison. Normality of data distribution was determined by D’Agostino and Pearson test normality test. All analyses were performed using GraphPad Prism software (GraphPad Prism, San Diego, CA, USA). A value of p < 0.05 was considered statistically significant. Page 4/18 Treatment with KBP-089 and liraglutide reduces overall adiposity in high fat diet rats At study end adipose tissues were isolated and weighed. In conjugation with the significant reduction in body weight, the weight of epididymal white adipose tissues was significantly reduced after treatment with 2.5 µg/kg of KBP-089 and combinations of KBP-089 and liraglutide, whereas only the combination therapy significantly reduced the weights of inguinal and perirenal adipose tissue (Figure 2A-C). KBP-089 in combination with liraglutide delay gastric emptying rate Gastric emptying was assessed in the treatment groups receiving the highest doses of KBP-089, high dose liraglutide and the combination of the two. The rate of gastric emptying during OGTT was assessed after 4 and 8 weeks of treatment (Figure 5A,B). After both 4 and 8 weeks of treatment administration of KBP-089 (2.5 µg/kg) resulted in a significant reduction of gastric emptying rate 30 min after acetaminophen administration, while liraglutide (L 400 µg/kg) had no pronounced effect on gastric emptying (Figure 5A,B). After 4 weeks of treatment liraglutide reduced gastric emptying by approximately 10% compared to vehicle (Figure 5A), while liraglutide increased gastric emptying by approximately 18% compared to vehicle after 8 weeks of treatment (Figure 5B). Additionally, the combination of high dose KBP-089 and liraglutide (L 400 µg/kg + KBP 2.5 µg/kg) significantly delayed gastric emptying, but equally to KBP-089 treatment alone. This effect on gastric emptying rate was unchanged from 4 (Figure 5A) to 8 (Figure 5B) weeks of treatment. High dose Liraglutide, KBP-089 and the combination reduced fasting plasma glucagon levels Fasting plasma glucagon levels and glucagon levels during OGTT were assessed after 8 weeks of treatment. High dose liraglutide (L 400 µg/kg), KBP-089 (1.25 and 2.5 µg/kg) and the combination of slightly reduced fasted plasma glucagon levels compared to vehicle (Figure 4A). Plasma glucagon levels during OGTT did not differ significantly between treatment (Figure 4B). Treatment with KBP-089 and liraglutide improves oral glucose tolerance with reduced insulin levels OGTTs were performed after 4 and 8 weeks of treatment. After 4 weeks of treatment, KBP-089 (1.25 µg/kg) and the combination of high KBP-089 (2.5 µg/kg) and liraglutide (400 µg/kg) had decreased blood glucose levels, though only the combination significantly (Figure 3A and supplementary figure 2A). During the OGTT after 8 weeks of treatment (Figure 3C and supplementary figure 2C), the two combination groups (L 200 µg/kg + KBP 2.5 µg/kg and L 400 µg/kg + KBP 2.5 µg/kg) were able to significantly improve oral glucose tolerance considering the iAUC (Figure 3C). After both short and long- term treatment (4 and 8 weeks) insulin levels were reduced in rats treated with KBP-089 (1.25 and 2.5 µg/kg) while unchanged in rats treated with liraglutide (200 and 400 µg/kg) compared to vehicle, resulting in significantly different iAUC values in KBP-089 and liraglutide treated rats (Figure 3B,D). All combinations of the two treatments resulted in insulin levels in the same range as the KBP-089 treated rats, being significantly lower compared to vehicle. In addition, the combination of combination groups receiving the highest dose of KBP-089 (L 200 µg/kg + KBP 2.5 µg/kg and L 400 µg/kg + KBP 2.5 µg/kg) resulted in significantly lower insulin levels compared to groups treated with liraglutide alone (Figure 3B,D and supplementary figure 2B,D). Page 5/18 Page 5/18 Discussion It is likely that the majority of these effects is explained by the massive weight loss; however, DACRAs are known to directly suppress insulin secretion in an IVGTT, as well as directly on the pancreatic islets (Andreassen et al, 2014; Hjuler et al, 2016), confirming weight independent effects. Secondly, studies applying pair-fed and pair-weighed controls, as well as studies in ZDF rats, which are insensitive to amylin receptor mediated weight loss (Duffy et al, 2018), have documented glucose regulatory capacities beyond what is observed with weight loss (Hjuler et al, 2016; Gydesen et al, 2017). This together with the significant weight loss suggest potential not only as anti-obesity therapy, but also in treatment of obesity related co-morbidities such as type 2 diabetes and NASH (Kahn et al., 2006; Guh et al., 2009; Milić et al., 2014). Surprisingly, liraglutide did not increase plasma insulin as expected for a GLP-1 receptor agonist. Other studies in obese rats found similar lack of liraglutide induced increase in plasma insulin during OGTT (Raun et al., 2007; Gydesen, Andreassen, et al., 2017), suggesting that the lack of effect observed here might be explained by the animal model that is non-diabetic. Plasma glucagon levels were assessed after 8 weeks of treatment. All treatments, except liraglutide (200 µg/kg), tended towards a lowering of fasting plasma glucagon levels compared to vehicle. Though, all treatment groups had nearly constant glucagon levels during OGTT and no significant differences between groups were observed. This suggests that the HFD rat model does not show inappropriate elevated glucagon levels as seen in diabetic conditions and might explain why there is no clear effect of the therapies post glucose challenge. GLP-1 and amylin analogues are both known to delay gastric emptying (Roth et al., 2012), hence gastric emptying rates were assessed. In accordance with earlier studies using DACRAs (Hjuler et al., 2015, 2016), KBP-089 (2.5 µg/kg) markedly reduced gastric emptying after both short- and long-term treatment. A similar effect was observed in the group receiving high-dose combination therapy. Perhaps surprisingly, liraglutide alone only had minor effect on gastric emptying, even trending towards increasing vehicle- corrected gastric emptying after 8 weeks of treatment. Several clinical studies have shown that chronic treatment with liraglutide delays gastric emptying (Flint et al., 2011; Horowitz et al., 2012; Van Can et al., 2014). Discussion As there is a continuous need for increased potency on weight loss, we investigated the pharmacological potential of combination therapy using the highly potent DACRA KBP-089 and the GLP-1 analog liraglutide for obesity. . In this study, we found a significant effect on appetite suppression and body weight loss when combining the two peptides over a period of nine weeks, an effect superior to either monotherapy alone. Importantly, this also manifested in reduction in food efficiency and overall adiposity. Generally, KBP-089 was superior to liraglutide therapy, and the effects on body weight and food intake dose dependently followed KBP-089 concentrations when combing the two therapies. This suggests that KBP-089 is responsible for the majority of the efficacy of the combination therapy in this study. These findings correspond well with earlier observations using lower doses of the two peptides (Gydesen, Andreassen, et al., 2017), and demonstrate an additive effect. Previous studies of KBP-089 using pair-feeding have demonstrated a weight loss beyond what is obtained through the reduction of appetite, and have clearly indicated that this effect likely entails increased energy expenditure, or at least a maintenance of energy expenditure, despite the reduction of food intake, a parameter known to reduce energy expenditure (Hjuler et al, 2016; Gydesen et al, 2017; Wielinga et al, 2010; Mack et al, 2007). Page 6/18 Page 6/18 In terms of glucoregulatory actions both amylin receptor agonism (Ratner et al., 2004; Ryan et al., 2009; Mack et al., 2011) and GLP-1R agonism (Vilsbøll et al., 2007; Klein et al., 2014; Van Can et al., 2014) have shown potential. However, the glucose-lowering effect of GLP-1 receptor agonists involves increased post-prandial insulin secretion (Vilsbøll et al., 2008; Ladenheim, 2015). During OGTT, both short and long- term treatment with KBP-089 improved glucose tolerance in accordance with previous studies performed with KBP-089 (Gydesen, Andreassen, et al., 2017; Gydesen, Hjuler, et al., 2017). Interestingly, the effect on blood glucose during OGTT was especially pronounced in combination therapy groups, particularly after eight weeks of treatment, supporting that the peptides act though complimentary pathways, and possibly that the combination leads to increased durability of the glucoregulatory effects compared to stand-alone treatment, consistent with the study by Liberini et al. (2019). Importantly, along with improved glucose clearance, significantly lower insulin levels during OGTT were observed in KBP-089 (1.25 and 2.5 µg/kg) and combination therapy groups, indicating improved insulin sensitivity. Discussion Page 8/18 Abbreviations ANOVA: Analysis of Variance DACRA: Dual Amylin and Calcitonin Receptor Agonists ELISA: Enzyme-linked immunosorbent assay GLP-1: Glucagon-like peptide 1 HFD: High Fat Diet (i)AUC: (incremental) Area Under the Curve OGTT: Oral Glucose Tolerance Test Discussion However, in a pre-clinical setting the ability of liraglutide to reduce gastric emptying markedly diminished within 14 days of treatment, explaining the lack of effect observed here (Jelsing et al., 2012). Page 7/18 Page 7/18 The inhibited gastric emptying can positively affect postprandial blood glucose levels by delaying entry of glucose into circulation, a central factor in diabetes treatment. From a mechanistic point-of-view, a series of studies have looked into co-administration of either amylin or the DACRA salmon calcitonin (SCT) in combination with incretin-based therapies (Liberini et al, 2019; Bello et al, 2010; Gydesen et al, 2017). These studies have highlighted that both amylin and GLP-1 activate receptors in the same areas of the hind brain, i.e. the dorsal-vagal-complex (DVC), which contains the area postrema and the nucleus tractus solitarius (Liberini et al, 2019). These studies showed a combined effect of SCT and liraglutide on c-fos activation in the DVC, consistent with a combined suppression of food intake and gastric emptying (Liberini et al, 2019; Gydesen et al, 2017). Furthermore, earlier work indicated that this effect may entail a local upregulation of brain IL-6 in the hypothalamus, by both amylin and GLP-1 (Jansson and Palsdottir, 2015). Hence, while the complete picture of how the combination works is still unclear, there is evidence supporting that it entails common signaling pathways. Importantly, there are some limitations to the study presented here. The weight lowering and glucoregulatory actions of both the mono- and the combination therapies are limited by model, as the HFD rat model does not develop diabetes, but only modest insulin resistance due to obesity. Furthermore, despite previous studies in diabetic model systems showing suppression of hyperglucagonemia (Hjuler et al, 2017, we only detected trends towards suppression of glucagon levels, most likely due to the model system only representing a mild disease. This is also seen for the weight loss, where the differences are rather small in the combination therapy arms of the study, as these seem to have reached maximal weight loss, albeit the lack of a lean control group confounds this conclusion. All in all, further studies in a diabetic model would be of importance. In conclusion, KBP-089 acts complementary with the GLP-1 analogue, liraglutide, on food consumption, weight loss and glucose tolerance, indicating the potential for an add-on therapy causing additional improvement in metabolic profile. SEM: Standard error of the mean SEM: Standard error of the mean Author contributions: Author contributions: Participated in the research design: SGY, KH, MK Conducted experiments: ATL, SGY Performed data analysis: ATL, SGY Performed data analysis: ATL, SGY All authors have read and approved the final manuscript. Competing interests: MK and KH own stocks in Nordic Bioscience. MK and KH hold patents on KBPs. All authors are employees of Nordic Bioscience. MK and KH own stocks in Nordic Bioscience. MK and KH hold patents on KBPs. All authors are employees of Nordic Bioscience. - Acknowledgements - Acknowledgements Not applicable - Acknowledgements Not applicable - Funding ATL, SGY and NIS received funding from the Danish Research Foundation (Den Danske Forskningsfond) to support their PhD-programs. ATL, SGY and NIS received funding from the Danish Research Foundation (Den Danske Forskningsfond) to support their PhD-programs. (Grant numbers N/A). (Grant numbers N/A). (Grant numbers N/A). Author contributions: Declarations Ethics approval and consent to participate All studies were approved by the Danish Animal Inspectorate (approval number: 2016−15−0201−00910). - Consent to publish Ethics approval and consent to participate All authors and their employer have consented to the publication of these data. All authors and their employer have consented to the publication of these data. - Availability of data and materials - Availability of data and materials The data is available for sharing upon request. The data is available for sharing upon request. - Funding Abbreviations ANOVA: Analysis of Variance DACRA: Dual Amylin and Calcitonin Receptor Agonists Page 8/18 References Page 9/18 Andreassen K V, Feigh M, Hjuler ST, Gydesen S, Henriksen JE, Beck-Nielsen H, Christiansen C, Karsdal M a, and Henriksen K (2014) A novel oral dual amylin and calcitonin receptor agonist (KBP-042) exerts antiobesity and antidiabetic effects in rats. Am J Physiol Endocrinol Metab 307:E24-33. Andreassen K V, Feigh M, Hjuler ST, Gydesen S, Henriksen JE, Beck-Nielsen H, Christiansen C, Karsdal M a, and Henriksen K (2014) A novel oral dual amylin and calcitonin receptor agonist (KBP-042) exerts antiobesity and antidiabetic effects in rats. Am J Physiol Endocrinol Metab 307:E24-33. Aronne L, Fujioka K, Aroda V, Chen K, Halseth A, Kesty NC, Burns C, Lush CW, and Weyer C (2007) Progressive reduction in body weight after treatment with the amylin analog pramlintide in obese subjects: A phase 2, randomized, placebo-controlled, dose-escalation study. J Clin Endocrinol Metab 92:2977–2983. Aronne L, Fujioka K, Aroda V, Chen K, Halseth A, Kesty NC, Burns C, Lush CW, and Weyer C (2007) Progressive reduction in body weight after treatment with the amylin analog pramlintide in obese subjects: A phase 2, randomized, placebo-controlled, dose-escalation study. J Clin Endocrinol Metab 92:2977–2983. Astrup A, Carraro R, Finer N, Harper A, Kunesova M, Lean MEJ, Niskanen L, Rasmussen MF, Rissanen A, Rossner S, Savolainen MJ, and Van Gaal L (2012) Safety, tolerability and sustained weight loss over 2 years with the once-daily human GLP-1 analog, liraglutide. Int J Obes (Lond) 36:843–854, England. Bello NT, Kemm MH, Ofeldt EM, and Moran TH (2010) Dose combinations of exendin-4 and salmon calcitonin produce additive and synergistic reductions in food intake in nonhuman primates. Am J Physiol Integr Comp Physiol 299:R945–R952. Crane J, and McGowan B (2016) The GLP-1 agonist, liraglutide, as a pharmacotherapy for obesity. Ther Adv Chronic Dis 7:92–107. Cusi K (2009) Role of Insulin Resistance and Lipotoxicity in Non-Alcoholic Steatohepatitis. Clin Liver Dis 13:545–563. Davies MJ, Bergenstal R, Bode B, Kushner RF, Lewin A, Skjoth TV, Andreasen AH, Jensen CB, and DeFronzo RA (2015) Efficacy of Liraglutide for Weight Loss Among Patients With Type 2 Diabetes: The SCALE Diabetes Randomized Clinical Trial. JAMA 314:687–699, United States. Duffy S, Lutz TA, and Boyle CN (2018) Rodent models of leptin receptor deficiency are less sensitive to amylin. weight. Am J Physiol Endocrinol Metab ajpendo.00514.2015. weight. Am J Physiol Endocrinol Metab ajpendo.00514.2015. Gydesen S, Andreassen KV, Hjuler ST, Hellgren LI, Karsdal MA, and Henriksen K (2017) Optimization of Tolerability and Efficacy of Dual Amylin and Calcitonin Receptor Agonist, KBP-089, through Dose Escalation and Combination with a GLP-1 Analogue. Am J Physiol - Endocrinol Metab ajpendo.00419.2016. Gydesen S, Andreassen KV, Hjuler ST, Hellgren LI, Karsdal MA, and Henriksen K (2017) Optimization of Tolerability and Efficacy of Dual Amylin and Calcitonin Receptor Agonist, KBP-089, through Dose Escalation and Combination with a GLP-1 Analogue. Am J Physiol - Endocrinol Metab ajpendo.00419.2016. aslam DW, and James WPT (2005) Obesity. Lancet (London, England) 366: Hjuler ST, Gydesen S, Andreassen KV, Lund S, Pedersen K, Hellgren LI, Karsdal MA, and Henriksen K (2016) The Dual Amylin- and Calcitonin-Receptor Agonist KBP-042 Increases Insulin Sensitivity and Induces Weight Loss in Rats with Obesity. Obesity 24:1712–1722. Hjuler ST, Andreassen K V, Gydesen S, Karsdal MA, and Henriksen K (2015) KBP-042 improves bodyweight and glucose homeostasis with indices of increased insulin sensitivity irrespective of route of administration. Eur J Pharmacol 762:229–238. Hjuler ST, Gydesen S, Andreassen KV, Karsdal MA, and Henriksen K (2017) The Dual Amylin- and Calcitonin Receptor Agonist KBP-042 Works as Adjunct to Metformin on Fasting Hyperglycemia and HbA1c in a Rat Model of Type 2 Diabetes. J Pharmacol Exp Ther 362: 24-30. Horowitz M, Flint A, Jones KL, Hindsberger C, Rasmussen MF, Kapitza C, Doran S, Jax T, Zdravkovic M, and Chapman IM (2012) Effect of the once-daily human GLP-1 analogue liraglutide on appetite, energy intake, energy expenditure and gastric emptying in type 2 diabetes. Diabetes Res Clin Pract 97:258–266, Elsevier Ireland Ltd. Jansson JO, and Palsdottir V (2015) Brain IL-6 - Where amylin and GLP-1 antiobesity signaling congregate. Diabetes 64:1498–1499. Jansson JO, and Palsdottir V (2015) Brain IL-6 - Where amylin and GLP-1 antiobesity signaling congregate. Diabetes 64:1498–1499. Jelsing J, Vrang N, Hansen G, Raun K, Tang-Christensen M, and Bjerre Knudsen L (2012) Liraglutide: Short-lived effect on gastric emptying-long lasting effects on body weight. Diabetes, Obes Metab 14:531– 538. Kahn SE, Hull RL, and Utzschneider KM (2006) Mechanisms linking obesity to insulin resistance and type 2 diabetes. Nature 444:840–846. Kahn SE, Hull RL, and Utzschneider KM (2006) Mechanisms linking obesity to insulin resistance and type 2 diabetes. Nature 444:840–846. References Am J Physiol Regul Integr Comp Physiol 315: R856-R865 Flint A, Kapitza C, Hindsberger C, and Zdravkovic M (2011) The once-daily human Glucagon-Like Peptide- 1 (GLP-1) analog liraglutide improves postprandial glucose levels in type 2 diabetes patients. Adv Ther 28:213–226. Guh DP, Zhang W, Bansback N, Amarsi Z, Birmingham L, and Anis AH (2009) The incidence of co- morbidities related to obesity and overweight : A systematic review and meta-analysis. 20:1–20. Guh DP, Zhang W, Bansback N, Amarsi Z, Birmingham L, and Anis AH (2009) The incidence of co- morbidities related to obesity and overweight : A systematic review and meta-analysis. 20:1–20. Gydesen S, Hjuler ST, Freving Z, Andreassen KV, Sonne N, Hellgren LI, Karsdal MA, and Henriksen K (2017) A novel Dual Amylin and Calcitonin Receptor Agonist (DACRA), KBP-089, induces weight loss through a reduction in fat, but not lean mass, while improving food preference. Br J Pharmacol, doi: 10.1111/bph.13723. Gydesen S, Hjuler ST, Freving Z, Andreassen KV, Sonne N, Hellgren LI, Karsdal MA, and Henriksen K (2017) A novel Dual Amylin and Calcitonin Receptor Agonist (DACRA), KBP-089, induces weight loss through a reduction in fat, but not lean mass, while improving food preference. Br J Pharmacol, doi: 10.1111/bph.13723. Page 10/18 Gydesen S, Andreassen KV, Hjuler ST, Christensen JM, Karsdal MA, and Henriksen K (2016) KBP-088, a novel DACRA with prolonged receptor activation, is superior to davalintide in terms of efficacy on body weight. Am J Physiol Endocrinol Metab ajpendo.00514.2015. Kanoski SE, Rupprecht LE, Fortin SM, De Jonghe BC, and Hayes MR (2012) The role of nausea in food intake and body weight suppression by peripheral GLP-1 receptor agonists, exendin-4 and liraglutide. Neuropharmacology 62:1916–1927, Elsevier Ltd. Klein DJ, Battelino T, Chatterjee DJ, Jacobsen L V., Hale PM, and Arslanian S (2014) Liraglutide’s Safety, Tolerability, Pharmacokinetics, and Pharmacodynamics in Pediatric Type 2 Diabetes: A Randomized, Double-Blind, Placebo-Controlled Trial. Diabetes Technol Ther 16:679–687. Klein DJ, Battelino T, Chatterjee DJ, Jacobsen L V., Hale PM, and Arslanian S (2014) Liraglutide’s Safety, Tolerability, Pharmacokinetics, and Pharmacodynamics in Pediatric Type 2 Diabetes: A Randomized, Double-Blind, Placebo-Controlled Trial. Diabetes Technol Ther 16:679–687. Page 11/18 Page 11/18 Knudsen LB (2010) Liraglutide: The therapeutic promise from animal models. Int J Clin Pract 64:4–11, Blackwell Publishing Ltd. Ladenheim EE (2015) Liraglutide and obesity: A review of the data so far. Drug Des Devel Ther 9:1867– 1875. Lean MEJ, Carraro R, Finer N, Hartvig H, Lindegaard ML, Rössner S, Van Gaal L, and Astrup A (2014) Tolerability of nausea and vomiting and associations with weight loss in a randomized trial of liraglutide in obese, non-diabetic adults. Int J Obes 38:689–697. Liberini CG, Koch-Laskowski K, Shaulson E, McGrath LE, Lipsky RK, Lhamo R, Ghidewon M, Ling T, Stein LM, Hayes MR (2019). Combined Amylin/GLP-1 pharmacotherapy to promote and sustain long-lasting weight loss. Sci Rep 9: 8447 Mack CM, Smith PA, Athanacio JR, Xu K, Wilson JK, Reynolds JM, Jodka CM, Lu MGW, and Parkes D. (2011) Glucoregulatory effects and prolonged duration of action of davalintide: a novel amylinomimetic peptide. Diabetes,Obesity Metab 1105–1113. Mack CM, Soares CJ, Wilson JK, Athanacio JR, Turek VF, Trevaskis JL, Roth JD, Smith PA, Gedulin B, Jodka CM, Roland BL, Adams SH, Lwin A, Herich J, Laugero KD, Vu C, Pittner R, and Jr JRP (2010) Davalintide ( AC2307 ), a novel amylin-mimetic peptide : enhanced pharmacological properties over native amylin to reduce food intake and body weight. 385–395. Mack C, Wilson J, Athanacio J, Reynolds J, Laugero K, Guss S, Vu C, Roth J, Parkes D (2007) Pharmacological actions of the peptide hormone amylin in the long-term regulation of food intake, food preference, and body weight. Am J Physiol Regul Integr Comp Physiol 293:R1855-R1863. Milić S, Lulić D, and Štimac D (2014) Non-alcoholic fatty liver disease and obesity: Biochemical, metabolic and clinical presentations. World J Gastroenterol 20:9330–9337. Pories WJ (2008) Bariatric surgery: risks and rewards. weight. Am J Physiol Endocrinol Metab ajpendo.00514.2015. Vilsbøll T, Zdravkovic M, Le-Thi T, Krarup T, Schmitz O, Courreges J-P, Verhoeven R, Buganova I, and Madsbad S (2007) Liraglutide , a Long-Acting Human Glucagon-Like Peptide-1 Analog, Given as Monotherapy Significantly Improves Glycemic Control and Lowers Body Weight Without Risk of Hypoglycemia in Patients With Type 2 Diabetes. Diabetes Care 1608–10. Vilsbøll T, Zdravkovic M, Le-Thi T, Krarup T, Schmitz O, Courreges J-P, Verhoeven R, Buganova I, and Madsbad S (2007) Liraglutide , a Long-Acting Human Glucagon-Like Peptide-1 Analog, Given as Monotherapy Significantly Improves Glycemic Control and Lowers Body Weight Without Risk of Hypoglycemia in Patients With Type 2 Diabetes. Diabetes Care 1608–10. Weyer C, Maggs DG, Young AA, and Kolterman OG (2001) Amylin replacement with pramlintide as an adjunct to insulin therapy in type 1 and type 2 diabetes mellitus: A physiological approach toward improved metabolic control. Curr Pharm Des 7:1353–1373. Weyer C, Maggs DG, Young AA, and Kolterman OG (2001) Amylin replacement with pramlintide as an adjunct to insulin therapy in type 1 and type 2 diabetes mellitus: A physiological approach toward improved metabolic control. Curr Pharm Des 7:1353–1373. Wielinga PY, Lowenstein C, Muff S, Munz M, Woods SC, Lutz TA (2010) Central amylin acts as an adiposity signal to control body weight and energy expenditure. Physiol Behav 101:45-52 Wielinga PY, Lowenstein C, Muff S, Munz M, Woods SC, Lutz TA (2010) Central amylin acts as an adiposity signal to control body weight and energy expenditure. Physiol Behav 101:45-52 Wielinga PY, Lowenstein C, Muff S, Munz M, Woods SC, Lutz TA (2010) Central amylin acts as an adiposity signal to control body weight and energy expenditure. Physiol Behav 101:45-52 weight. Am J Physiol Endocrinol Metab ajpendo.00514.2015. J Clin Endocrinol Metab 93:S89-96, United States. Ratner RE, Dickey R, Fineman M, Maggs DG, Shen L, Strobel SA, Weyer C, and Kolterman OG (2004) Amylin replacement with pramlintide as an adjunct to insulin therapy improves long-term glycaemic and weight control in Type 1 diabetes mellitus : a 1-year , randomized controlled trial. Diabet Med 21:1204– 1212. Raun K, Von Voss P, Gotfredsen CF, Golozoubova V, Rolin B, and Knudsen LB (2007) Liraglutide, a long- acting glucagon-like peptide-1 analog, reduces body weight and food intake in obese candy-fed rats, whereas a dipeptidyl peptidase-IV inhibitor, vildagliptin, does not. Diabetes 56:8–15. Raun K, Von Voss P, Gotfredsen CF, Golozoubova V, Rolin B, and Knudsen LB (2007) Liraglutide, a long- acting glucagon-like peptide-1 analog, reduces body weight and food intake in obese candy-fed rats, whereas a dipeptidyl peptidase-IV inhibitor, vildagliptin, does not. Diabetes 56:8–15. Roth JD, Erickson MR, Chen S, and Parkes DG (2012) GLP-1R and amylin agonism in metabolic disease: Complementary mechanisms and future opportunities. Br J Pharmacol 166:121–136. Page 12/18 Page 12/18 Ryan G, Briscoe TA, and Jobe L (2009) Review of pramlintide as adjunctive therapy in treatment of type 1 and type 2 diabetes. Drug Des Devel Ther 2:203–14. Smith SR, Aronne LJ, Burns CM, Kesty NC, Halseth AE, and Weyer C (2008) Sustained weight loss following 12-month pramlintide treatment as an adjunct to lifestyle intervention in obesity. Diabetes Care 31:1816–23. Van Can J, Sloth B, Jensen CB, Flint A, Blaak EE, and Saris WHM (2014) Effects of the once-daily GLP-1 analog liraglutide on gastric emptying, glycemic parameters, appetite and energy metabolism in obese, non-diabetic adults. Int J Obes 38:784–793. Van Can J, Sloth B, Jensen CB, Flint A, Blaak EE, and Saris WHM (2014) Effects of the once-daily GLP-1 analog liraglutide on gastric emptying, glycemic parameters, appetite and energy metabolism in obese, non-diabetic adults. Int J Obes 38:784–793. Vilsbøll T, Brock B, Perrild H, Levin K, Lervang H, Kølendorf K, Krarup T, Schmitz O, Zdravkovic M, and Madsbad S (2008) Original Article : Treatment Liraglutide , a once-daily human GLP-1 analogue , improves pancreatic B-cell function and arginine-stimulated insulin secretion during hyperglycaemia in patients with Type 2 diabetes mellitus. Diabet Med 152–156. Figures Page 13/18 Fi 1 Figure 1 Accumulated food intake for the initial 7 day (A) and for the entire duration of the study (B). Body weights at study end (C). Calculated food efficiency (D). N=8-10 rats per group. Statistical analysis between groups were performed as a one-way ANOVA followed by Tukey’s post-hoc test with the following annotations: P < 0.05, P < 0.01, *P < 0.001 vs. vehicle, #P < 0.05, ##P < 0.01 vs. liraglutide (200 µg/kg), §P < 0.05, §§P < 0.01 vs. liraglutide (400 µg/kg), ¤ P < 0.05, ¤¤ P < 0.01, ¤¤¤ P < 0.001 vs. KBP- 089 (0.625 µg/kg), † P < 0.05, †† P < 0.01, ††† P < 0.001 vs. KBP-089 (1.25 µg/kg ) and ‡‡P<0.01 vs. KBP- 089 (2.5 µg/kg ). All data are means ± SEM. Figure 1 Accumulated food intake for the initial 7 day (A) and for the entire duration of the study (B). Body weights at study end (C). Calculated food efficiency (D). N=8-10 rats per group. Statistical analysis between groups were performed as a one-way ANOVA followed by Tukey’s post-hoc test with the following annotations: P < 0.05, P < 0.01, P < 0.001 vs. vehicle, #P < 0.05, ##P < 0.01 vs. liraglutide (200 µg/kg), §P < 0.05, §§P < 0.01 vs. liraglutide (400 µg/kg), ¤ P < 0.05, ¤¤ P < 0.01, ¤¤¤ P < 0.001 vs. KBP- 089 (0.625 µg/kg), † P < 0.05, †† P < 0.01, ††† P < 0.001 vs. KBP-089 (1.25 µg/kg ) and ‡‡P<0.01 vs. KBP- 089 (2.5 µg/kg ). All data are means ± SEM. Page 14/18 Page 14/18 Page 14/18 Figure 2 Weights of epididymal (A), inguinal (B) and perirenal (C) white adipose tissue (AT) at study end (n=8-10 rats per group). Statistical analysis between groups (A and B) were performed as a one-way ANONA followed by Tukey’s post-hoc test and as Kruskal Wallis test followed by Dunn’s post-hoc test with the following annotations: P < 0.05, P < 0.01, P < 0.001 vs. vehicle, §P < 0.05, §§P < 0.01 vs. liraglutide (400 µg/kg). All data are means ± SEM. Figure 2 Figure 2 Weights of epididymal (A), inguinal (B) and perirenal (C) white adipose tissue (AT) at study end (n=8-10 rats per group). Statistical analysis between groups (A and B) were performed as a one-way ANONA followed by Tukey’s post-hoc test and as Kruskal Wallis test followed by Dunn’s post-hoc test with the following annotations: P < 0.05, P < 0.01, P < 0.001 vs. vehicle, §P < 0.05, §§P < 0.01 vs. liraglutide (400 µg/kg). All data are means ± SEM. Page 15/18 Page 15/18 Figure 3 Oral glucose tolerance test (OGTT) after 4 and 8 weeks of treatment. The incremental area under the curve (iAUC) shown for blood glucose (A and C) and plasma insulin (B and D) during OGTT after 4 and 8 weeks, respectively. n=8-10 rats per group. Statistical analysis between groups were performed as a one- way ANONA followed by Tukey’s post-hoc test (C) and as Kruskal Wallis test followed by Dunn’s post-hoc test (A,B,D) with the following annotations: P < 0.05, P < 0.01, P < 0.001 vs. vehicle, #P < 0.05, ##P < Figure 3 Oral glucose tolerance test (OGTT) after 4 and 8 weeks of treatment. The incremental area under t curve (iAUC) shown for blood glucose (A and C) and plasma insulin (B and D) during OGTT after 4 weeks, respectively. n=8-10 rats per group. Statistical analysis between groups were performed as way ANONA followed by Tukey’s post-hoc test (C) and as Kruskal Wallis test followed by Dunn’s po test (A B D) with the following annotations: P < 0 05 P < 0 01 P < 0 001 vs vehicle #P < 0 05 Figure 3 Oral glucose tolerance test (OGTT) after 4 and 8 weeks of treatment. The incremental area under the curve (iAUC) shown for blood glucose (A and C) and plasma insulin (B and D) during OGTT after 4 and 8 weeks, respectively. n=8-10 rats per group. Statistical analysis between groups were performed as a one- way ANONA followed by Tukey’s post-hoc test (C) and as Kruskal Wallis test followed by Dunn’s post-hoc test (A,B,D) with the following annotations: P < 0.05, P < 0.01, P < 0.001 vs. vehicle, #P < 0.05, ##P < Page 16/18 0.01 vs. liraglutide (200 µg/kg), §P < 0.05, §§P < 0.01 vs. liraglutide (400 µg/kg), ¤ P < 0.05 vs. KBP-089 (0.625 µg/kg). All data are means ± SEM. Figure 5 Gastric emptying after 4 (A) and 8 (B) weeks of treatment. Gastric emptying was estimated by the appearance of acetaminophen in plasma 30 min post dosing and calculated as % change relative vehicle. Statistical analysis between groups were performed as a one-way ANONA followed by Tukey’s post-hoc test with the following annotations: P < 0.01, P < 0.001 vs. vehicle and §§§P < 0.001 vs. liraglutide (400 µg/kg). All data are means ± SEM. Gastric emptying after 4 (A) and 8 (B) weeks of treatment. Gastric emptying was estimated by the appearance of acetaminophen in plasma 30 min post dosing and calculated as % change relative vehicle. Statistical analysis between groups were performed as a one-way ANONA followed by Tukey’s post-hoc test with the following annotations: P < 0.01, *P < 0.001 vs. vehicle and §§§P < 0.001 vs. liraglutide (400 µg/kg). All data are means ± SEM. Figure 4 Fasting plasma glucagon levels (A) and plasma glucagon levels during OGTT after 8 weeks of treatment (B). n=8-10 rats per group. Statistical analysis between groups were performed as a one-way ANONA followed by Tukey’s post-hoc test. All data are means ± SEM. Fasting plasma glucagon levels (A) and plasma glucagon levels during OGTT after 8 weeks of treatment (B). n=8-10 rats per group. Statistical analysis between groups were performed as a one-way ANONA followed by Tukey’s post-hoc test. All data are means ± SEM. Page 17/18 Figure 5 Gastric emptying after 4 (A) and 8 (B) weeks of treatment. Gastric emptying was estimated by the appearance of acetaminophen in plasma 30 min post dosing and calculated as % change relative vehicle. Statistical analysis between groups were performed as a one-way ANONA followed by Tukey’s post-hoc test with the following annotations: P < 0.01, ***P < 0.001 vs. vehicle and §§§P < 0.001 vs. liraglutide (400 µg/kg). All data are means ± SEM. Figure 5 Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. Supplementaryfigure1.docx Supplementaryfigure2.docx NC3RsARRIVEGuidelinesChecklistfillablecompleted.pdf Page 18/18
https://openalex.org/W3000026723	https://europepmc.org/articles/pmc6978286?pdf=render	English	null	Runting and Stunting Syndrome Is Associated With Mitochondrial Dysfunction in Sex-Linked Dwarf Chicken	Frontiers in genetics	2,020	cc-by	8,735	Keywords: runting and stunting syndrome, chicken, mitochondrial dysfunction, oxidative phosphorylation, ATP synthesis, vacuoles Citation: Li H, Hu B, Luo Q, Hu S, Luo Y, Zhao B, Gan Y, Li Y, Shi M, Nie Q, Zhang D and Zhang X (2020) Runting and Stunting Syndrome Is Associated With Mitochondrial Dysfunction in Sex-Linked Dwarf Chicken. Front. Genet. 10:1337. doi: 10.3389/fgene.2019.01337 Runting and Stunting Syndrome Is Associated With Mitochondrial Dysfunction in Sex-Linked Dwarf Chicken Hongmei Li1,2†, BowenHu1,2†,Qingbin Luo1,2, Shuang Hu1,2, YabiaoLuo1,2, Bojing Zhao1,2, Yanmin Gan1,2, YingLi3, MeiqingShi4, QinghuaNie1,2, DexiangZhang1,2and Xiquan Zhang1,2 1 Department of Animal Genetics, Breeding and Reproduction, College of Animal Science, South China Agricultural University, Guangzhou, China, 2 Guangdong Provincial Key Lab of Agro-Animal Genomics and Molecular Breeding and Key Lab of Chicken Genetics, Breeding and Reproduction, Ministry of Agriculture, College of Animal Science, South China Agricultural University, Guangzhou, China, 3 Institute of Animal Science, Guangdong Academy of Agricultural Sciences, Guangzhou, China, 4 Division of Immunology, Virginia-Maryland Regional College of Veterinary Medicine, University of Maryland, College Park, College Park, MD, United States ORIGINAL RESEARCH published: 17 January 2020 doi: 10.3389/fgene.2019.01337 Reviewed by: Reviewed by: Dinko Novosel, University of Zagreb, Croatia Carlo Fiore Viscomi, University of Cambridge, United Kingdom Correspondence: Dexiang Zhang zhangdexiang0001@sina.com Xiquan Zhang xqzhang@scau.edu.cn †These authors have contributed equally to this work Specialty section: This article was submitted to Livestock Genomics, a section of the journal Frontiers in Genetics Specialty section: This article was submitted to Livestock Genomics, a section of the journal Frontiers in Genetics Received: 16 December 2018 Accepted: 09 December 2019 Published: 17 January 2020 Received: 16 December 2018 Accepted: 09 December 2019 Published: 17 January 2020 Edited by: Ino Curik, Runting and stunting syndrome (RSS) in chicken are commonly known as “frozen chicken.” The disease is characterized by lower body weight and slow growth and the incidence rate is widely 5%–20% in sex-linked dwarf (SLD) chickens. However, the etiology of RSS in chickens has plagued researchers for several decades. In this study, histopathology studies demonstrated that the hepatocytes of the RSS chickens contain many mitochondria with damaged and outer and inner membrane along with vacuolar hydropic degeneration. No mtDNA mutation was detected, but our microarray data showed that RSS chickens exhibited abnormal expression of genes, many of which are involved in oxidative phosphorylation (OXPHOS) and fatty acid metabolism. In particular, nuclear gene IGF2BP3 was upregulated in RSS chickens' liver cells. The abnormal expression of these genes is likely to impair the OXPHOS, resulting in reduced ATP synthesis in the hepatocytes of the RSS chickens, which may in turn leads to poor weight gain and retarded growth or stunting of chicks. Our ﬁndings suggest that mitochondria dysfunction rather than chronic inﬂammation is responsible for the reduced growth and RSS in SLD chickens. Mutations in GHR have been shown to compromise mitochondrial function in SLD chickens. Since the mitochondrial damage in the RSS chicken is more severe, we suggest that extra genes are likely to be affected to exacerbate the phenotype. y Ino Curik, University of Zagreb, Croatia University of Zagreb, Croatia Reviewed by: Dinko Novosel, University of Zagreb, Croatia Carlo Fiore Viscomi, University of Cambridge, United Kingdom Correspondence: Dexiang Zhang zhangdexiang0001@sina.com Xiquan Zhang xqzhang@scau.edu.cn †These authors have contributed equally to this work INTRODUCTION The chicks are frequently pale and may exhibit distention of the abdomen. In addition to smaller size and reduced body weight, other clinical signs were also observed in RSS chickens include poor feather development, listlessness, and diarrhea. The comb develops slowly, which is associated with impaired hearing and vision. enzyme activities in RSS chickens and SLD chickens, suggesting that the OXPHOS function was compromised. Ethics Standards All of the animal experiments performed in this study were approved by South China Agriculture University's Institutional Animal Care and Use Committee (approval number SCAU#0017), according to the regulations established by this committee and international standards for animal welfare. Frontiers in Genetics \| www.frontiersin.org Animals The chickens were purchased from XinXing Poultry Breeding Company near Guangzhou, China. Three groups of chickens were used: RSS affected SLD chickens (RSS chickens) along with normal SLD chicken (SLD chickens) in 301 strain, and this strain is characterized by point mutations in exon 5 of the GHR gene as previously reported (Ouyang et al., 2012); normal chickens in 202 strain, which have a wild-type GHR gene. All of the chicks were provided with standard poultry feed and fresh water and reared at an optimum temperature (27°C–37°C). After 7 weeks, six RSS chickens (three males and three females) and 6 SLD chickens (3 males and 3 females) from the 301 strain group, along with six normal chickens (three males and three females) from the 202 strain group, were collected. All of the experimental chicks were monitored periodically for signs of RSS, including body weight, abnormal feathering, uneven growth rate, poor performance, beak and leg color, lameness, and reluctance to move. Why are RSS chickens easily observed in SLD chickens? Our study demonstrated the occurrence of mitochondrial abnormalities in the 7-week-old chicken's livers of RSS chickens and SLD chickens. RSS chicken's mitochondria dysfunction is more seriousness than SLD chickens, and we observed the abnormal nuclear genes related to mitochondrial function in 7-week-old RSS chickens and normal SLD chickens by expression patterns (Table 2), and found these genes involved in oxidative phosphorylation (OXPHOS) and other metabolic pathways. The chicken mitochondrial genome is a 16.775-kb circle of double-stranded DNA (Desjardins and Morais, 1990; Zavala, 2006) that encodes only 13 proteins, 2 rRNAs, and 22 tRNAs, which is very similar to the human mitochondrial genome (Desjardins and Morais, 1990). It has been reported that mutation or deletion of mitochondrial DNA (mtDNA) can induce mitochondrial disorders in humans (Zeviani et al., 1989; Moraes et al., 1991). As over 99% of mitochondrial proteins are encoded by the nuclear genome, mitochondrial disorders can also be caused by mutations or/and abnormal expression of nuclear-encoded mitochondrial proteins (Bourgeron et al., 1995). In this study, we detected no mtDNA mutations in RSS chickens and SLD chickens. Transmission Electron Microscopy py Liver tissues were ﬁxed in 2.5% glutaraldehyde for 4 h at 4°C and then cultured as previously described (Luo et al., 2016). Transmission electron microscope (Hitachi HT7700, Japan) was used to examine and photograph mitochondria, and ﬁve randomly selected areas were photographed at × 2500 magniﬁcation and counted as previously reported (Hu et al., 2019). Liver Histology Liver tissues were removed from each experimental chicken and ﬁxed in formalin and 2.5% glutaraldehyde, respectively. The samples were stained using hematoxylin and eosin (H&E) and Oil Red O, the slices were observed by optical microscopy, and photographed. We also used TEM (Hitachi HT-7700, Japan) to study the mitochondrial structure. Very little is currently known about mitochondrial diseases of chickens, and the etiology of RSS in chickens has plagued researchers for several decades. OXPHOS is the metabolic pathway in which cells use enzymes to oxidize nutrients, thereby releasing energy that is used to reform adenosine triphosphate (ATP). This pathway is a highly efﬁcient way of generating energy, compared to alternative fermentation processes such as anaerobic glycolysis. OXPHOS also produces reactive oxygen species (ROS) which lead to the propagation of free radicals, damaging cells, and contributing to disease and possibly aging. The enzymes responsible for this metabolic pathway are also the targets of many drugs that inhibit their activities. INTRODUCTION Runting and stunting syndrome (RSS) is a condition in which a number of the birds in a ﬂock are considerably smaller owing to delayed growth (Zavala, 2006), and RSS chickens are easily observed in sex-linked dwarf (SLD) chickens. It is known that the SLD chickens are caused by the mutation of growth hormone receptor (GHR) gene and characterized by shorter shanks, and lower body weight Runting and stunting syndrome (RSS) is a condition in which a number of the birds in a ﬂock are considerably smaller owing to delayed growth (Zavala, 2006), and RSS chickens are easily observed in sex-linked dwarf (SLD) chickens. It is known that the SLD chickens are caused by the mutation of growth hormone receptor (GHR) gene and characterized by shorter shanks, and lower body weight January 2020 \| Volume 10 \| Article 1337 Frontiers in Genetics \| www.frontiersin.org Chicken's Mitochondrial Dysfunction Li et al. (Ouyang et al., 2012). But the RSS affected SLD chickens are smaller and lower body weight than SLD chickens. RSS has been reported in poultry ﬂocks around the world, including in the Netherlands (Kouwenhoven et al., 1978) and the UK (Bracewell and Wyeth, 1981). RSS causes economic hardship in the poultry industry through reduced uniformity, increased mortality, decreased body weight, poor feed conversion rate, and numerous secondary diseases. The ﬁrst clinical signs may be observed as early as three days of age, although they are most commonly observed in 6- to 12-day-old chicks and may occur until 3 weeks of age. The chicks are frequently pale and may exhibit distention of the abdomen. In addition to smaller size and reduced body weight, other clinical signs were also observed in RSS chickens include poor feather development, listlessness, and diarrhea. The comb develops slowly, which is associated with impaired hearing and vision. (Ouyang et al., 2012). But the RSS affected SLD chickens are smaller and lower body weight than SLD chickens. RSS has been reported in poultry ﬂocks around the world, including in the Netherlands (Kouwenhoven et al., 1978) and the UK (Bracewell and Wyeth, 1981). RSS causes economic hardship in the poultry industry through reduced uniformity, increased mortality, decreased body weight, poor feed conversion rate, and numerous secondary diseases. The ﬁrst clinical signs may be observed as early as three days of age, although they are most commonly observed in 6- to 12-day-old chicks and may occur until 3 weeks of age. ATP Concentration The ATP level was measured using an ATP assay kit (Beyotime, China) according to the manufacturer's instructions. A Fluorescence/Multi-Detection Microplate Reader (BioTek, USA) was used to determine the ATP level in gastrocnemius muscle and cells. Data were normalized to the control group and expressed as percentage of control levels. ROS Production The production of ROS was measured by the change in absorbance of formazan measured at 560 nm as previously described (Kim et al., 2003). Absorbance was determined using a Fluorescence/ Multi-Detection Microplate Reader (BioTek, USA). Mitochondrial Membrane Potential Mitochondrial Membrane Potential The mitochondrial membrane potential (DYm) was measured using a JC-1 kit (Beyotime, China) according to the manufacturer's instructions. The ﬂuorescence was determined using a Fluorescence/Multi-Detection Microplate Reader (BioTek, USA). Rotenone was used as standard inhibitor of DYm. The DYm of mitochondria were represented as the ratio of JC-1 aggregated and JC-1 monomeric, and data were normalized to the control group and expressed as percentage of control levels. Mitochondrial Membrane Potential The mitochondrial membrane potential (DYm) was measured using a JC-1 kit (Beyotime, China) according to the manufacturer's instructions. The ﬂuorescence was determined using a Fluorescence/Multi-Detection Microplate Reader (BioTek, USA). Rotenone was used as standard inhibitor of DYm. The DYm of mitochondria were represented as the ratio of JC-1 aggregated and JC-1 monomeric, and data were normalized to the control group and expressed as percentage of control levels. TABLE 1 \| Primers for qPCR analysis of nuclear gene expression and mtDNA content. Gene Primer sequence (5′ to 3′) Annealing temperature (°C) F-NDUFB1 TGTACAAGAGAGAGCTGAAGCC 57.5 R-NDUFB1 AGCAGTGACAAGTTGTAGGTGT F-NDUFB2 GCTGGGCCACTTCCCGTA 64 R-NDUFB2 AGTTCAAAACCCGCCCCTAC F-NDUFB8 CGATGCACTGGGACTTTGAC 65 R-NDUFB8 TTGTAATGCGTCACAACCGG F-NDUFB9 GGCAGAAAGAGGTGAAGCAG 65 R-NDUFB9 CATACGTTCACTGGCACACC F-NDUFB10 CCAGCAAGCCAAGAACAAGT 64.3 R-NDUFB10 TTGGTAACCTGTGCAAGCT F-NDUFB5 TGAGACAGAGGGGAGATGGA 64.3 R-NDUFB5 CTCCACAGACAAGCCACAAT F-NDUFB6 GGTGTTCAACGCTTACCAGA 64.3 R-NDUFB6 CCGTCTCTAAAATTCTGTCCCC F-IGF2BP3 CAAGCTCTACATCGGCAACC 65 R-IGF2BP3 GGGACCGAATGCTCAACTTC F-NDUFA8 ACAAGGAGTTCATGCTGTGC 57.5 R-NDUFA8 CACCCAGCCCAACTTCTCTA F-NDUFA9 ATCCACTTCCACGTCCTCTC 65 R-NDUFA9 CTTCGCTGGTTTTGCTTCCT F-SDHA GGCACTGCTATGGTTACACG 65 R-SDHA CCCCTTCCTTCCCGTATCTC F-NDUFS4 GGACTGGACTTCTCTGTGCT 57 R-NDUFS4 TGGCAGCAGGGAATACAGAA TABLE 1 \| Primers for qPCR analysis of nuclear gene expression and mtDNA content. TABLE 1 \| Primers for qPCR analysis of nuclear gene expression and mtDNA content. Statistical Analysis All the experiments were performed at least three times. The data were presented as means ± standard error of the mean (S.E.M.), and the statistical analyses were performed using Student's t-test and the signiﬁcance was represented by P-values. P value < 0.05 was considered to be statistically signiﬁcant. Quantitative Real-Time PCR To verify the data from microarray analysis (Table 2), quantitative real-time PCR (qRT-PCR) analysis was conducted to determine the relative expression levels of genes involved in the OXPHOS pathway in the livers of RSS chickens, SLD chickens, and normal chickens. Total RNA was extracted from the tissue samples using the RNAiso reagent (Takara, Japan) In this study, we demonstrated that mitochondrial membranes are destroyed in RSS chicken hepatocytes using transmission electron microscopy (TEM). Activity assays revealed that a series of complexes proteins (complex I, complex II, complex III, and complex IV) showed decreased January 2020 \| Volume 10 \| Article 1337 Chicken's Mitochondrial Dysfunction Li et al. Li et al. activities of complex III and complex IV were determined by the change in absorbance of reduced cytochrome c measured at 550 nm. according to the manufacturer's instructions. The RNA integrity and concentration were determined using 1.5% agarose gel electrophoresis and a Nanodrop 2000c spectrophotometer (Thermo, USA), respectively. cDNA was carried out using a PrimeScript RT Reagent Kit (Takara) for RT-qPCR. The MonAmp™ChemoHS qPCR Mix (Monad, China) was used for qRT-PCR in a Bio-Rad CFX96 Real-Time Detection instrument (Bio-Rad, USA) according to the manufacturer's protocol. Relative gene expression was measured by qRT-PCR twice for each reaction and nuclear gene b-actin was used as a control. The primers are listed in Table 1. Mitochondrial Respiratory Control Ratio Mitochondrial Respiratory Control Ratio The mitochondrial respiratory control ratio (RCR) was determined using an RCR assay kit (Genmed Scientiﬁcs Inc., USA) according to the manufacturer's instructions as previously described (Su et al., 2016). Oxygen consumption was measured using a Clarke-type oxygen electrode (Hansatech Oxytherm, UK). RCR was represented as the ratio of State III to State IV respiration rate. Data were normalized to the control group and expressed as percentage of control levels. Mitochondrial Protein Concentration The mitochondrial protein concentration was measured using a BCA protein assay kit (Beyotime, China) carried out in a Fluorescence/Multi-Detection Microplate Reader (BioTek, USA) according to the manufacturer's instructions. Enzyme Activities of Mitochondrial Respiratory Complexes Liver tissues were dissected, frozen in liquid nitrogen, and then stored at −80°C (Thorburn et al., 2004). Enzyme activities of mitochondrial respiratory complexes were measured using commercial assay kits (Solarbio, China) according to the manufacturer's instructions carried out in a Fluorescence/ Multi-Detection Microplate Reader (BioTek, USA) according to the manufacturer's protocol. The enzyme activity of complex I was determined by the change in absorbance of NADH measured at 340 nm. The enzyme activity of complex II was determined by the change in absorbance of 2, 6- dichlorophenolindophenol measured at 600 nm. The enzyme H&E Staining and Oil Red O Staining of RSS Chickens, SLD Chickens and Normal Chickens FIGURE 1 \| Photographs of runting and stunting syndrome (RSS) chickens and sex-linked dwarf (SLD) chickens at the same age but different growth stages. The RSS chickens are smaller and possess villi faded slowly compared with the SLD chickens, regardless of sex. Several viruses, bacteria, and other pathogens are believed to be responsible for RSS in broiler chickens. Bacteria are frequently isolated from RSS birds, which include Escherichia coli, Proteus mirabilis, Enterococcus faecium, Staphylococcus cohnii, Clostridium perfringens, Bacteroides fragilis, and Bacillus licheniformis. They are commonly found in the intestinal tract and may cause secondary infections that aggravate the initial lesions (Rebel et al., 2006). Some researchers have demonstrated that astrovirus may cause RSS in broilers (Kang et al., 2018). In this study, vacuolation of the hepatocytes was observed in both RSS and SLD chickens (Figures 2A–F). Hydrophic degeneration in the hepatocytes was also seen in the RSS chickens, which may be responsible for the liver cell vacuoles (Figures 2A, D). The hydrophic degeneration in the hepatocytes of RSS chickens may disrupt the structure and inhibited the function of mitochondria. This may further result in the destruction of the energy supply functions of the mitochondria. FIGURE 1 \| Photographs of runting and stunting syndrome (RSS) chickens and sex-linked dwarf (SLD) chickens at the same age but different growth stages. The RSS chickens are smaller and possess villi faded slowly compared with the SLD chickens, regardless of sex. RESULTS RSS Chickens and SLD Chickens At the Same Age Exhibit Different Growth Stages As shown in Figure 1, RSS chickens were smaller with villi faded slowly compared with SLD chickens. The cockscomb, legs, and beaks of the RSS chickens appeared pale in color, indicating a decline in blood production. Some of the chickens were found to have rickets, broken legs, or fractured toes. In addition, all RSS January 2020 \| Volume 10 \| Article 1337 Frontiers in Genetics \| www.frontiersin.org 3 Chicken's Mitochondrial Dysfunction Li et al. Abnormally Expressed Genes in the Livers of RSS Chickens Compared With SLD Chickens Are Mainly in the OXPHOS Pathway, Fatty-Acid Metabolism Pathway, and Other Metabolic Pathways We next examined gene expression differences in the livers using expression proﬁle chip analysis. Gene Set Enrichment Analysis results revealed that the RSS chickens exhibited abnormal expression of numerous genes involved in metabolic pathways (Table 2). We identiﬁed that many of the abnormally expressed genes were involved in OXPHOS pathway. These genes are associated with metabolic function in chickens. The results indicate a defective energy metabolism in RSS chickens compared with SLD chickens. Mitochondrial Volume and Mitochondrial Structure of RSS Chickens, SLD Chickens, and Normal Chickens chickens were ALV-J and REV virus negative showed in Figure S1. We next analyzed the structure of the mitochondria using TEM. The results revealed that the total volume and average volume of mitochondria which were reduced in RSS chickens compared with SLD chickens and normal chickens (Figures 3A, B). Furthermore, excessive amounts of vacuoles were present in the hepatocytes of RSS chickens. This may induce a decrease in the number of mitochondria. The inner and outer mitochondrial membranes exhibited different degrees of damage, and even disappeared, and the endoplasmic reticulum (ER) structure was also damaged (Figure 3C). Abnormally Expressed Genes in the Livers of RSS Chickens Compared With SLD Chickens Are Mainly in the OXPHOS Pathway, Fatty-Acid Metabolism Pathway, and Other Metabolic Pathways Expression of Genes Involved in the OXPHOS Assessed by qRT-PCR To verify the consequences of expression proﬁle chip analysis, the relative expression levels of genes involved in mitochondrial OXPHOS were determined in RSS chickens, SLD chickens, and normal chickens. These three groups of chickens were evaluated at 7 weeks of age to verify the microarray data. As shown in Figure 4, comparison of the female RSS chickens with SLD chickens and SLD chickens with normal chickens revealed that the IGF2BP3 and NDUFB2 genes were upregulated and the NDUFA8, NDUFA9, NDUFB1, NDUFB5, NDUFB6, NDUFB8, Complex I is the ﬁrst complex in the electron transport chain (Hirst, 2005). The results revealed that the RSS chickens exhibited altered expression of the complex I genes. The change of the enzyme complex function directly or indirectly reﬂects the change of mitochondrial function. Furthermore, the IGF2BP3 and SDHA genes were abnormally expressed in RSS chicken livers. TABLE 2 \| Abnormally expressed genes in the livers of runting and stunting syndrome (RSS) chickens [relative to sex-linked dwarf (SLD) chickens]. Pathway Gene Oxidative phosphorylation NDUFB5, NDUFB6, NDUFB8, NDUFB9, ATP6V1G1, NDUFB1, NDUFB2, ATP6V0C, NDUFS4, ATP5I, TCIRG1, NDUFB10, NDUFA8, NDUFA9, NDUFA7, ATP5F1, ATP6V1D, LOC770190, PPA1, COX6C, SDHA, ATP6V1E1, COX6A1, ATP6V0A2, LOC770879 Fatty-acid metabolism ALDH7A1, CPT2, ADH5, ACADL, ACAT2, ALDH3A2, ACSL5 Metabolism of xenobiotics by cytochrome P450 CYP3A37, ADH5, EPHX1, LOC421447, LOC396380, UGT1A1, CYP3A80 Frontiers in Genetics \| www.frontiersin.org January 2020 \| Volume 10 \| Article 1337 4 TABLE 2 \| Abnormally expressed genes in the livers of runting and stunting syndrome (RSS) chickens [relative to sex-linked dwarf (SLD) chickens]. Pathway Gene Oxidative phosphorylation NDUFB5, NDUFB6, NDUFB8, NDUFB9, ATP6V1G1, NDUFB1, NDUFB2, ATP6V0C, NDUFS4, ATP5I, TCIRG1, NDUFB10, NDUFA8, NDUFA9, NDUFA7, ATP5F1, ATP6V1D, LOC770190, PPA1, COX6C, SDHA, ATP6V1E1, COX6A1, ATP6V0A2, LOC770879 Fatty-acid metabolism ALDH7A1, CPT2, ADH5, ACADL, ACAT2, ALDH3A2, ACSL5 Metabolism of xenobiotics by cytochrome P450 CYP3A37, ADH5, EPHX1, LOC421447, LOC396380, UGT1A1, CYP3A80 Frontiers in Genetics \| www.frontiersin.org January 2020 \| Volume 10 \| Article 1337 4 Frontiers in Genetics \| www.frontiersin.org January 2020 \| Volume 10 \| Article 1337 Chicken's Mitochondrial Dysfunction Li et al. FIGURE 2 \| Liver tissues stained with H&E and Oil Red O. (A) Liver section stained with H&E from runting and stunting syndrome (RSS) chicken. Bar, 50 mm. As indicated by the arrow, the hepatocytes possess a sparse cytoplasm and contain a large number of vacuoles. The hepatocytes are not only abnormal in terms of morphology and structure but also present in reduced number. Expression of Genes Involved in the OXPHOS Assessed by qRT-PCR (B) Liver section stained with H&E from sex-linked dwarf (SLD) chicken. Bar, 50 mm. Compared with the hepatocytes from the RSS chicken, the hepatocytes of the normal SLD chicken contain fewer vacuoles and less sparse cytoplasm. However, compared with the hepatocytes from the normal chicken, the hepatocytes of the SLD chicken possess a sparse cytoplasm and contain a larger number of vacuoles. (C) Liver section stained with H&E from normal chicken. Bar, 50 mm. The hepatocytes possess a normal shape and structure, and no vacuoles and sparse cytoplasm are observed. (D, E) Liver sections stained with Oil red O from RSS and SLD chickens, respectively, showing vacuolar hydropic degeneration, which leads to an incomplete structure of hepatocytes. Bar, 50 mm. These phenomena were more severe observed in the RSS chickens. (F) Liver section stained with Oil red O from normal chicken. Bar, 50 mm. No abnormal vacuoles in the hepatocytes is evident. FIGURE 2 \| Liver tissues stained with H&E and Oil Red O. (A) Liver section stained with H&E from runting and stunting syndrome (RSS) chicken. Bar, 50 mm. As indicated by the arrow, the hepatocytes possess a sparse cytoplasm and contain a large number of vacuoles. The hepatocytes are not only abnormal in terms of morphology and structure but also present in reduced number. (B) Liver section stained with H&E from sex-linked dwarf (SLD) chicken. Bar, 50 mm. Compared with the hepatocytes from the RSS chicken, the hepatocytes of the normal SLD chicken contain fewer vacuoles and less sparse cytoplasm. However, compared with the hepatocytes from the normal chicken, the hepatocytes of the SLD chicken possess a sparse cytoplasm and contain a larger number of vacuoles. (C) Liver section stained with H&E from normal chicken. Bar, 50 mm. The hepatocytes possess a normal shape and structure, and no vacuoles and sparse cytoplasm are observed. (D, E) Liver sections stained with Oil red O from RSS and SLD chickens, respectively, showing vacuolar hydropic degeneration, which leads to an incomplete structure of hepatocytes. Bar, 50 mm. These phenomena were more severe observed in the RSS chickens. (F) Liver section stained with Oil red O from normal chicken. Bar, 50 mm. No abnormal vacuoles in the hepatocytes is evident. NDUFB9, NDUFB10, NDUFS4, and SDHA genes were downregulated. Moreover, some differences were also observed in the male RSS chickens. Mitochondrial Function Measurement The activities of mitochondrial enzymes in the liver, including OXPHOS complexes, are summarized in Figure 6. The enzyme activities of complex I, complex II, complex III, and complex IV in the RSS chickens were reduced by 45%, 24%, 21%, and 41%, respectively, compared with SLD chickens (Figures 6A–D). Similarly, the enzyme activities of complex I, complex II, complex III, and complex IV in the SLD chickens were reduced by 56%, 22%, 21%, and 61%, respectively, compared with normal chickens. To determine whether these changes in the activities of the respiratory complexes inﬂuenced the function of the mitochondria, we next assessed the mitochondrial function by measuring the RCR, ATP level, DYm, and ROS production. The RCR values of the RSS chickens was reduced by 15% compared with SLD chickens, and that of SLD chickens was reduced by 35% compared with normal chickens (Figure 6E), indicating a lower oxygen consumption in the RSS chickens and SLD chickens. The ATP level of the RSS chickens was reduced by 37% compared with Expression of Genes Involved in the OXPHOS Assessed by qRT-PCR In particular, the NDUFA8, NDUFA9, NDUFB1, NDUFB5, NDUFB6, NDUFB8, NDUFB9, NDUFB10, NDUFS4, and SDHA genes were upregulated and the NDUFB2 genes were downregulated in the livers of male RSS chickens, which is contrary to the consequences of female RSS chickens except for IGF2BP3 gene (Figure 5). These results suggest that the IGF2BP3 and SDHA genes, as major genes related to growth and energy supply, are associated with the development of RSS in chickens. SLD chickens, and that of SLD chickens was reduced by 25% compared with normal chickens (Figure 6F), indicating a lower efﬁciency of ATP production in the RSS chickens and SLD chickens. The DYm measured by JC-1 of the RSS chickens was reduced by 16% compared with SLD chickens, and that of SLD chickens was reduced by 21% compared with normal chickens (Figure 6G), indicating a dissipative DYm in the RSS chickens and SLD chickens. Finally, the production of ROS in the RSS chickens was increased by 14% compared with SLD chickens, and that of SLD chickens was increased by 22% compared with normal chickens (Figure 6H). DISCUSSION In this group, normal mitochondria (indicated by white circles) and other organelles are observed. They are not only complete in structure but also present in large numbers. Data are represented as the mean ± SEM; p < 0.01; p < 0.001; ns, no signiﬁcan difference. FIGURE 3 \| Total and average volumes of mitochondria and transmission electron microscopy (TEM) images. Total volume (A) and average volume of mitochondria (B) in runting and stunting syndrome (RSS) chickens, sex-linked dwarf (SLD) chickens, and normal chickens. To examine the mitochondrial morphology, ﬁve randomly selected areas were imaged at 25,000× magniﬁcation and analyzed. (C) RSS 1–3: TEM images of liver sections from 7-week-old RSS chickens. In this group, vacuoles in the hepatocytes had destroyed the mitochondria and other organelles, as indicated by the yellow circles. This not only caused a severe decrease in the number of mitochondria, leading to the destruction of the interactions between mitochondria and other organelles, but also seriously affected the structure and function of the mitochondria. As indicated by the red circles, the mitochondria were small in size and their inner and outer membranes had been destroyed or even disappeared. The mitochondrial cristae had also disappeared. SLD 1–3: TEM images of liver sections from 7-week-old SLD chickens. In this group, the mitochondria in the hepatocytes are surrounded by a large number of vacuoles, and the number of mitochondria is also reduced. The mitochondria are small in size and their structure is incomplete, although the degree of damage is less than that in the RSS chickens. Vacuoles and parts of destroyed mitochondria are indicated by blue and purple circles, respectively. Normal 1–3: TEM images of liver sections from 7-week-old normal chickens. In this group, normal mitochondria (indicated by white circles) and other organelles are observed. They are not only complete in structure but also present in large numbers. Data are represented as the mean ± SEM; p < 0.01; *p < 0.001; ns, no signiﬁcan difference. tissues of the RSS chickens by viruses detection as previously described (Zavala, 2006; Zhang et al., 2015). is typically expressed in embryonic tissue, muscles, and the placenta. In fact, the expression of IGF2BP3 is low in the liver tissues of normal chickens. IGF2BP3 is an oncofetal protein with high expression during embryogenesis, low expression in adult tissues, and re-expression in malignant tissues (Palanichamy et al., 2016a). DISCUSSION The signs of RSS are usually observed in meat-type chickens between 3 and 6 weeks of age. Previous work has demonstrated that certain strains of birds appear to be more susceptible to the effects of RSS than others, and that male birds are more severely affected than females (Zavala, 2006). However, it is interesting to note that RSS-resistant broiler strains have stronger immunological responses against bacterial and viral infections than RSS-susceptible strains (Rebel et al., 2006). Some researchers have suggested that the poor growth and retarded feathering consistently observed in RSS-affected birds are due to a chronic infection. However, we found that SLD chickens were more susceptible to the effects of RSS and exhibited weaker immunological responses than normal chickens. Therefore, we studied 7-week-old yellow-feather broilers with RSS like features. Of note, No indication of viral was also detected in the liver January 2020 \| Volume 10 \| Article 1337 Frontiers in Genetics \| www.frontiersin.org 5 Li et al. Chicken's Mitochondrial Dysfunction FIGURE 3 \| Total and average volumes of mitochondria and transmission electron microscopy (TEM) images. Total volume (A) and average volume of mitochondria (B) in runting and stunting syndrome (RSS) chickens, sex-linked dwarf (SLD) chickens, and normal chickens. To examine the mitochondrial morphology, ﬁve randomly selected areas were imaged at 25,000× magniﬁcation and analyzed. (C) RSS 1–3: TEM images of liver sections from 7-week-old RSS chickens. In this group, vacuoles in the hepatocytes had destroyed the mitochondria and other organelles, as indicated by the yellow circles. This not only caused a severe decrease in the number of mitochondria, leading to the destruction of the interactions between mitochondria and other organelles, but also seriously affected the structure and function of the mitochondria. As indicated by the red circles, the mitochondria were small in size and their inner and outer membranes had been destroyed or even disappeared. The mitochondrial cristae had also disappeared. SLD 1–3: TEM images of liver sections from 7-week-old SLD chickens. In this group, the mitochondria in the hepatocytes are surrounded by a large number of vacuoles, and the number of mitochondria is also reduced. The mitochondria are small in size and their structure is incomplete, although the degree of damage is less than that in the RSS chickens. Vacuoles and parts of destroyed mitochondria are indicated by blue and purple circles, respectively. Normal 1–3: TEM images of liver sections from 7-week-old normal chickens. DISCUSSION Another study revealed that IGF2BP3, as a Further, we revealed that the mRNA of the insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) was upregulated in the RSS chicken's liver tissue. IGF2BP3 plays an important role in the formation and growth of many tumors and January 2020 \| Volume 10 \| Article 1337 Frontiers in Genetics \| www.frontiersin.org 6 Li et al. Chicken's Mitochondrial Dysfunction FIGURE 4 \| Relative expression of IGF2BP3 and other genes involved in oxidative phosphorylation (OXPHOS) in the livers of female chickens. Comparison of the female runting and stunting syndrome (RSS) chickens with sex-linked dwarf (SLD) chickens and SLD chickens with normal chickens revealed that the IGF2BP3 and NDUFB2 genes were upregulated and the NDUFA8, NDUFA9, NDUFB1, NDUFB5, NDUFB6, NDUFB8, NDUFB9, NDUFB10, NDUFS4, and SDHA genes were downregulated. Data are represented as the mean ± SEM; p < 0.01; p < 0.01; p < 0.001; ns, no signiﬁcant difference. FIGURE 4 \| Relative expression of IGF2BP3 and other genes involved in oxidative phosphorylation (OXPHOS) in the livers of female chickens. Comparison of the female runting and stunting syndrome (RSS) chickens with sex-linked dwarf (SLD) chickens and SLD chickens with normal chickens revealed that the IGF2BP3 and NDUFB2 genes were upregulated and the NDUFA8, NDUFA9, NDUFB1, NDUFB5, NDUFB6, NDUFB8, NDUFB9, NDUFB10, NDUFS4, and SDHA genes were downregulated. Data are represented as the mean ± SEM; p < 0.01; p < 0.01; p < 0.001; ns, no signiﬁcant difference. human ontogenic master switch, restricts megakaryocyte development by modulating a lineage-speciﬁc P-TEFb activation mechanism, revealing potential strategies toward enhancing platelet production (Elagib et al., 2017). In this study, the expression of IGF2BP3 in RSS chickens was examined by qRT-PCR. We found that IGF2BP3 expression was increased in the livers of RSS chickens and SLD chickens. This raises the question of the nature of the association between enhanced IGF2BP3 expression and RSS in chickens. Minchenko et al. demonstrated that the IGF2BP3 gene is expressed in hypoxic tissues, indicating its participation in the regulation of metabolic and proliferative processes via IGF/INS receptors (Minchenko et al., 2015). In this study, we found that both the livers of RSS and SLD chickens exhibited signs of mitochondrial dysfunction, and the livers exhibited black or yellow in color. This suggests that the livers of RSS chickens are hypoxic. This would explain why IGF2BP3 expression was increased in the livers of the RSS chickens. Frontiers in Genetics \| www.frontiersin.org DISCUSSION Comparison of the male RSS chickens with SLD chickens the IGF2BP3, NDUFA8, NDUFA9, NDUFB1, NDUFB5, NDUFB6, NDUFB8, NDUFB9, NDUFB10, NDUFS4, and SDHA genes were upregulated and the NDUFB2 genes were downregulated. Comparison of the male SLD chickens with normal chickens the IGF2BP3 were upregulated and the NDUFA8, NDUFA9, NDUFB1, NDUFB2, NDUFB5, NDUFB6, NDUFB8, NDUFB9, NDUFB10, NDUFS4, and SDHA genes were downregulated. Data are represented as the mean ± SEM; p < 0.01; p < 0.01; p < 0.001. consumption in CHO cells transfected with cDNA coding for the full-length GHR (Perret-Vivancos et al., 2006a). GH affects mitochondrial metabolism by indirectly regulating the activities of OXPHOS enzyme complexes (Brown-Borg et al., 2012). We found that the abnormal interaction between GH and GHR can inﬂuence mitochondrial oxidation and respiratory functions. The ﬁve multimeric enzyme complexes that drive OXPHOS and cellular respiration are contained within the inner mitochondrial membrane (Barrientos et al., 2009). Determining the activities of individual enzymes in the respiratory chain complex provides an important diagnostic indicator of mitochondrial disease (Frazier and Thorburn, 2012). In mitochondrial disease, the activities of single or multiple complexes are reduced in the affected tissue (El- Hattab and Scaglia, 2013). Activity assays typically reveal decreased activities of complex I, complex III, and complex IV in liver tissues (El-Hattab and Scaglia, 2013). In this study, we observed decreased enzyme activities of complex I, complex II, complex III, and complex IV in RSS and SLD chickens. SDHA activity, likely reducing the overall growth rates in vitro and in vivo that have been observed in experimental models of SDHA deﬁciency (Guzy et al., 2008). Another study demonstrated that knockdown of the SDHA/B genes in HeLa cells led to the accumulation of succinate (Xiao et al., 2012). Konieczna et al. found that partial silencing of SDHA with speciﬁc siRNAs resulted in either less efﬁcient or delayed entrance of the cells to the S phase (Konieczna et al., 2015). However, the cells remained viable. The results of this study revealed that SDHA was abnormal expression in the livers of RSS chickens compared with SLD chickens, but no mutation was detected. Lower OXPHOS activity will lead to insufﬁcient energy supply, which appears to be one of the reasons for the development of RSS in chickens. Decreased OXPHOS activities, along with higher ROS production in RSS and SLD chickens, are indicative of damaged mitochondrial function and efﬁciency. DISCUSSION The RCR results further demonstrated that the livers of RSS chickens were hypoxic compared with SLD chickens. The abnormal expression of IGF2BP3 conﬁrmed that oxidative respiratory dysfunction occurs in the mitochondria of both RSS chickens and SLD chickens. Under hypoxic conditions, NADH and FADH2 cannot participate in respiratory chain regeneration, thereby interrupting the OXPHOS and preventing the normal synthesis of ATP. Our results also demonstrated that the ATP production was reduced in the livers of RSS chickens. Moreover, we also revealed that the amounts of several components of the OXPHOS pathway in RSS chickens. NDUFA8, NDUFA9, NDUFB1, NDUFB5, NDUFB6, NDUFB8, NDUFB9, NDUFB10, and NDUFS4 are downregulated in female RSS chickens compared to SLD chickens. However, the expression of these genes of female RSS chickens is contrary to that of male RSS chickens, which may indicate that the regulatory mechanisms of male and female RSS chickens are different. In particular, succinate dehydrogenase complex subunit A (SDHA) was abnormal expression in the livers of RSS chickens compared with those of SLD chickens. SDHA is anchored to the inner mitochondrial membrane by one hydrophobic subunit of SDHC (Sun et al., 2005). The catalytic subunit of SDHA is hydrophilic and extends into the mitochondrial matrix (Lancaster and Simon, 2002). SDHA contains a covalently attached ﬂavin adenine dinucleotide (FAD) cofactor and the succinate-binding site. Flavination is important for succinate oxidation (Robinson et al., 1994). A previous study conﬁrmed that ﬂavination defects interfere with January 2020 \| Volume 10 \| Article 1337 Frontiers in Genetics \| www.frontiersin.org 7 Li et al. Chicken's Mitochondrial Dysfunction FIGURE 5 \| Relative expression of IGF2BP3 and other genes involved in oxidative phosphorylation (OXPHOS) in the livers of male chickens. Comparison of the male RSS chickens with SLD chickens the IGF2BP3, NDUFA8, NDUFA9, NDUFB1, NDUFB5, NDUFB6, NDUFB8, NDUFB9, NDUFB10, NDUFS4, and SDHA genes were upregulated and the NDUFB2 genes were downregulated. Comparison of the male SLD chickens with normal chickens the IGF2BP3 were upregulated and the NDUFA8, NDUFA9, NDUFB1, NDUFB2, NDUFB5, NDUFB6, NDUFB8, NDUFB9, NDUFB10, NDUFS4, and SDHA genes were downregulated. Data are represented as the mean ± SEM; p < 0.01; p < 0.01; p < 0.001. FIGURE 5 \| Relative expression of IGF2BP3 and other genes involved in oxidative phosphorylation (OXPHOS) in the livers of male chickens. Frontiers in Genetics \| www.frontiersin.org DISCUSSION The vacuolar hydropic degeneration may induce the enlargement of hepatocytes and compress adjacent organelles, leading to damage of the nuclei and mitochondria of liver cells. This phenomenon was observed not only in the RSS chickens but also in SLD chickens with GHR mutation. Previous studies have conﬁrmed that growth hormone (GH) stimulates cellular oxygen Inefﬁcient mitochondrial function was also observed upon measuring the RCR, ATP level, DYm, and ROS production. As mitochondria are responsible for the majority of oxygen consumption, the RCR is an essential parameter for studying January 2020 \| Volume 10 \| Article 1337 8 Chicken's Mitochondrial Dysfunction Li et al. FIGURE 6 \| Enzymatic activities of mitochondrial respiratory complexes and mitochondrial function analysis of 7-week-old runting and stunting syndrome (RSS) (RSS) chickens, sex-linked dwarf (SLD) chickens, and normal chickens. The enzymatic activities of (A) complex I, (B) complex II, (C) complex III, and (D) complex IV in the livers of RSS chickens, SLD chickens, and normal chickens expressed a percentage of normal chickens. Respiratory control ratio (RCR) (E), adenosine triphosphate (ATP) level (F), DYm (G), and reactive oxygen species (ROS) production (H) in the livers of RSS, SLD, and normal chickens expressed as a percentage of normal chickens. Data are represented as the mean ± SEM; p < 0.01; *p < 0.01; ns, no signiﬁcant diffrerence. FIGURE 6 \| Enzymatic activities of mitochondrial respiratory complexes and mitochondrial function analysis of 7-week-old runting and stunting syndrome (RSS) (RSS) chickens, sex-linked dwarf (SLD) chickens, and normal chickens. The enzymatic activities of (A) complex I, (B) complex II, (C) complex III, and (D) complex IV in the livers of RSS chickens, SLD chickens, and normal chickens expressed a percentage of normal chickens. Respiratory control ratio (RCR) (E), adenosine triphosphate (ATP) level (F), DYm (G), and reactive oxygen species (ROS) production (H) in the livers of RSS, SLD, and normal chickens expressed as a percentage of normal chickens. Data are represented as the mean ± SEM; p < 0.01; **p < 0.01; ns, no signiﬁcant diffrerence. mitochondrial function (Frezza et al., 2007). Meanwhile, DYm is essential for mitochondrial function, and mitochondrial dysfunction is normally accompanied by a decrease in DYm (Javadov et al., 2006). Further, excessive ROS production can impair the activities of mitochondrial respiratory complexes and reduced ATP production (Sudheesh et al., 2013). DISCUSSION Here, we observed alterations in the RCR, ATP level, DYm, and ROS production in both RSS chickens and SLD chickens. However, the mitochondrial function was lowest in RSS chickens, indicating further mitochondrial dysfunction in RSS chickens compared with SLD chickens. This may further impair immunity, causing greater mortality in RSS chickens. mitochondrial membranes of the RSS chickens exhibited varying degrees of damage or had even disappeared. Consequently, the ﬁve multimeric enzyme complexes within the inner mitochondrial membrane are unable to drive OXPHOS and cellular respiration. The GHR mutation, along with contributions from the additional factors mentioned above, may lead to the hindered growth and development in SLD chickens. If mitochondrial damage becomes more severe in SLD chickens, these may exhibit the signs of RSS. The methods reported in this study can also be used as a model for inner mitochondrial membrane GHR mutation. Together, all of these changes in mitochondrial functions could account for mitochondria dysfunction, resulting in poor weight gain and retarded growth or stunting of chicks. Our ﬁndings revealed that the RSS in chicken is caused by mitochondria dysfunction. We didn't ﬁnd mtDNA mutation in RSS and SLD chickens. The GHR mutation may cause mitochondria dysfunction in SLD chicken which is induced by GHR mutation, but RSS chicken's mitochondria dysfunction is more serious. We hypothesize that RSS chicken's mitochondrial disorders are caused by other nuclear gene mutations. Mitochondria, the only organelles in animal cells that contain their own genome, exchange Ca2+ and ROS with the endoplasmic reticulum (Pacher and Hajnoczky, 2001; Zorov et al., 2014; Booth et al., 2016). An evolutionarily conserved machinery enables the complete and sequential fusion of the outer and inner mitochondrial membranes (Robinson et al., 1994). In this study, no inﬂammation was detected by H&E staining and no lymphocyte inﬁltration was observed. Vacuolation of hepatocytes was observed, and the cytoplasm was disappeared in RSS and SLD chickens. The hydropic degeneration in the hepatocytes was the main cause of the liver cell vacuoles. GHR mutation induces an abnormal interaction between GH and GHR. As GHR gene is associated with oxidative respiration (Perret-Vivancos et al., 2006b), this may further inﬂuences mitochondrial oxidation and respiratory functions. Here, the TEM results revealed that the inner and outer REFERENCES mitochondrial-derived reactive oxygen species. J. Pharmacol. Exp. Ther. 317, 1036–1043. doi: 10.1124/jpet.105.100107 mitochondrial-derived reactive oxygen species. J. Pharmacol. Exp. Ther. 317, 1036–1043. doi: 10.1124/jpet.105.100107 Barrientos, A., Fontanesi, F., and Diaz, F. (2009). Evaluation of the mitochondrial respiratory chain and oxidative phosphorylation system using polarography and spectrophotometric enzyme assays. Curr. Protoc. Hum. Genet. 19, 4. doi: 10.1002/0471142905.hg1903s63 Kang, K. I., Linnemann, E., Icard, A. H., Durairaj, V., Mundt, E., and Sellers, H. S. (2018). Chicken astrovirus as an aetiological agent of runting-stunting syndrome in broiler chickens. J. Gen. Virol. 99, 512–524. doi: 10.1099/ jgv.0.001025 Kim, T. S., Yun, B. Y., and Kim, I. Y. (2003). Induction of the mitochondrial permeability transition by selenium compounds mediated by oxidation of the protein thiol groups and generation of the superoxide. Biochem. Pharmacol. 66, 2301–2311. doi: 10.1016/j.bcp.2003.08.021 Booth, D. M., Enyedi, B., Geiszt, M., Varnai, P., and Hajnoczky, G. (2016). Redox nanodomains are induced by and control calcium signaling at the ER- mitochondrial interface. Mol. Cell 63, 240–248. doi: 10.1016/j.molcel.2016.05.040 Bourgeron, T., Rustin, P., Chretien, D., Birch-Machin, M., Bourgeois, M., Viegas- Pequignot, E., et al. (1995). Mutation of a nuclear succinate dehydrogenase gene results in mitochondrial respiratory chain deﬁciency. Nat. Genet. 11, 144– 149. doi: 10.1038/ng1095-144 Konieczna, A., Szczepanska, A., Sawiuk, K., Wegrzyn, G., and Lyzen, R. (2015). Effects of partial silencing of genes coding for enzymes involved in glycolysis and tricarboxylic acid cycle on the enterance of human ﬁbroblasts to the S phase. BMC Cell Biol. 16, 16. doi: 10.1186/s12860-015-0062-8 Bracewell, C. D., and Wyeth, P. (1981). Infectious stunting of chickens. Vet. Rec. 109, 64. doi: 10.1136/vr.109.3.64-b Kouwenhoven, B., Davelaar, F. G., and Van Walsum, J. (1978). Infectious proventriculitis causing runting in broilers. Avian Pathol. 7, 183–187. doi: 10.1080/03079457808418269 Brown-Borg, H. M., Johnson, W. T., and Rakoczy, S. G. (2012). Expression of oxidative phosphorylation components in mitochondria of long-living Ames dwarf mice. Age (Dordr) 34, 43–57. doi: 10.1007/s11357-011-9212-x Lancaster, C. R., and Simon, J. (2002). Succinate:quinone oxidoreductases from epsilon-proteobacteria. Biochim. Biophys. Acta 1553, 84–101. doi: 10.1016/ S0005-2728(01)00230-4 Desjardins, P., and Morais, R. (1990). Sequence and gene organization of the chicken mitochondrial genome. A novel gene order in higher vertebrates. J. Mol. Biol. 212, 599–634. doi: 10.1016/0022-2836(90)90225-B Luo, W., Lin, S., Li, G., Nie, Q., and Zhang, X. (2016). Integrative analyses of miRNA-mRNA interactions reveal let-7b, miR-128 and MAPK pathway involvement in muscle mass loss in sex-linked dwarf chickens. Int. J. Mol. Sci. 17, 276. REFERENCES doi: 10.3390/ijms17030276 Elagib, K. E., Lu, C. H., Mosoyan, G., Khalil, S., Zasadzinska, E., Foltz, D. R., et al. (2017). Neonatal expression of RNA-binding protein IGF2BP3 regulates the human fetal-adult megakaryocyte transition. J. Clin. Invest. 127, 2365–2377. doi: 10.1172/JCI88936 Meldau, S., De Lacy, R. J., Riordan, G., Goddard, E. A., Pillay, K., Fieggen, K. J., et al. (2018). Identiﬁcation of a single MPV17 nonsense-associated altered splice variant in 24 South African infants with mitochondrial neurohepatopathy. Clin. Genet. 93, 1093–1096. doi: 10.1111/cge.13208 El-Hattab, A. W., and Scaglia, F. (2013). Mitochondrial DNA depletion syndromes: review and updates of genetic basis, manifestations, and therapeutic options. Neurotherapeutics 10, 186–198. doi: 10.1007/s13311-013-0177-6 Minchenko, D. O., Kharkova, A. P., Karbovskyi, L. L., and Minchenko, O. H. (2015). Expression of insulin-like growth factor binding protein genes and its hypoxic regulation in U87 glioma cells depends on ERN1 mediated signaling pathway of endoplasmic reticulum stress. Endocr. Regul. 49, 73–83. doi: 10.4149/endo_2015_04_185 Frazier, A. E., and Thorburn, D. R. (2012). Biochemical analyses of the electron transport chain complexes by spectrophotometry. Methods Mol. Biol. 837, 49– 62. doi: 10.1007/978-1-61779-504-6_4 Frezza, C., Cipolat, S., and Scorrano, L. (2007). Organelle isolation: functional mitochondria from mouse liver, muscle and cultured ﬁbroblasts. Nat. Protoc. 2, 287–295. doi: 10.1038/nprot.2006.478 Moraes, C. T., Shanske, S., Tritschler, H. J., Aprille, J. R., Andreetta, F., Bonilla, E., et al. (1991). mtDNA depletion with variable tissue expression: a novel genetic abnormality in mitochondrial diseases. Am. J. Hum. Genet. 48, 492–501. doi: 10.1016/0378-1119(91)90142-X Guzy, R. D., Sharma, B., Bell, E., Chandel, N. S., and Schumacker, P. T. (2008). Loss of the SdhB, but Not the SdhA, subunit of complex II triggers reactive oxygen species-dependent hypoxia-inducible factor activation and tumorigenesis. Mol. Cell. Biol. 28, 718–731. doi: 10.1128/mcb.01338-07 Ouyang, J., Xie, L., Nie, Q., Zeng, H., Peng, Z., Zhang, D., et al. (2012). The effects of different sex-linked dwarf variations on chinese native chickens. J. Integr. Agr. 11, 1500–1508. umorigenesis. Mol. Cell. Biol. 28, 718–731. doi: 10.1128/mcb.0133 Hirst, J. (2005). Energy transduction by respiratory complex I–an evaluation of current knowledge. Biochem. Soc. Trans. 33, 525–529. doi: 10.1042/ BST0330525 Pacher, P., and Hajnoczky, G. (2001). Propagation of the apoptotic signal by mitochondrial waves. EMBO J. 20, 4107–4121. doi: 10.1093/emboj/20.15.4107 Hu, B., Hu, S., Yang, M., Liao, Z., Zhang, D., Luo, Q., et al. (2019). Growth hormone receptor gene is essential for chicken mitochondrial function in vivo and in vitro. Int. J. Mol. Sci. FUNDING regulations established by this committee. Animals involved in this study were humanely sacriﬁced as necessary. This work was supported by grants from the National Natural Science Fund Committee National Youth Project (Project grant no: 31401046), Guangdong Provincial Promotion Project on Preservation and Utilization of Local Breed of Livestock and Poultry and Guangdong Youth Talent Project. This work was supported by grants from the National Natural Science Fund Committee National Youth Project (Project grant no: 31401046), Guangdong Provincial Promotion Project on Preservation and Utilization of Local Breed of Livestock and Poultry and Guangdong Youth Talent Project. AUTHOR CONTRIBUTIONS HL and BH contributed equally to this manuscript. HL designed the study, and wrote the paper. BH carried out experiments, and analyzed data. QL and SH participated in the design of the experiment and data analysis. YLu, BZ, and YG participated in data collection and interpretation, and helped with performing some of the manuscripts' experiments. YLi, MS, and QN helped for useful discussion and language correction. XZ and DZ participated in the design, manuscript writing and ﬁnal approval of the manuscript. All authors read and approved the ﬁnal manuscript. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fgene.2019. 01337/full#supplementary-material ETHICS STATEMENT All procedures involving animals were approved by South China Agriculture University's Institutional Animal Care and Use Committee (approval number SCAU#0017), according to the January 2020 \| Volume 10 \| Article 1337 Frontiers in Genetics \| www.frontiersin.org 9 Chicken's Mitochondrial Dysfunction Li et al. Li et al. REFERENCES 20, 7. doi: 10.3390/ijms20071608 Palanichamy, J. K., Tran, T. M., Howard, J. M., Contreras, J. R., Fernando, T. R., Sterne-Weiler, T., et al. (2016a). RNA-binding protein IGF2BP3 targeting of oncogenic transcripts promotes hematopoietic progenitor proliferation. J. Clin. Invest. 126, 1495–1511. doi: 10.1172/JCI80046 Javadov, S., Baetz, D., Rajapurohitam, V., Zeidan, A., Kirshenbaum, L. A., and Karmazyn, M. (2006). Antihypertrophic effect of Na+/H+ exchanger isoform 1 inhibition is mediated by reduced mitogen-activated protein kinase activation secondary to improved mitochondrial integrity and decreased generation of Perret-Vivancos, C., Abbate, A., Ardail, D., Raccurt, M., Usson, Y., Lobie, P. E., et al. (2006a). Growth hormone activity in mitochondria depends on GH January 2020 \| Volume 10 \| Article 1337 Frontiers in Genetics \| www.frontiersin.org 10 Chicken's Mitochondrial Dysfunction Li et al. receptor Box 1 and involves caveolar pathway targeting. Exp. Cell Res. 312, 215–232. doi: 10.1016/j.yexcr.2005.10.027 receptor Box 1 and involves caveolar pathway targeting. Exp. Cell Res. 312, 215–232. doi: 10.1016/j.yexcr.2005.10.027 Wong, L. J., Brunetti-Pierri, N., Zhang, Q., Yazigi, N., Bove, K. E., Dahms, B. B., et al. (2007). Mutations in the MPV17 gene are responsible for rapidly progressive liver failure in infancy. Hepatology 46, 1218–1227. doi: 10.1002/ hep.21799 Perret-Vivancos, C., Abbate, A., Ardail, D., Raccurt, M., Usson, Y., Lobie, P. E., et al. (2006b). Growth hormone activity in mitochondria depends on GH receptor Box 1 and involves caveolar pathway targeting. Exp. Cell Res. 312, 215–232. doi: 10.1016/j.yexcr.2005.10.027 Xiao, M., Yang, H., Xu, W., Ma, S., Lin, H., Zhu, H., et al. (2012). Inhibition of alpha-KG-dependent histone and DNA demethylases by fumarate and succinate that are accumulated in mutations of FH and SDH tumor suppressors. Genes Dev. 26, 1326–1338. doi: 10.1101/gad.191056.112 Rebel, J. M. J., Balk, F. R. M., Post, J., Hemert, S. V., and Stockhofe, B. Z. N. (2006). Malabsorption syndrome in broilers. World. Poultry Sci. J. 62, 17–30. doi: 10.1079/WPS200481 pp , g Zavala, G. (2006). Runting Stunting Syndrome (RSS) in Broilers. In Vivo Stud. Zavala, G. (2006). Runting Stunting Syndrome (RSS) in Broilers. In Vivo Stud. Zeviani M Bonilla E DeVivo D C and DiMauro S (1989) Mitochondrial Zavala, G. (2006). Runting Stunting Syndrome (RSS) in Broilers. In Vivo Stud. Zeviani, M., Bonilla, E., DeVivo, D. C., and DiMauro, S. (1989). Mitochondrial diseases. Neurol. Clin. 7, 123–156. doi: 10.1053/beog.2002.0315 Robinson, K. M., Rothery, R. A., Weiner, J. H., and Lemire, B. D. (1994). Frontiers in Genetics \| www.frontiersin.org January 2020 \| Volume 10 \| Article 1337 REFERENCES The covalent attachment of FAD to the ﬂavoprotein of Saccharomyces cerevisiae succinate dehydrogenase is not necessary for import and assembly into mitochondria. Eur. J. Biochem. 222, 983–990. doi: 10.1111/j.1432-1033.1994.tb18949.x Zhang, L., Li, Y., Xie, X., Xu, H., Xu, Z., Ma, J., et al. (2015). A Systematic Analysis on mRNA and MicroRNA expression in runting and stunting chickens. PloS One 10, e0127342. doi: 10.1371/journal.pone.0127342 Biochem. 222, 983–990. doi: 10.1111/j.1432-1033.1994.tb18949.x Sarzi, E., Goffart, S., Serre, V., Chretien, D., Slama, A., Munnich, A., et al. (2007). Twinkle helicase (PEO1) gene mutation causes mitochondrial DNA depletion. Ann. Neurol. 62, 579–587. doi: 10.1002/ana.21207 Zorov, D. B., Juhaszova, M., and Sollott, S. J. (2014). Mitochondrial reactive oxygen species (ROS) and ROS-induced ROS release. Physiol. Rev. 94, 909– 950. doi: 10.1152/physrev.00026.2013 Su, Y., Zhu, L., Yu, X., Cai, L., Lu, Y., Zhang, J., et al. (2016). Mitochondrial transplantation attenuates airway hyperresponsiveness by inhibition of cholinergic hyperactivity. Theranostics 6, 1244–1260. doi: 10.7150/thno.13804 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. Sudheesh, N. P., Ajith, T. A., and Janardhanan, K. K. (2013). Ganoderma lucidum ameliorate mitochondrial damage in isoproterenol-induced myocardial infarction in rats by enhancing the activities of TCA cycle enzymes and respiratory chain complexes. Int. J. Cardiol. 165, 117–125. doi: 10.1016/j.ijcard.2011.07.103 Copyright © 2020 Li, Hu, Luo, Hu, Luo, Zhao, Gan, Li, Shi, Nie, Zhang and Zhang. 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. Sun, F., Huo, X., Zhai, Y., Wang, A., Xu, J., Su, D., et al. (2005). Crystal structure of mitochondrial respiratory membrane protein complex II. Cell 121, 1043–1057. doi: 10.1016/j.cell.2005.05.025 Thorburn, D. R., Chow, C. W., and Kirby, D. M. (2004). Respiratory chain enzyme analysis in muscle and liver. Mitochondrion 4, 363–375. doi: 10.1016/ j.mito.2004.07.003 January 2020 \| Volume 10 \| Article 1337 Frontiers in Genetics \| www.frontiersin.org 11
https://openalex.org/W2903251157	https://europepmc.org/articles/pmc6267080?pdf=render	English	null	Prognostic factors for survival with nab-paclitaxel plus gemcitabine in metastatic pancreatic cancer in real-life practice: the ANICE-PaC study	BMC cancer	2,018	cc-by	7,230	Prognostic factors for survival with nab- paclitaxel plus gemcitabine in metastatic pancreatic cancer in real-life practice: the ANICE-PaC study Prognostic factors for survival with nab- paclitaxel plus gemcitabine in metastatic pancreatic cancer in real-life practice: the ANICE-PaC study Ana Fernández1, Mercedes Salgado2, Adelaida García3, Elvira Buxò4, Ruth Vera5, Jorge Adeva6, Paula Jiménez-Fonseca7, Guillermo Quintero8, Cristina Llorca9, Mamen Cañabate10, Luis Jesús López11, Andrés Muñoz12, Patricia Ramírez13, Paula González14, Carlos López15, Margarita Reboredo16, Elena Gallardo17, Manuel Sanchez-Cánovas18, Javier Gallego19, Carmen Guillén20, Nuria Ruiz-Miravet21, Víctor Navarro-Pérez22, Juan De la Cámara23, Inmaculada Alés-Díaz24, Roberto Antonio Pazo-Cid25 and Alberto Carmona-Bayonas18 Abstract Background: Treatment with nab-paclitaxel plus gemcitabine increases survival in patients with metastatic pancreatic cancer. However, the assessment of treatment efficacy and safety in non-selected patients in a real-life setting may provide useful information to support decision-making processes in routine practice. Methods: Retrospective, multicenter study including patients with metastatic pancreatic cancer, who started first- line treatment with nab-paclitaxel plus gemcitabine between December 2013 and June 2015 according to routine clinical practice. In addition to describing the treatment pattern, overall survival (OS) and progression-free survival (PFS) were assessed for the total sample and the exploratory subgroups based on the treatment and patients’ clinical characteristics. Results: All 210 eligible patients had a median age of 65.0 years (range 37–81). Metastatic pancreatic adenocarcinoma was recurrent in 46 (21.9%) patients and de novo in 164 (78.1%); 38 (18%) patients had a biliary stent. At baseline, 33 (18.1%) patients had an ECOG performance status ≥2. Patients received a median of four cycles of treatment (range 1–21), with a median duration of 3.5 months; 137 (65.2%) patients had a dose reduction of nab-paclitaxel and/or gemcitabine during treatment, and 33 (17.2%) discontinued treatment due to toxicity. Relative dose intensity (RDI) for nab-paclitaxel, gemcitabine, and the combined treatment was 66.7%. Median OS was 7.2 months (95% CI 6.0–8.5), and median PFS was 5.0 months (95% CI 4.3–5.9); 50 patients achieved either a partial or complete response (ORR 24.6%). OS was influenced by baseline ECOG PS, NLR and CA 19.9, but not by age ≥70 years and/or the presence of hepatobiliary stent or RDI < 85%. All included variables, computed as dichotomous, showed a significant contribution to the Cox regression model to build a nomogram for predicting survival in these patients: baseline ECOG 0–1 vs. 2–3 (p = 0.030), baseline NLR > 3 vs. ≤3 (p = 0.043), and baseline CA 19.9 > 37 U/mL vs. ≤37 U/mL (p = 0.004). Correspondence: ana.fernandez.montes@sergas.es 1Complejo Hospitalario Universitario Ourense, Calle Ramon Puga Noguerol, 54, 32005 Ourense, Spain Full list of author information is available at the end of the article Fernández et al. BMC Cancer (2018) 18:1185 https://doi.org/10.1186/s12885-018-5101-3 Fernández et al. BMC Cancer (2018) 18:1185 https://doi.org/10.1186/s12885-018-5101-3 Open Access © 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. Background clinical picture of randomized trials. As such, observa- tional trials can provide useful information for clinicians to support decision-making processes in their routine practice, especially when treating patient groups under- represented in clinical trials, as are elderly patients and those with poorer performance status [14, 15]. In line with this unmet need, Ellenrieder et al. highlighted the importance of real-life data regarding first-line regimens in metastatic pancreatic cancer to select the most suitable treatment for each patient [7]. The aim of the ANICE study was to assess the effectiveness and tolerability of gemcitabine and nab-paclitaxel as first-line therapy for metastatic pancreatic cancer in a real-life setting. Pancreatic adenocarcinoma, the most common form of pancreatic cancer, is currently the fourth cause of cancer-related mortality in Europe and the United States, with a 5-year survival rate in the range of 6–10% [1–4]. This has been attributed, among other causes, to the premature vascular, lymphatic and perineural spread of these tumors, which makes that 85% of patients present disseminated disease at diagnosis. Only between 15 and 20% of tumors are resectable and, within these, 50 to 86% will experience local failure despite curative resection, with a resulting 5-year survival rate of 10–20% [5]. Gemcitabine has been the standard first-line treat- ment for advanced pancreatic cancer for 15 years, and it is associated with median overall survivals (OS) ranging from 5.6 to 6.8 months [6]. Its combination with a wide range of other agents such as capecita- bine, oxaliplatin, cisplatin, irinotecan, and erlotinib has unfortunately shown little impact on survival in this population [6, 7]. Nonetheless, two combination regimens, FOLFIRINOX (folinic acid, fluorouracil, iri- notecan, oxaliplatin) and more recently, nab-paclitaxel plus gemcitabine, have been associated with a median OS of 11.1 months and 8.5 months in the ACCORD4/ PRODIGE11 and MPACT phase III clinical trials, re- spectively [8, 9]. These encouraging results have set a new standard and international guidelines now recom- mend FOLFIRINOX and nab-paclitaxel plus gemcitabine as first-line treatments in patients with metastatic pancre- atic cancer [10, 11]. Study design and patients The ANICE trial was an observational, retrospective, multicenter study focused on patients with metastatic pancreatic adenocarcinoma (recurrent or de novo) treated according to routine clinical practice at 20 Spanish hospitals between December 2013 and June 2015. All adult patients (≥18 years) with measurable metastatic disease at baseline in at least one dimension (per Response Evaluation Criteria in Solid Tumors [RECIST], version 1.1, [16]) who received at least one dose of nab-paclitaxel (Abraxane®, Celgene Europe Limited) plus gemcitabine as first-line chemotherapy were included. Data were obtained from clinical medical records with a cut-off date of 16 March 2017. All patients provided written informed consent. The study protocol was approved by the local independent ethics committee, and was conducted in accordance with the Spanish personal data protection law (LOPD 15/1999). A questionnaire-based study showed that clinicians are likely to adhere to the selection criteria of these trials, being the patient’s performance status one of the most influential factors in the decision-making process in the real-life setting [12]. As randomized trials have strict selection criteria, particularly regarding performance sta- tus and age [8, 9], reported data might not capture the scenario faced in the real-life setting, with non-selected patients. A prospective registry-based study showed that fewer than half of the patients treated in routine clinical practice would have been eligible for the PRODIGE or MPACT trials and that meeting the eligibility criteria for either trial is associated with longer survival [13]. Fernández et al. BMC Cancer (2018) 18:1185 Page 2 of 11 (Continued from previous page) Conclusions: Nab-Paclitaxel plus gemcitabine remain effective in a real-life setting, despite the high burden of dose reductions and poorer performance of these patients. A nomogram to predict survival using baseline ECOG performance status, NLR and CA 19.9 is proposed. Keywords: Metastatic pancreatic adenocarcinoma, Gemcitabine, Nab-paclitaxel, Real-life, First-line chemotherapy, Survival Patient characteristics Of the 216 patients recruited, 6 were excluded for one or more of the following reasons: not initiating com- bined treatment with gemcitabine plus nab-paclitaxel (n = 4), absence of measurable disease at baseline per RECIST (n = 2), and being enrolled in a clinical trial (n = 1). Table 1 summarizes the demographic and clinical characteristics of study patients. The 210 eligible pa- tients had a median age of 65.0 years (range 37–81). Metastatic pancreatic adenocarcinoma was recurrent in 46 (21.9%) patients and de novo in 164 (78.1%). For patients with recurrent metastatic disease, the primary tumor was resectable in 38 cases (82.6%), borderline in 3 (6.5%), and locally advanced unresectable in 5 (10.9%), and median time to recurrence was 11.0 months. Assessment of eligibility for the MPACT and ACCORD4/ PRODIGE11 trials could be assessed in 78 (37.1%) and 172 (81.9%) patients, respectively, with 37 meeting the criteria for entering the MPACT trial and 109 the ACCORD4/PRODIGE11 trial. Variables and endpoints Variables collected from the patient’s medical records included demographic data (sex and age), clinical data, characteristics of the metastatic disease and treatment. Among the registered clinical and disease characteristics were performance status (PS) (ECOG and Karnofsky scales), relevant comorbidities, initial diagnosis, number and localization of metastases, time between diagnosis of the primary tumor and recurrence, presence of a hepatobiliary stent, serum bilirubin, neutrophil/lymphocyte Clinicians are increasingly interested in obtaining real-life data from daily practice, which completes the Fernández et al. BMC Cancer (2018) 18:1185 Page 3 of 11 Page 3 of 11 ratio (NLR), and CA 19.9 antigen levels. Treatment charac- teristics included concomitant treatments, relative dose intensity (RDI) for nab-paclitaxel plus gemcitabine (i.e., per the summary of product characteristics), treatment duration, cause of treatment discontinuation, number of cycles, and dose reductions and interruptions. Decisions regarding the initial dose and subsequent dose reductions were made at physicians’ discretion, based on patient’s PS and toxicity, and following the routine practice of each participating sit. included in a Cox regression model as dichotomous variables to build a nomogram for predicting OS in real-life practice. The obtained hazard ratios (HR) were used to associate each variable with a survival score. In addition to the predicted survival, a probability for 3-, 6-, 12, and 18-month survival was estimated. Based on the range of final scores resulting from the possible combinations of variables, low-, medium-, and high-risk groups were defined. All analyses were per- formed using the statistical package SAS system for Windows version 9.4. The primary objective was to describe the treatment pattern in terms of extent of exposure and reductions of treatment with nab-paclitaxel plus gemcitabine in real-life clinical practice. Secondary objectives included objective response rate (ORR), progression-free survival (PFS) defined as the time from the start of treatment to disease progression or all-cause death, overall survival (OS) defined as the time from the start of treatment to death from any cause, and the 12-month survival rate defined as the percentage of patients alive at 12 months after starting treatment. Safety was assessed in terms of adverse events (AE), coded according to the preferred term of the Medical Dictionary for Regulatory Activities (MedDRA), and graded according to the National Cancer Institute-Common Toxicity Criteria (version 4.0) [17]. Statistical analyses Given the descriptive nature of the statistical analyses, the sample size was calculated based on the confidence interval (CI) of the 12-month survival rate. Considering the 12-month survival rate previously reported by Goldstein et al. [18], and assuming that nearly 20% of patients present with recurrent metastatic cancer, a sample of 225 patients was deemed necessary to esti- mate a 12-month survival rate of 30% with a ± 6% preci- sion and a 95% CI. Treatment characteristics and outcome eat e t c a acte st cs a d outco e Table 2 summarizes treatment characteristics. Median time from diagnosis to treatment start was 28 days. Patients received a median of 4 cycles (range 1–21) of treatment, with a median treatment duration of 3.5 months. Sixty-eight patients (32%) started treatment with dose reduction for either nab-paclitaxel, gemci- tabine or both drugs. Table 3 summarizes the base- line characteristics of the 68 patients with dose reduction and treatment start. The median RDIs were 66.7% for nab-paclitaxel, gemcitabine, and the com- bined treatment. Overall, 137 patients (65.2%) had a dose reduction in nab-paclitaxel and/or gemcitabine during treatment. Thirty-four (17%) patients received ≤30 days of treatment, mainly due to toxicity (n = 11) or disease progression (n = 10). There were no signifi- cant differences between patients receiving ≤30 days of treatment and those receiving > 30 days in terms of baseline clinical characteristics including perform- ance status, the presence of a hepatobiliary stent, Categorical variables were summarized as frequencies and percentages, and quantitative variables as the mean and standard deviation (SD) and/or median and inter- quartile range (IQR). The quantitative variables NLR and CA 19.9 were transformed into dichotomous variables with the cut-offs values of 3 and 37 U/mL, re- spectively. Categorical values were compared using the Fisher’s exact test or the chi-square test when the requirements for a Fisher’s exact test could not be assumed. Quantitative values were compared using the T-test, ANOVA test, and their non-parametric counter- parts, the Wilcoxon and Kruskal-Wallis tests. OS and PFS curves were plotted using the Kaplan-Meier esti- mate, and compared according to selected parameters using the Log-rank test and the Cox regression model. The significance threshold for all bivariate analyses was set at a two-sided α = 0.05. All factors showing a signifi- cant influence on OS in the bivariate analysis were Fernández et al. BMC Cancer (2018) 18:1185 Page 4 of 11 NLR, CA 19.9 and bilirubin levels, or weight loss of more than 10%. At the time of data collection, median follow-up was 7.2 months (IQR 3.5–13.3). A total of 193 pa- tients (91.9%) had died, with a 12-month survival rate of 30.1%. Median OS was 7.2 months (95% CI 5.9). Among 203 patients eligible for response, 50 patients achieved either a partial or complete re- sponse (ORR 24.6%), and 72 patients (35.5%) a stable disease. Treatment characteristics and outcome Ninety-seven patients (46.9%) received further treatment lines, most of whom (n = 63, 64.9%) had one or two lines of treatment (n = 25, Table 1 Demographic and clinical characteristics of study patients Numbera Overall Recurrent De novo Demographic characteristics Age, n (%) 210 < 65 years 99 (47.1%) 22 (47.8%) 77 (47.0%) 65–69 years 58 (27.6%) 8 (17.4%) 50 (30.5%) ≥70 years 53 (25.2%) 16 (34.8%) 37 (22.6%) Sex, n (%) 210 Males 127 (60.5) 33 (71.7) 94 (57.3) Females 83 (39.5) 13 (28.3) 70 (42.7) Clinical characteristics Weight loss > 10%, n (%) 208 81 (38.9) 9 (20.5) 72 (43.9) ECOG PS, n (%) 182 0–1 149 (81.9) 28 (77.8) 121 (82.9) 2–3 33 (18.1) 8 (22.2) 25 (17.1) Karnofsky PS, n (%) 55 < 70 3 (5.5) – 3 (6.8) 70–80 33 (60.0) 7 (63.6%) 26 (59.1%) 90–100 19 (34.5) 4 (36.4%) 15 (34.1%) Common comorbidities, n (%) 210 Hypertension 56 (26.7) 8 (17.4) 48 (29.3) Diabetes 26 (12.4) 4 (8.7) 22 (13.4) Dyslipemia 29 (13.8) 6 (13.0) 22 (13.4) Hepatobiliary stent, n (%) 209 38 (18.2) 3 (6.5) 35 (21.5) Platelet count, median (IR) 178 231.0 (172.0, 318.0) 207.0 (170.0, 303.0) 237.0 (181.0, 318.0) Bilirubin (mg/dL), median (IR) 175 0.70 (0.50, 1.00) 0.57 (0.50, 0.80) 0.79 (0.50, 1.10) NLR, n (%) 170 > 3 91 (53.5) 18 (52.9) 73 (53.7) ≤3 79 (46.5) 16 (47.1) 63 (46.3) CA 19.9, n (%) 173 > 37 U/mL 142 (82.1) 32 (82.1) 110 (82.1) ≤37 U/mL 31 (17.9) 7 (17.9) 24 (17.9) Number of metastatic sites, n (%) 210 1–3 205 (97.6) 46 (100.0) 159 (97.0) > 3 5 (2.4) 5 (3.0) Concommitant treatment, n (%) 210 Analgesics 76 (36.2) 12 (26.1) 63 (38.4) Corticosteroids 25 (11.9) 4 (8.7) 20 (12.2) IR Interquartile range (percentile 25, percentile 75) anumber of evaluable patients (no-missing) Table 1 Demographic and clinical characteristics of study patients IR Interquartile range (percentile 25, percentile 75) anumber of evaluable patients (no-missing) NLR, CA 19.9 and bilirubin levels, or weight loss of more than 10%. NLR, CA 19.9 and bilirubin levels, or weight loss of more than 10%. 5.9). Among 203 patients eligible for response, 50 patients achieved either a partial or complete re- sponse (ORR 24.6%), and 72 patients (35.5%) a stable disease. Safety Grade ≥3 treatment-related AEs were reported in 78 patients (37.1%), the most common being neutro- penia, thrombocytopenia, and fatigue (Table 4). One treatment-related event of paralytic ileus led to treat- ment interruption and death. Patients aged < 70 years received a median of 5 cycles of combined treatment and patients aged ≥70 years received a median of 3. Of 53 elderly patients (i.e., aged ≥70 years), 38 (71.7%) experienced at least one treatment-related AE, compared to 78.1% in the overall population, with similar profile of grade ≥3 events (Table 4). No significant differ- ences were found in the frequency of treatment-related AEs of grade 3 or higher between patients aged ≥70 years and < 70 (39.6% vs. 36.3%; p = 0.789). Treatment characteristics and outcome Ninety-seven patients (46.9%) received further treatment lines, most of whom (n = 63, 64.9%) had one or two lines of treatment (n = 25, 25.8%). At the time of data collection, median follow-up was 7.2 months (IQR 3.5–13.3). A total of 193 pa- tients (91.9%) had died, with a 12-month survival rate of 30.1%. Median OS was 7.2 months (95% CI 6.0–8.5), and median PFS 5.0 months (95%CI 4.3– Fernández et al. BMC Cancer (2018) 18:1185 Page 5 of 11 Page 5 of 11 Table 2 Treatment characteristics No. (%) Started treatment with dose reduction, n (%) Only gemcitabine 1 (0.5) Only nab-Paclitaxel 41 (19.5) Both 26 (12.4) Dose reduction during treatment, n (%) Nab-Paclitaxel 91 (43.3) Gemcitabine 75 (35.7) Either of the two drugs 96 (45.7) Received ≤30 days of treatment, n (%) 34 (16.5) Reasons for treatment discontinuationa, n (%) Progression 134 (69.8) Toxicity 33 (17.2) Death 24 (12.5) Patient’s request 7 (3.6) aA patient could have more than one reason for treatment discontinuation Table 2 Treatment characteristics Table 3 Demographic and clinical characteristics of patients who started treatment with dose reduction Table 3 Demographic and clinical characteristics of patients who started treatment with dose reduction Demographic characteristics Age, n (%) (n = 68) < 65 years 33 (48.5%) 65–79 years 14 (20.6%) ≥70 years 21 (30.9%) Sex, n (%) (n = 68) Males 41 (60.3%) Females 27 (39.7%) Clinical characteristics Weight loss > 10%, n (%) (n = 66) 18 (27.3%) ECOG PS, n (%) (n = 62) 0–1 46 (74.2%) 2-Mar 16 (25.8%) Karnofsky PS, n (%) (n = 23) < 70 – 70–80 13 (56.5%) 90–100 10 (43.5%) Common comorbidities, n (%) (n = 68) Hypertension 15 (22.1%) Diabetes 8 (11.8%) Dyslipemia 4 (5.9%) Hepatobiliary stent, n (%) (n = 68) 10 (14.7%) Platelet count, median (IR) (n = 57) 252.0 (172.0, 339.0) Bilirubin (mg/dL), median (IR) (n = 58) 0.70 (0.51, 1.10) NLR, n (%) (n = 55) > 3 29 (52.7%) ≤3 26 (47.3%) CA 19.9, n (%) (n = 62) > 35 52 (83.9%) ≤35 10 (16.1%) Number of metastatic sites, n (%) (n = 68) 1–3 67 (98.5%) > 3 1 (1.5%) Concommitant treatment, n (%) (n = 68) Analgesics 22 (32.4%) Corticosteroids 9 (13.2%) IR Interquartile range (percentile 25, percentile 75) anumber of evaluable patients (no-missing) Prognostic factors (%) Overall (n = 210) < 70 years (n = 157) ≥70 years (n = 53) Hematological toxicities Neutropenia 38 (18.1) 33 (21.0) 5 (9.4) Thrombocytopenia 13 (6.2) 9 (5.7) 4 (7.5) Anemia 7 (3.3) 2 (1.3) 5 (9.4) Febrile neutropenia 4 (1.9) 2 (1.3) 2 (3.8) Non-hematological toxicities Fatigue 13 (6.2) 10 (6.4) 3 (5.7) Vomiting 3 (1.4) 1 (0.6) 2 (3.8) Colangitis 3 (1.4) 3 (1.9) – Neurotoxicity 3 (1.4) 1 (0.6) 2 (3.8) Peripheral neuropathy 5 (2.4) 3 (1.9) 2 (3.8) Alopecia 9 (4.3) 6 (3.8) 3 (5.7) Table 4 Common treatment-related adverse events (> 1% of patients overall) of grade 3. No. (%) treatment within 30 days of treatment, 33% of them due toxicity. Despite the poor baseline characteristics in our cohort, median OS and PFS were of 7.2 months (95% CI 6.0, 8.5) and 5.0 months (4.3, 5.9), respectively, indicating that this combination is effective in the real-life setting. OS was influenced by the baseline ECOG PS, NLR, and CA 19.9, but not by age ≥70 years, the presence of hepatobiliary stent or RDI < 85%. p y Randomized controlled trials investigating therapies for metastatic pancreatic cancer have restrictive selection criteria, particularly regarding the patient’s age and per- formance status [8, 9]. In the case of the pivotal study of the combined treatment (MPACT trial), a Karnofsky index of 70 or more was required [9]. In Spain the preferred scale to assess the performance status in most centers is the ECOG score, thus, in our study, the Karnofsky index could only be assessed in a limited number of patients. However, although the Karnofsky index and the ECOG score lack a linear relationship for direct comparisons between study populations, the fact that 18% of patients had ECOG ≥2 (roughly ≥70 in the Karnofsky index) indicates a trend towards a poorer average performance status in our study population than those in the pivotal MPACT trial. In the case of the ACCORD4/PRODIGE11 study, recruitment was limited to patients under 76 years with ECOG per- formance status ≤1; [8] based on these criteria alone, nearly 40% of our study patients would have been excluded. In addition to the patients’ baseline charac- teristics, treatment patterns in real-life practice often differ from those used in pivotal trials. In our study, 66.2% of patients had a dose reduction in either nab-paclitaxel and/or gemcitabine. Prognostic factors Overall, the me- dian RDI was 67% for both nab-paclitaxel and gemci- tabine, which is well below those reported in the MPACT trial (81 and 75% for nab-paclitaxel and gemcitabine, respectively). Thus, the demographic, clinical and treatment characteristics of this large co- hort of real-life patients underscore fundamental dif- ferences between RCTs and routine practice settings. for ECOG improvement, stable, and worsening, re- spectively (p = 0.020). Neither age ≥70 years, the presence of an hepatobiliary stent, nor an RDI of gemcitabine plus nab-paclitaxel < 85% showed a significant influence on median PFS and OS at 12 months (Fig. 2). The 12-month survival rate was 28.3 and 30.8% for patients aged ≥70 and < 70 years, respectively (p = 0.863); 36.8 and 28.8% for patients with and without hepatobiliary stent, respectively (p = 0.335); and 28.0 and 31.5% for patients with RDI of the com- bined treatment ≥85 and < 85%, respectively (p = 0.645). Factors significantly influencing OS (ECOG PS, NLR, and CA 19.9) were used to build a nomogram to predict survival of patients with pancreatic adenocarcinoma treated in the real-life setting (Fig. 3a). All included vari- ables, computed as dichotomous, showed a significant contribution to the Cox regression model: baseline ECOG PS 0 or 1 vs 2 or 3 (p = 0.030), baseline NLR > 3 vs ≤3 (p = 0.043), and baseline CA 19.9 > 37 U/mL vs ≤37 U/mL (p = 0.004) (Fig. 3a). Based on the scale ob- tained with the nomogram, three risk groups were de- fined: low-risk group (n = 21, 15.1%), medium-risk group (n = 93, 66.9%), and high-risk group (n = 25, 18.0%). Figure 3b shows the survival curves (Kaplan-Meier estimates for these groups). Despite the inclusion of elderly patients, the trend to- wards a poorer performance status, and adjusted treat- ment schedule in our study, our results confirmed the effectiveness of combined treatment with gemcitabine plus nab-paclitaxel in the real-life setting. The estimated median OS (7.2 months) was slightly lower than that ob- served in the MPACT trial (8.5 months) and those re- ported by Giordano et al. (11 months), Lo Re et al. (9.2 months), and De Vita et al. (10 months) in three series of 118, 37, and 41 real-life patients, respectively [19–21]. Prognostic factors Analysis of the influence of baseline characteristics on survival showed that only ECOG, NLR, and CA 19.9 sig- nificantly influenced OS, PFS (Fig. 1), and/or 12-month survival. Baseline CA 19.9 had a significant influence on the three analyzed outcomes: patients with CA 19.9 ≤37 U/mL had longer OS (p = 0.004) (Fig. 1e), PFS (p = 0.011) (Fig. 1f), and a higher 12-month survival rate (45.2% vs. 24.8%; p = 0.030). On the other hand, patients with baseline NLR ≤3 had longer OS (p = 0.024) (Fig. 1c) and a higher 12-month survival rate than those with NLR > 3 (38% vs. 23.3%; p = 0.045) but showed similar PFS (Fig. 1d). Patients with a baseline ECOG PS of 0 or 1 had longer OS than those with an ECOG PS of 2 or 3 (p = 0.018) (Fig. 1a), although this trend was not ob- served in PFS (Fig. 1b). Likewise, the 12-month survival for patients with ECOG PS 0 or 1 and 2 or 3 was 31.1 and 21.2%, respectively (p = 0.297). Patients with stable ECOG (n = 68) or ECOG improvement (n = 22) during the first three treatment cycles had longer OS than those with a worsening ECOG (n = 19), with a median OS of 12.9 months (95% CI 6.7–15.4), 10.6 months (95% CI 8.1–13.3), and 7.1 months (95% CI 4.7–9.2) and 21.2%, respectively (p = 0.297). Patients with stable ECOG (n = 68) or ECOG improvement (n = 22) during the first three treatment cycles had longer OS than those with a worsening ECOG (n = 19), with a median OS of 12.9 months (95% CI 6.7–15.4), 10.6 months (95% CI 8.1–13.3), and 7.1 months (95% CI 4.7–9.2) Fernández et al. BMC Cancer (2018) 18:1185 Page 6 of 11 Page 6 of 11 Table 4 Common treatment-related adverse events (> 1% of patients overall) of grade 3. No. Prognostic factors Likewise, the median PFS of our cohort (5.0 months) was comparable to that reported in the MPACT trial (5.5 months), but lower than that reported by Giordano et al. (7 months), Lo Re et al. (6.2 months), and of Discussion In this observational retrospective study including all the patients with metastatic pancreatic cancer treated at the participating centers with first-line nab-paclitaxel plus gemcitabine in a real-life setting, 25% of patients were aged 70 years or more and 18% had a baseline ECOG score of 2 or 3. Furthermore, 32% of patients started combined treatment with a dose reduction of nab-paclitaxel, and 17% of patients discontinued Fernández et al. BMC Cancer (2018) 18:1185 Page 7 of 11 bl l ( h ) f ( ) [ ] d l h l l h f Fig. 1 Overall survival (a, c and e) and progression-free survival (b, d and f) depending on baseline ECOG (a and b), NLR (c and d) and CA 19.9 (e and f). Survival is presented as median (95% CI); p-values correspond to the Log-rank test for inter-curve differences Fig. 1 Overall survival (a, c and e) and progression-free survival (b, d and f) depending on baseline ECOG (a and b), NLR (c and d) and CA 19.9 (e and f). Survival is presented as median (95% CI); p-values correspond to the Log-rank test for inter-curve differences notably Da Vitta et al. (9.2 months). Of note, patients of our cohort had poorer PS than that reported in these studies. (38%) [9] and slightly lower than in previous series of real-life patients (20 to 24%) [14, 19, 20]. Remarkably, being aged over 70 years was not associated with a worse toxicity profile in our study, consistent with the trend reported by Giordano et al. in a retrospect- ive study addressing the safety of this treatment in the elderly [14]. As reported in previous studies, neutropenia was the most common treatment-related adverse event of grade 3 or higher, however its 18% incidence was substantially below that reported in the MPACT trial Fernández et al. BMC Cancer (2018) 18:1185 Page 8 of 11 In addition to assessing the safety and effectiveness of previous analyses of real-life patients, [19, 21] stratifying Fig. 2 Overall survival (a, c and e) and progression-free survival (b, d and f) depending on baseline age (a and b), presence of hepatobiliary stent (c and d), and relative dose intensity (RDI) (e and f). Survival is presented as median (95% CI); p-values correspond to the Log-rank test for inter-curve differences Fig. Discussion 2 Overall survival (a, c and e) and progression-free survival (b, d and f) depending on baseline age (a and b), presence of hepatobiliary stent (c and d), and relative dose intensity (RDI) (e and f). Survival is presented as median (95% CI); p-values correspond to the Log-rank test for inter-curve differences In addition to assessing the safety and effectiveness of first-line nab-paclitaxel plus gemcitabine in real-life patients, we also analyzed prognostic factors and their influence on survival in this setting. The influence of the patient’s performance status has been consistently reported by various authors. [18, 19, 21, 22] As in previous analyses of real-life patients, [19, 21] stratifying patients according to the baseline ECOG score of 0–1 or > 1, shows an influence of ECOG on OS. Similarly, the inflammation-based NLR score, identified as a prog- nostic factor for OS and PFS in patients receiving nab-paclitaxel plus gemcitabine in pivotal studies [18] Fernández et al. BMC Cancer (2018) 18:1185 Page 9 of 11 and real-life practice [20, 21, 23], influenced OS in our patients but did not reach the significance threshold in the PFS analysis. In both localized and metastatic pan- OS and PFS with a cut-off of 37 U/mL. Other factors, such as age, the presence of a hepatobiliary stent, and the RDI did not significantly influence either OS or PFS. Fig. 3 Survival estimate of patients with metastatic pancreatic cancer starting combined treatment with nab-paclitaxel plus gemcitabine in real-life practice. a Nomogram for predicting overall survival and the probability of 3-month, 6-month, 12-month in real-life practice. b Survival (Kaplan Meier estimate) for low-, medium-, and high-risk groups Fig. 3 Survival estimate of patients with metastatic pancreatic cancer starting combined treatment with nab-paclitaxel plus gemcitabine in real-life practice. a Nomogram for predicting overall survival and the probability of 3-month, 6-month, 12-month in real-life practice. b Survival (Kaplan Meier estimate) for low-, medium-, and high-risk groups Fig. 3 Survival estimate of patients with metastatic pancreatic cancer starting combined treatment with nab-paclitaxel plus gemcitabine in real-life practice. a Nomogram for predicting overall survival and the probability of 3-month, 6-month, 12-month in real-life practice. b Survival (Kaplan Meier estimate) for low-, medium-, and high-risk groups and real-life practice [20, 21, 23], influenced OS in our patients but did not reach the significance threshold in the PFS analysis. Competing interests AG has received a grant for attending an international oncology congress and fees as scientific consultant from Celgene. AM has received research support grants and fees as consultant from Celgene, and fees as speaker from Shire. CL2 has received fees as speaker and consultant from Celgene. AF, MS, EB, RV, JA, PJ, GQ, CL1, LJL, PR, PG, MR, EG, MSC, AC, JG, CG, NR, VN, JC, IA, RAP declare that they have no conflicts of interest regarding the content of this manuscript. Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Consent for publication Consent for publication Not applicable. Funding The study coordination, data analysis, and medical writing assistance were supported by the non-profit Galician Group for Research on Gastrointestinal Tumors (GITuD) with funding from Celgene Corporation. The funding body was not directly involved in the study design, data analysis, and manuscript writing, which were performed by either the authors or the contract research organization BioClever, S.L. as stated in the acknowledgements section. Abbreviations AE: Adverse events; CI: Confidence interval; HR: Hazard ratio; IQR: Interquartile range; NLR: Neutrophil lymphocyte rate; ORR: Objective response rate; OS: Overall survival; PFS: Progression-free survival; PS: Performance status; RDI: Relative dose intensity; RECIST: Response Evaluation Criteria in Solid Tumors p The prognostic factors identified in our cohort (i.e., ECOG PF, NLR, and CA 19.9) allowed us to develop a nomogram for predicting survival of real-life patients treated with first-line nab-paclitaxel plus gemcitabine. A similar tool for predicting survival in real-life patients receiving gemcitabine-based chemotherapy was presented by Hamada et al., and included age, sex, PS, tumor size, and the presence of nodal or distant metastases [25]. As in their analysis, PS carried a notable weight in our nomo- gram, with patients having an ECOG score of 2 or more dramatically reducing the predicted survival. On the other hand, variables not routinely assessed in standard practice, such as tumor size were not considered. In addition to the nomogram by Hamada et al., Goldstein et al. presented a similar tool based on the cohort of the MPACT trial [26]. The nomogram by Goldstein et al. was similar to ours in terms of the inclusion of performance status and NLR, but included other variables such as albumin, tumor size, and the presence of liver metastasis. Future studies shall validate the proposed nomogram as a tool for predicting survival in real-life patients. Authors’ contributions AF and MS significantly contributed to the study design. AF, AG, EB, RV, JA, PJ, GQ, CLl, MC, LJL, AM, PR, PG, CLó, MR, EG, MSC, AC, JG, CG, NR, VN, JC, IA, RAP, and AC contributed to data collection, and AF, AG, PJ, and AC analyzed and interpreted the results. The manuscript was drafted by AF, AG, JA, PJ, and AC. AF, MS, AG, EB, RV, JA, PJ, GQ, CLl, MC, LJL, AM, PR, PG, CLó, MR, EG, MSC, JG, CG, NR, VN, JC, IA, RAP, and AC revised the manuscript drafts critically and approved the final version of the manuscript. Our results should be interpreted in the context of the intrinsic limitations of retrospective studies. Thus, in addition to the risk of reporting bias associated with ob- servational designs, missing data in the medical records could not be considered for the analysis, leading uneven sample sizes across analyses. The retrospective design also precluded the inclusion of variables not recorded in routine clinical practice in Spain, notably the Karnofsky index, which was reported in very few patients and pre- vented a direct comparison with the study sample of the pivotal trial MPACT. Another limitation of the retro- spective design was the lack of pre-defined criteria for dose reductions, which were established at physician’s discretion, according to the routine practice in each center. Finally, the reduced size of some patient sub- groups in the comparative analyses limited the investiga- tion of baseline factors with potential influence on patient survival. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Acknowledgements The authors would like to thank the Galician Group for Research on Gastrointestinal Tumors (GITuD), as well as all study patients and their relatives. Statistical and medical writing support were provided by Josep Puig and Gerard Carot-Sans (PhD) on behalf of BioClever S.L. Discussion In both localized and metastatic pan- creatic adenocarcinoma, a proinflammatory status of the tumor results in worse prognosis, and therefore, influ- ences treatment response and consequently, survival. Fi- nally, the antigen CA 19.9 significantly influenced both OS and PFS with a cut-off of 37 U/mL. Other factors, such as age, the presence of a hepatobiliary stent, and the RDI did not significantly influence either OS or PFS. This finding is particularly controversial for age, which has been identified as a major prognostic factor for all patients with metastatic pancreatic cancer [22], and was subsequently confirmed for patients treated specifically Fernández et al. BMC Cancer (2018) 18:1185 Page 10 of 11 with gemcitabine plus nab-paclitaxel [18]. It is worth noting that the cut-off age considered as a prognostic factor is not homogeneous across studies, and while risk analyses were traditionally based on patients over 60 or 65 years [18, 22], there is increasing interest in investi- gating patients over 70 years as a risk group in real-life practice [14, 24]. a nomogram was developed to predict survival of patients starting treatment with the combination. It was based on three variables routinely assessed in real-life practice, ECOG performance status, NLR, and CA 19.9. Ethics approval and consent to participate The study was approved by the ethics committee of the autonomous community of Galicia (SERGAS) (Spain). All patients provided written informed consent before data collection. References Goldstein D, Von Hoff DD, Chiorean EG, Reni M, Tabernero J, Ramanathan RK, et al. Nomogram for predicting overall survival (OS) in patients (pts) treated with nab-paclitaxel (nab-P) plus gemcitabine (Gem) or Gem alone for metastatic pancreatic cancer (MPC). J Clin Oncol. 2017;35(15_suppl):4109. http://ascopubs.org/doi/abs/10.1200/JCO.2017.35.15_suppl.4109. 6. Conroy T, Bachet J-BB, Ayav A, Huguet F, Lambert A, Caramella C, et al. Current standards and new innovative approaches for treatment of pancreatic cancer. Eur J Cancer. 2016;57:10–22. 7. Ellenrieder V, König A, Seufferlein T, Konig A, Seufferlein T. Current standard and future perspectives in first- and second-line treatment of metastatic pancreatic adenocarcinoma. Digestion. 2016;94(1):44–9. 7. Ellenrieder V, König A, Seufferlein T, Konig A, Seufferlein T. Current standard and future perspectives in first- and second-line treatment of metastatic pancreatic adenocarcinoma. Digestion. 2016;94(1):44–9. 8. Conroy T, Desseigne F, Ychou M, Bouché O, Guimbaud R, Bécouarn Y, et al. FOLFIRINOX versus gemcitabine for metastatic pancreatic cancer. N Engl J Med. 2011;364(19):1817–25. 9. Von Hoff DD, Ervin T, Arena FP, Chiorean EG, Infante J, Moore M, et al. Increased survival in pancreatic cancer with nab-paclitaxel plus gemcitabine. N Engl J Med. 2013;369(18):1691–703. 10. Sohal DPS, Mangu PB, Khorana AA, Shah MA, Philip PA, O’Reilly EM, et al. Metastatic pancreatic cancer: american society of clinical oncology clinical practice guideline. J Clin Oncol. 2016;34(23):2784–96. 11. Ducreux M, Cuhna AS, Caramella C, Hollebecque A, Burtin P, Goere D, et al. Cancer of the pancreas: ESMO clinical practice guidelines for diagnosis, treatment and follow-up. Ann Oncol. 2015;26(Suppl 5):v56–68. 12. Le N, Vinci A, Schober M, Krug S, Javed MA, Kohlmann T, et al. Real-world clinical practice of intensified chemotherapies for metastatic pancreatic cancer: results from a pan-european questionnaire study. Digestion. 2017; 94(4):222–9. 13. Peixoto RD, Ho M, Renouf DJ, Lim HJ, Gill S, Ruan JY, et al. Eligibility of metastatic pancreatic cancer patients for first-line palliative intent nab-paclitaxel plus gemcitabine versus FOLFIRINOX. Am J Clin Oncol Cancer Clin Trials. 2017; 40(5):507–11. 14. Giordano G, Vaccaro V, Lucchini E, Musettini G, Bertocchi P, Bergamo F, et al. Nab-paclitaxel (Nab-P) and gemcitabine (G) as first-line chemotherapy (CT) in advanced pancreatic cancer (APDAC) elderly patients (pts): a “real-life” study. J Clin Oncol. 2015;33(3_suppl):424. 15. Ghosn M, Ibrahim T, Assi T, El Rassy E, Kourie HR, Kattan J. Dilemma of first line regimens in metastatic pancreatic adenocarcinoma. World J Gastroenterol. 2016;22(46):10124–30. 16. References 1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2015. CA Cancer J Clin. 2015;65(1):5–29. 1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2015. CA Cancer J Clin. 2015;65(1):5–29. 23. Montes A, Villarroel P, Ayerbes M, la Gómez JC, Aldana G, Tuñas L, et al. Prognostic and predictive markers of response to treatment in patients with locally advanced unresectable and metastatic pancreatic adenocarcinoma treated with gemcitabine/nab-paclitaxel: results of a retrospective analysis. J Cancer Res Ther. 2017;13(2):240. 2. De Angelis R, Sant M, Coleman MP, Francisci S, Baili P, Pierannunzio D, et al. Cancer survival in Europe 1999–2007 by country and age: results of EUROCARE-5—a population-based study. Lancet Oncol. 2014;15(1):23–34. 2. De Angelis R, Sant M, Coleman MP, Francisci S, Baili P, Pierannunzio D, et al. Cancer survival in Europe 1999–2007 by country and age: results of EUROCARE-5—a population-based study. Lancet Oncol. 2014;15(1):23–34. 3. Bouvier A-M, Bossard N, Colonna M, Garcia-Velasco A, Carulla M, Manfredi S, et al. Trends in net survival from pancreatic cancer in six European Latin countries: results from the SUDCAN population-based study. Eur J Cancer Prev. 2017:S63–9. https://doi.org/10.1097/CEJ.0000000000000303. 3. Bouvier A-M, Bossard N, Colonna M, Garcia-Velasco A, Carulla M, Manfredi S, et al. Trends in net survival from pancreatic cancer in six European Latin countries: results from the SUDCAN population-based study. Eur J Cancer Prev. 2017:S63–9. https://doi.org/10.1097/CEJ.0000000000000303. 24. Huang L, Jansen L, Balavarca Y, Molina-Montes E, Babaei M, van der Geest L, et al. Resection of pancreatic cancer in Europe and USA: an international large-scale study highlighting large variations. Gut. 2017. https://doi.org/10. 1136/gutjnl-2017-314828. 4. Malvezzi M, Carioli G, Bertuccio P, Boffetta P, Levi F, La Vecchia C, et al. European cancer mortality predictions for the year 2018 with focus on colorectal cancer. Ann Oncol Off J Eur Soc Med Oncol. 2018;29(4):1016–22. 4. Malvezzi M, Carioli G, Bertuccio P, Boffetta P, Levi F, La Vecchia C, et al. European cancer mortality predictions for the year 2018 with focus on colorectal cancer. Ann Oncol Off J Eur Soc Med Oncol. 2018;29(4):1016–22. 25. Hamada T, Nakai Y, Yasunaga H, Isayama H, Matsui H, Takahara N, et al. Prognostic nomogram for nonresectable pancreatic cancer treated with gemcitabine-based chemotherapy. Br J Cancer. 2014;110(8):1943–9. 5. De Felice F, Musio D, Raffetto N, Tombolini V. Neoadjuvant strategy as initial treatment in resectable pancreatic cancer: concrete evidence of benefit. Anticancer Res. 2014;34(9):4673–6. 26. Conclusions 1Complejo Hospitalario Universitario Ourense, Calle Ramon Puga Noguerol, 54, 32005 Ourense, Spain. 2Complejo Universitario Ourense, Ourense, Spain. 3Institut Català d’Oncologia (ICO) Hospital Dr. Trueta, Girona, Spain. 4Hospital Clínic de Barcelona, Barcelona, Spain. 5Complejo Hospitalario de Navarra, Navarra, Spain. 6Hospital 12 de Octubre, Madrid, Spain. 7Hospital Universitario Central de Asturias, Asturias, Spain. 8Hospital Lucus Agustí, Lugo, Spain. 9Hospital de Elda, Alicante, Spain. 10Hospital Público Lluis Alcanyis de Xátiva, Xátiva, Spain. 11Hospital Virgen de la Salud, Toledo, Spain. 12Hospital Gregorio Marañón, Madrid, Spain. 13Hospital Puerta del Mar, Cadiz, Spain. 14Hospital Our results, obtained from the largest published series of real-world patients with metastatic pancreatic cancer, show that nab-paclitaxel plus gemcitabine remains effective in this setting, despite the high burden of dose reductions and the poorer performance of these patients. Based on the exploratory analysis of prognostic factors, Page 11 of 11 Page 11 of 11 Fernández et al. BMC Cancer (2018) 18:1185 Fernández et al. BMC Cancer (2018) 18:1185 Fernández et al. BMC Cancer (2018) 18:1185 Fernández et al. BMC Cancer (2018) 18:1185 Universitario de Vigo, Vigo, Spain. 15Hospital Marqués de Valdecilla, Santander, Spain. 16Hospital A Coruña Teresa Herrera, A Coruña, Spain. 17Hospital de Pontevedra, Pontevedra, Spain. 18Hospital Morales Meseguer, Murcia, Spain. 19Hospital de Elche, Elche, Spain. 20Hospital Ramón y Cajal, Madrid, Spain. 21Hospital Provincial Castellón, Castellón de la Plana, Spain. 22Complejo Hospitalario de Jaén, Jaén, Spain. 23Hospital del Ferrol, Ferrol, Spain. 24Hospital Regional Universitario, Málaga, Spain. 25Hospital Miguel Servet, Zaragoza, Spain. 19. Lo Re G, Santeufemia DA, Foltran L, Bidoli E, Basso SMM, Lumachi F. Prognostic factors of survival in patients treated with nab- paclitaxel plus gemcitabine regimen for advanced or metastatic pancreatic cancer: a single institutional experience. Oncotarget. 2015;6(10):8255–60. a single institutional experience. Oncotarget. 2015;6(10):8255–60. 20. De Vita F, Ventriglia J, Febbraro A, Laterza MM, Fabozzi A, Savastano B, et al. NAB-paclitaxel and gemcitabine in metastatic pancreatic ductal adenocarcinoma (PDAC): from clinical trials to clinical practice. BMC Cancer. 2016;16(1):1–8. 21. Giordano G, Vaccaro V, Lucchini E, Bertocchi P, Bergamo F, Musettini G, et al. Analysis of prognostic factors in advanced pancreatic cancer (APDAC) patients (pts) undergoing to first-line nab-paclitaxel (Nab-P) and gemcitabine (G) treatment. J Clin Oncol. 2015;33(3_suppl):412. Received: 20 September 2018 Accepted: 16 November 2018 22. Tas F, Sen F, Keskin S, Kilic L, Yildiz I. Prognostic factors in metastatic pancreatic cancer: older patients are associated with reduced overall survival. Mol Clin Oncol. 2013;1(4):788–92. References Therasse P, Arbuck SG, Eisenhauer EA, Wanders J, Kaplan RS, Rubinstein L, et al. New guidelines to evaluate the response to treatment in solid tumors. J Natl Cancer Inst. 2000;92(3):205–16. 17. National Cancer Institute. CTCAE v4.0 Archive. Common Terminology Criteria for Adverse Events (CTCAE). 2009. Available from: https://evs.nci.nih. gov/ftp1/CTCAE/About.html. Cited 24 Jan 2018. 18. Goldstein D, El-Maraghi RH, Hammel P, Heinemann V, Kunzmann V, Sastre J, et al. Nab-paclitaxel plus gemcitabine for metastatic pancreatic cancer: long-term survival from a phase III trial. J Natl Cancer Inst. 2015;107(2):dju413. 18. Goldstein D, El-Maraghi RH, Hammel P, Heinemann V, Kunzmann V, Sastre J, et al. Nab-paclitaxel plus gemcitabine for metastatic pancreatic cancer: long-term survival from a phase III trial. J Natl Cancer Inst. 2015;107(2):dju413.
https://openalex.org/W2973820415	https://www.scielo.br/j/nec/a/bdXzdjtkMqG8gCNnMNpCbGm/?lang=pt&format=pdf	Portuguese	null	"Seeing Like a Social Movement": Institucionalização simbólica e capacidades estatais cognitivas	Novos estudos CEBRAP	2,019	cc-by	13,980	Institucionalização simbólica e capacidades estatais cognitivas1 José Szwako* Adrian Gurza Lavalle** http://dx.doi.org/10.25091/ S01013300201900020009 “Seeing like a social movement”: Symbolic Institutionalization and Cognitive State Capacities Abstract Inspired by Scott’s visual cognitive metaphor in Seeing Like a State, we develop the notion of symbolic institutionalization shedding light on the disputes around social movements’ categories and forms of classification, as well as their circulation. The attention given to such symbolic forms is fruitful to the comprehension of the genesis and transformation of the state cognitive capacities, institutionalized via interactions with social movements within the space of certain public policies. Keywords: symbolic institutionalization; social movements; cognitive state capacities; public policy. Resumo Inspirados na metáfora visual cognitiva de Scott em Seeing Like a State, propomos a noção de institucionalização simbólica. Nela, são enfatizadas as disputas e a circulação de categorias e formas de classificação do mundo dos movimentos sociais. Sugerimos que atentar para tais formas simbólicas é heuristi‑ camente proveitoso à compreensão da gênese e da transformação de capacidades cognitivas do Estado, institucionalizadas em políticas públicas via interações com os movimentos. Palavras‑chave: institucionalização simbólica; movimentos sociais; capacidades estatais cognitivas; políticas públicas. [] Universidade do Estado do Rio de Janeiro. Rio de Janeiro, rj, Brasil. E‑mail: zeszwako@iesp.uerj.br [] Universidade do Estado do Rio de Janeiro. Rio de Janeiro, rj, Brasil. E‑mail: zeszwako@iesp.uerj.br [] Universidade de São Paulo, sp, São Paulo, Brasil. E‑mail: gurzalaval leadrian@gmail.com Introdução Não seria exagero dizer que as teorias dos movimen‑ tos sociais, em suas vertentes hoje consagradas, privilegiaram as for‑ mas de ação e organização dos atores e grupos societais de maneira a negligenciar, conscientemente ou não, suas formas de classificação. Isto é, nas análises de movimentos sociais (ms) deu‑se menor atenção às categorias com que os grupos nomeiam, classificam e hierarquizam simbolicamente o mundo social e suas divisões, em favor da análise de táticas, repertórios de ação e, no caso mais próximo à dimensão sim‑ [] Universidade de São Paulo, sp, São Paulo, Brasil. E‑mail: gurzalaval leadrian@gmail.com [1] Os autores agradecem a Aril‑ son Favareto, Luiz Augusto Campos, Janine Mello dos Santos e Paulo Jannuzzi pelas valiosas sugestões. Declaram‑se, ainda assim, os únicos responsáveis por eventuais impreci‑ sões. Adrian Gurza Lavalle agradece Novos estud. ❙❙ CEBRAP ❙❙ SÃO PAULO ❙❙ V38n02 ❙❙ 411-434 ❙❙ MAI.–AGO. 2019 411 bólica, enquadramentos estratégicos (frames).2 Seeing Like a State, de James C. Scott (1998), é, a nosso ver, um livro inspirador para abordar ms e suas interações tanto com o Estado e seus agentes quanto com agentes do sistema político, tomando tais interações pelas categorias nelas em jogo, isto é, colocando no centro da análise as formas nativas de classificação vis‑à‑vis a construção sociopolítica das capacidades de ação do Estado. Em particular, interessa aqui recuperar a dimensão cognitiva das capacidades estatais. Quer se trate de taxação, conscri‑ ção e vigilância — funções clássicas dos Estados‑nação —, quer se trate das operações de regulação e controle efetivos da produção, circu‑ lação e reprodução da população e dos bens materiais, são fundamen‑ tais o conhecimento das dinâmicas do mundo social e a abertura aos saberes práticos que o organizam e lhe conferem sentido. “Conhecer como Estado” significa tornar visíveis ou legíveis essas dinâmicas e saberes práticos populacionais enraizados na experiência local (metis, conforme a denominação de Scott). Para tanto, é preciso simplificá‑los e traduzi‑los para esquemas (schemes) que estruturam a forma pela qual o Estado faz o que faz, ou seja, que operam constituindo suas capacidades (cf. Lindvall; Teorel, 2017, p. 5). o apoio do Centro de Estudos da Me‑ trópole (Cepid‑cem), financiado pela Fundação de Amparo à Pesquisa do Estado de São Paulo (Fapesp), pro‑ cesso n. 2013/07616‑7. As opiniões, hipóteses e conclusões ou recomen‑ dações expressas são de responsabi‑ lidade dos autores e não necessaria‑ mente refletem a visão da Fapesp. Introdução [2] Na literatura de ms que lança mão dos enquadramentos estratégi‑ cos, estes não se confundem com suas categorias — embora ambos, frames e categorias, eventualmente possam coincidir no plano empírico. A ênfa‑ se recai ora no caráter estratégico do frame (McAdam, 1996, pp. 340 ss.), ora numa versão despolitizada dos enquadramentos (Benford; Snow, 2000). Revisitar Seeing Like a State nos parece frutífero para uma agenda de pesquisa orientada às relações entre ms e políticas públicas por‑ quanto soma — em registro cognitivo e lançando mão de metáfora visual potente — dimensões caras aos processos de state‑building, marca registrada do neoinstitucionalismo histórico. É precisamen‑ te mostrando as vantagens analíticas desse referencial teórico que atentamos alhures, em uma revisão do debate sobre as relações Esta‑ do/sociedade no Brasil pós‑transição, para seus pressupostos e seu desenvolvimento conceitual (Gurza Lavalle; Szwako, 2015), bem como para as lacunas na compreensão dos processos de institucio‑ nalização das agendas, demandas e interesses dos ms no Estado, tratando‑os sob a ótica dos “domínios de agência”, “encaixes ins‑ titucionais” e “capacidades estatais” (Gurza Lavalle et al., 2019b). Voltar‑se aos processos de institucionalização permite aproximar os debates de políticas e ms, ampliando a compreensão das dinâ‑ micas de interação socioestatal por meio das quais as capacidades organizacionais societárias se transformam e são transformadas em capacidades estatais (cf. Bichir et al., 2017). Neste artigo, beneficiando‑nos do trabalho magistral de Scott, buscamos tratar da institucionalização de categorias, ideias e ideais dos ms (ou seja, suas formas de classificação e ordenação do mun‑ do) em capacidades de ação do Estado via políticas públicas. O de‑ safio analítico de abordar tal institucionalização e a correspondente dimensão cognitiva das capacidades estatais é triplo: em uma ponta, supõe desenvolvimentos teóricos que suplementem e/ou corrijam as 2 “Seeing like a social movement”: Institucionalização simbólica e capacidades estatais cognitivas ❙❙ José Szwako e Adria social movement”: Institucionalização simbólica e capacidades estatais cognitivas ❙❙ José Szwako e Adrian Gurza Laval abordagens disponíveis para compreender processos de instituciona‑ lização, notadamente as teorias dos ms; em outra, exige atentar para as categorias nativas de classificação, que são pouco trabalhadas não só na literatura de ms, mas também na agenda da institucionaliza‑ ção; e, no meio, alargar a agenda das capacidades estatais tendo em vista a natureza sociopolítica da sua construção. Ademais, o foco na institucionalização, conforme será visto, permite corrigir certa lógica dicotômica presente na proposta de Scott. Introdução Além de tomar a heterogeneidade estatal como parti pris,3 orienta‑ mo‑nos por uma perspectiva interacionista e genética das capacida‑ des de atuação da sociedade civil (sc) e do Estado, a qual toma como pressuposto teórico fundamental o caráter mutuamente constitutivo da relação entre ambos (Gurza Lavalle et al., 2019a). Assim, formas movimentalistas de classificação e formas estatais de classificação se constituem mutuamente por meio de interações socioestatais. Nos‑ so argumento é que as categorias e ideias implicadas nessa produção interpelam a maneira pela qual o Estado apreende — ou conhece, nos termos de Scott — as divisões do mundo social, veiculando formas de hierarquização e simbolização do mundo que buscam transformar a visão que orienta suas ações. O mundo social não é transparente, e a capacidade do Estado de enxergá‑lo e ler as dinâmicas que o cons‑ tituem é uma construção passível de modelagem e disputa. Incidir na construção da legibilidade do mundo social pelo Estado dista de ser trivial para a distribuição de serviços e bens públicos — e para aqueles afetados por tal distribuição —, mesmo que essa disputa seja pouco afeita à dramaticidade dos repertórios de ação mais usualmente estu‑ dados na literatura. Ao utilizar categorias sedimentadas nas intera‑ ções e tensões com ms, o Estado muda parcialmente seus esquemas e a cognição de seus modos de intervenção e ordenação, e, ao ajustar essas categorias, passa a “enxergar como um movimento social”. [3] No conjunto da produção na‑ cional, Dagnino (2011; Dagnino et. al., 2006) é referência central na de‑ fesa de uma concepção não monolíti‑ ca de Estado, distinguido, portanto, por regimes e formas de acesso variá‑ veis, desiguais e em múltiplas escalas. Noutro âmbito, a definição de Skoc pol (1992) encerra lógica análoga por conceber o Estado como série de organizações e agentes eleitos ou de carreira. Segundo essas perspec‑ tivas, se mencionamos Estado, não pressupomos qualquer coerência in‑ terna e nem sequer que algo como “o Estado” possa ser apreendido in totum et totaliter. Para nós, falar da mútua constituição entre Estado e sociedade civil é falar de partes e de relações entre partes de um e de outra: suas lógicas de formação derivam de processos de in‑ teração e mútua constituição ao longo do tempo, e seu funcionamento resi‑ de, por sua vez, em instâncias recípro‑ cas e assimétricas de contato e acesso. Introdução [4] A noção de “‘aparelho de captu‑ ra’ designa basicamente os dispositi‑ vos de apropriação das ações huma‑ nas heterogêneas […] e de sua con‑ versão em atividades homogêneas, reguláveis […]. Na verdade, são os próprios autores [Deleuze e Guatta‑ ri] que chamam a atenção para o fato de que é o Estado, em bloco, que po‑ deria ser considerado o aparelho de captura por excelência” (Goldman, 2006, p. 264). Certamente há mais de uma abordagem para se compreender as relações entre Estado e sociedade civil. Este artigo, conforme men‑ cionado, visa contribuir ao campo de estudos da institucionalização e ampliar o debate sobre movimentos sociais e políticas públicas par‑ tindo da contribuição visual‑cognitiva de J. Scott e somando ao de‑ bate o foco voltado para a institucionalização de categorias gestadas nas relações que grupos da sociedade civil travam com outros atores e discursos. O foco na institucionalização, como veremos, permite cor‑ rigir certa lógica dicotômica presente quer na proposta de Scott, quer numa antropologia deleuze‑guattariana que vê as relações com partes do Estado como “aparelhos de captura” dos ms4 e, assim, reproduz o viés aqui criticado. É evidente, porém, que o escopo da análise an‑ tropológica dedicada ao Estado e a políticas públicas ultrapassa em muito essa perspectiva (cf. Teixeira; Souza Lima, 2010),5 destacando, [5] Como nossa interlocução teórica se situa na fronteira da ciência política com a sociologia histórica, não há aqui espaço para dar conta da quantidade — nem faríamos jus à qualidade — da reflexão antropológica sobre política, políticos, políticas e afins. A despeito dessa limitação, vale notar que a re‑ flexão sobre o tema teve na produção do Núcleo de Antropologia da Política (ou seja, no Museu Nacional e em suas relações com a UnB), com sua veia et‑ nográfica distintiva, um de seus prin‑ cipais marcos nacionais. Novos estud. ❙❙ CEBRAP ❙❙ SÃO PAULO ❙❙ V38n02 ❙❙ 411-434 ❙❙ MAI.–AGO. 2019 413 como nós, que as “ações e tecnologias de governo [são] formuladas não só desde organizações administrativas de Estados nacionais, mas também a partir de diferentes modalidades de organizações [civis e internacionais]” (Souza Lima; Castro, 2015, p. 35). Introdução Vamos nos debruçar sobre dois casos de institucionalização sim‑ bólica: a disputa de atrizes e redes feministas paraguaias pela insti‑ tucionalização da categoria “gênero”, forjando identidades, alianças e coalizões para engendrar uma política de saúde; e as relações do movimento dos agricultores familiares com o Programa Nacional de Fortalecimento da Agricultura Familiar (Pronaf). A escolha dos casos não obedece a critérios de confirmação empírica nem de generaliza‑ ção teórica, mas de variabilidade, o que permite mostrar a pertinência analítica e o potencial heurístico dos argumentos aqui expostos. O caso das redes feministas, que se baseia em extensa pesquisa (Szwako, 2012), oferece traços comuns aos movimentos centrados na defesa de políticas e direitos de grupos sistematicamente marginalizados. Já o da agricultura familiar introduz variabilidade porque trata de ato‑ res vinculados ao mundo do trabalho, em que a lógica da economia, e não a dos direitos universais, constitui matriz incontornável para a elaboração de demandas. Tal variabilidade, note‑se, provém de fontes diversas: no caso da trajetória das interações da militância feminista paraguaia, trata‑se do alargamento de consequências analíticas inspi‑ rado por acúmulo de pesquisa (cf. Szwako, 2012); no da agricultura fa‑ miliar, caso informado por fontes secundárias, trata‑se de uma espécie de meta‑análise, cuja reconstrução atenta para lógicas características da institucionalização simbólica por nós defendida. Antes de passar aos casos, abordaremos brevemente a institu‑ cionalização e nossos pressupostos, bem como a relação dos ar‑ gumentos aqui apresentados com a literatura pertinente, a fim de iluminar a institucionalização de demandas e interesses de atores coletivos em políticas públicas, especialmente quando essa insti‑ tucionalização é de índole simbólica. Depois, discorreremos sobre a metáfora visual de Seeing Like a State e seus insights analíticos. Em diálogo com Scott, examinaremos em seguida os dois casos e es‑ boçaremos, ao fim, linhas e questões para uma agenda de pesquisa centrada no aspecto simbólico das dinâmicas de institucionaliza‑ ção de categorias em e de políticas públicas. Institucionalização e capacidades cognitivas do Estado Para os campos de estudo dos movimentos sociais, da sociedade civil e da participação, a institucionalização das reivindicações dos atores neles pesquisados é fenômeno dos mais notáveis no Brasil pós‑transição democrática e pré‑impeachment de Dilma Rousseff. A atenção às interações socioestatais na evolução das políticas pú‑ blicas é fundamental para compreender as feições adquiridas por parte significativa dessas políticas em setores como saúde, assistên‑ cia social ou habitação, bem como em áreas de políticas transversais cas é u da e ta pa a co p ee de as e ções adqu das po parte significativa dessas políticas em setores como saúde, assistên‑ cia social ou habitação, bem como em áreas de políticas transversais — direitos humanos, direitos da criança e adolescente, gênero, raça, entre outras.6 É certo que o interesse nos processos de institucio‑ nalização foi propiciado por momento recente na história do país (o pós‑transição) e, nesse sentido, responda a motivações que hoje possam parecer datadas, à luz do impeachment e da emergência de um governo de forças conservadoras no cenário político nacional. Mesmo assim, a permeabilidade do Estado às demandas de atores societais tradicionalmente considerados outsiders evidenciou, nos anos pós‑transição, não apenas lacunas no conhecimento empírico mas a urgência de se revisar os pressupostos teóricos da literatura e empreender esforços de construção conceitual (inter alia Gurza La‑ valle et al., 2019a). Assim, se a compreensão de processos específicos de institucionalização obedece a processos temporal e espacialmen‑ te delimitados, as questões analíticas são de relevância geral. Afinal, sejam quais forem as orientações políticas e as adesões ideológicas de governos e atores societais, processos de institucionalização de interesses permanecem, malgré tout, centrais. Direta ou obliquamente, diagnósticos afins têm surgido não só de um amplo conjunto de análises que inquirem, a partir da perspectiva societal, os condicionantes, os fatores e os atores im‑ plicados na produção de políticas (em especial, as sociais), mas também de estudos do campo de políticas públicas orientados por abordagens de governança (Marques, 2013). Além da presen‑ ça reiterada de movimentos sociais e organizações da sociedade civil, as análises constatam que as capacidades organizacionais societárias acumuladas em redes e agremiações civis têm ganhado lugar tanto na gênese de determinadas políticas públicas quanto em seus desenvolvimentos institucionais. [6] Além da literatura mobilizada em Gurza Lavalle e Szwako (2015), ver, dentre outros, Bichir et al. (2017). Institucionalização e capacidades cognitivas do Estado Assim, ao aproximar os debates de políticas e movimentos sociais, amplia‑se a com‑ preensão das dinâmicas de interação socioestatal mediante as quais aquelas capacidades organizacionais societárias se trans‑ formam e são transformadas em capacidades estatais. Mais uma vez, também aqui no registro próprio às análises da construção de capacidades estatais, o cenário mais abrangente e imediato de tom fortemente regressivo — no Brasil como alhures — não deve obliterar a compreensão da lógica sociopolítica na raiz das dispu‑ tas sobre determinadas políticas, isto é, de posições contra ou a favor da institucionalização de determinadas ideias e categorias tornadas instituição por força e via das interações Estado/atores sociais — movimentos ou outros. Novos estud. ❙❙ CEBRAP ❙❙ SÃO PAULO ❙❙ V38n02 ❙❙ 411-434 ❙❙ MAI.–AGO. 2019 415 Em sintonia com o neoinstitucionalismo histórico, buscamos nes‑ te texto alargar os registros em que a institucionalização de demandas e interesses dos ms e das organizações da sociedade civil (osc) é pen‑ sada, articulando‑a com a produção de capacidades estatais de tipo cognitivo. Ao examinar as implicações dessa ampliação, entende‑ mos que a institucionalização ocorre mediante processos históricos de interação entre atores organizados e instituições administrativas e políticas do Estado e que, uma vez em andamento, ela altera a um só tempo a atuação dos atores sociais e as capacidades estatais, alargando seus repertórios e, em alguma medida, potencializando‑lhes o alcance (Gurza Lavalle; Szwako, 2015; Gurza Lavalle et al., 2019a). De modo mais preciso, capacidades estatais podem responder a processos en‑ dógenos — desenvolvimento institucional, consolidação e mudança institucional —, mas podem também ser produto da interação com ms e osc, resultando na alteração das capacidades de ação das insti‑ tuições, dos agentes estatais e dos atores sociais. A institucionalização compreende, assim, a sociogênese das capa‑ cidades estatais, das políticas e de seus instrumentos, e, simultanea mente, a gênese institucional das capacidades de ação, demandas e repertórios de ação dos ms. Por isso, pressupomos a mútua constitui‑ ção entre sociedade civil e Estado, entendendo que as instituições e os agentes estatais não são mero contexto externo das estratégias de ms e vice‑versa (Gurza Lavalle et al., 2019a). A institucionalização não ocorre in toto, mas como progressiva sedimentação institucional que traduz seletivamente certas reivindicações ou partes delas. Tal sedi‑ mentação institucional cristaliza encaixes, os quais fazem funcionar a seletividade das instituições a favor dos atores sociais que os construí‑ ram. [7] Para uma exposição das confi‑ gurações nos planos analítico e empí‑ rico, ver: Gurza Lavalle et al, 2019b. Institucionalização e capacidades cognitivas do Estado Note‑se que, embora o foco seja a institucionalização, certamente outras questões podem demandar recortes mais centrados no Estado e em seus agentes ou nos movimentos e em seus atores — e, portanto, exigir atenção a outros processos e interações. Todavia, quando o foco é esse, a análise se debruça sobre sedimentações institucionais para lhes rastrear o processo (Gurza Lavalle et al., 2019b). p Processos de interação nem sempre produzem institucionaliza‑ ção; quando a produzem, ela pode assumir configurações variadas e ocorrer em diversos modos.7 Aqui nos centraremos apenas em um modo. Sem o intuito de arrolar as instâncias estudadas na fronteira dos debates de ms e políticas públicas, é possível dizer que, além da institucionalização simbólica centrada em categorias, há outros três modos de institucionalização correlatos àquilo que é o objeto a ser institucionalizado. Os dois mais conhecidos na literatura brasilei‑ ra são o modo posicional (cf. Abers e Oliveira, 2015; Dowbor, 2012), distinguido pelos cargos oficiais como instâncias a serem ocupadas e eventualmente transformadas, e o modo programático (cf. Gutierres, 6 “Seeing like a social movement”: Institucionalização simbólica e capacidades estatais cognitivas ❙❙ José Szwako e Adria social movement”: Institucionalização simbólica e capacidades estatais cognitivas ❙❙ José Szwako e Adrian Gurza Laval 2015; Carlos, 2012; Albuquerque, 2015), relativo à institucionaliza‑ ção de projetos e programas específicos em políticas. Outro modo de institucionalização, bem menos reconhecido, é o técnico‑prático, que diz respeito a instrumentos de políticas instituídos por meio de inte‑ rações socioestatais.8 [8] A institucionalização técnico‑prá‑ tica passou despercebida na literatura sobre movimentos sociais, mas vem recebendo tratamento acurado na literatura de políticas públicas e sua instrumentação. Ver: Marques, 2013; Minarelli, no prelo; Hoyler, 2016. Quanto à institucionalização simbólica, é notório que a forma pela qual os ms articulam suas demandas e tornam públicas suas denún‑ cias envolve a produção de formas simbólicas de classificação. A so‑ ciologia clássica abandonou a noção de categorias como formas puras, independentes de qualquer experiência e que operam como condição de possibilidade da cognição (Bourdieu, 1979). Assim, categorias, noções ou sistemas de classificação (doravante apenas categorias)9 passaram a ser entendidos não apenas como construções sociais — em Durkheim e Mauss (2009 [1903]), geneticamente modelados por analogia à sociedade10 —, mas como mediações determinantes na construção do próprio mundo social (Berger; Luckmann, 2004 [1966]). Institucionalização e capacidades cognitivas do Estado A nomeação e classificação de seres e coisas, e suas relações segundo formas classificatórias hierarquizam o mundo, produzem‑no em sintonia com ideias e compreensões que os atores — neste caso, ms — têm dele. [9] Ver Durkheim e Mauss (2009 [1903]), pp. 48 ss. 2019 417 tornam parte central das formas pelas quais o mundo é cognitivamente apreendido por agentes políticos, podendo deslocar outras categorias. Ao dizer que a noção de capacidade estatal pode ser analisada por sua dimensão cognitiva, sugerimos ir além da posição ocupada por tal noção nos debates do neoinstitucionalismo histórico e em suas ressonâncias contemporâneas (Gomide; Boschi, 2016; Gomide et al., 2017). Para compreender o que o Estado faz e como o faz (cf. Cin‑ golani, 2013), propomos, inspirados em Scott, somar às dimensões técnico‑administrativa e político‑relacional de suas capacidades (Go‑ mide; Pires, 2014) a dimensão cognitiva como âmbito de análise para entender as dinâmicas de institucionalização de demandas e ideias de ms e outros atores civis em capacidades expressas nas categorias que estruturam políticas públicas. Perguntamo‑nos, então, pela gênese sociopolítica das capacidades es‑ tatais (Gurza Lavalle et al., 2019b), procurando nas interações com atores não estatais — nomeadamente, com ms e osc — e nos tecidos relacionais do Estado (cf. Marques, 2006) os fatores condicionan‑ tes e estruturantes daquilo que o Estado faz e como o faz. A ênfase na dimensão cognitiva das capacidades estatais se inspira tanto em Seeing Like a State como na chamada “virada ideacional” (inter alia Rues‑ chemeyer, 2006; Schmidt, 2010). Ao se perguntar como e quando as ideias importam, esse tipo discursivo de institucionalismo reconhece que as ideias dos atores a respeito de seus “contextos” importam e que a força cognitiva e valorativa das ideias veiculada em interações e em dispositivos institucionais, tais como as capacidades, é crucial para a compreensão daquilo que ocorre nas instituições. Quanto ao debate próprio aos ms, o leque de atores e questões privilegiados por esse campo teórico foi profundamente marcado pela relação umbilical entre o nascimento e crescimento das teorias dos movimentos sociais e a emergência de atores externos à incorpora‑ ção política ou à inclusão econômica (outsiders). Estes se caracterizam pela disrupção com os circuitos centrais do poder político e econômi‑ co — são antissistêmicos ou antiestablishment — e pelo engajamento em agendas de contestação mediante expedientes contenciosos em maior ou menor medida. A institucionalização é claramente um des‑ ses fenômenos secundários, ora empurrado para a penumbra pelas ênfases das lentes analíticas, ora caracterizado com valência negativa porque supostamente conduziria à burocratização dos movimentos e a sua desmobilização. [9] Ver Durkheim e Mauss (2009 [1903]), pp. 48 ss. [10] Se o aspecto simbólico das for mas de classificação foi em geral negligenciado nas fronteiras da so‑ ciologia com a ciência política, isso parece não valer para a reflexão an‑ tropológica. Também de inspiração mauss‑durkheimiana, a análise de Chaves (2000), por exemplo, enfati‑ zou a fabricação do social nos dramas públicos performados pelo MST em sua Marcha Nacional de 1997. [10] Se o aspecto simbólico das for mas de classificação foi em geral negligenciado nas fronteiras da so‑ ciologia com a ciência política, isso parece não valer para a reflexão an‑ tropológica. Também de inspiração mauss‑durkheimiana, a análise de Chaves (2000), por exemplo, enfati‑ zou a fabricação do social nos dramas públicos performados pelo MST em sua Marcha Nacional de 1997. Porém, as instituições e o Estado também produzem categorias e, mediante elas, conhecem e produzem os “problemas sociais” sobre os quais intervêm (Bourdieu; Wacquant; Farage, 1994). Conforme será visto, essa produção é objeto de disputa. Em registro institucionalista, a analogia antropomórfica inerente à asseveração “o Estado conhece” — ou “enxerga”, na metáfora visual de Scott — apenas pressupõe que os agentes das instituições de Estado operam animados por essas ca‑ tegorias e, ao tomar decisões, julgam correto aquilo que se adapta aos esquemas de simplificação e ao corpo de conhecimento codificados e chancelados pela instituição à qual são filiados (Douglas, 1986). Em sua expressão mais abstrata e estilizada, e antecipando o argumento de Scott (1998, pp. 309‑41), a oposição entre episteme ou tékhn-e e metis separa os corpos de conhecimento e as categorias classificatórias que alimentam os “esquemas” empregados pelo Estado, de um lado, das categorias e dos saberes práticos que conferem sentido ao mundo so‑ cial e a seus atores, de outro. Destarte, quando — mediante diferentes mecanismos e processos históricos de interação entre agentes e insti‑ tuições estatais, e ms e atores civis — as categorias destes últimos pas‑ sam a ser utilizadas por burocratas, partidos e políticos na concepção e na execução de políticas públicas, produzem efeitos no desenho e na implementação das políticas de interesse dos atores sociais. A insti‑ tucionalização altera, nesse caso, as capacidades estatais na dimensão cognitiva teorizada por Scott (1998). Em outras palavras, por meio de interações com atores não estatais, essas categorias simbólicas se Novos estud. ❙❙ CEBRAP ❙❙ SÃO PAULO ❙❙ V38n02 ❙❙ 411-434 ❙❙ MAI.–AGO. [9] Ver Durkheim e Mauss (2009 [1903]), pp. 48 ss. A teorização do confronto à la Tilly e Tarrow (2006) não nos oferece uma chave de leitura adequada para pensar a institucionalização na medida em que a aproximação não conflituosa entre Estado e movimentos sociais é lida, em geral, como cooptação, desmobilização ou desradicalização (cf. Clemens, 1997). Noutro dia‑ pasão, ainda que o aspecto simbólico da mobilização tenha sido 418 “Seeing like a social movement”: Institucionalização simbólica e capacidades estatais cognitivas ❙❙ José Szwako e A social movement”: Institucionalização simbólica e capacidades estatais cognitivas ❙❙ José Szwako e Adrian Gurza Laval enfatizado nas leituras dos chamados “novos movimentos sociais”, essa vertente pressupõe que tais movimentos “não se dirigiriam ao Estado”, inscrevendo sua “luta simbólica” numa hipótese “de domi‑ nação eminentemente cultural” (cf. Alonso, 2009, pp. 62‑7). Assim, ambas as vertentes, uma por superestimar o conflito com o Estado e outra por desligar os ms dele, parecem limitadas para tratar do aspecto simbólico implicado nas formas de interação e circulação de catego‑ rias entre ms e políticas públicas. Legibilidade do mundo social e ação do Estado Dados os pressupostos de nossa abordagem, o trabalho de Scott parece‑nos propício para indagar o caráter mutuamente constitutivo das relações entre sc e Estado quando as categorias ou sistemas de classificação dos ms são institucionalizados, moldando as capacida‑ des cognitivas dos agentes estatais que definem e operam as políticas. É certo que suas preocupações com o processo de construção dos Es‑ tados‑nação (statecraft) e com as catástrofes ambientais e sociais pro‑ duzidas por grandes projetos/programas de intervenção concebidos e implementados em contextos de Estados autoritários diferem das questões que aqui nos ocupam. Ainda assim, suas afinidades analíti‑ cas substantivas com tais questões nos permitem avançar a dimensão cognitiva das capacidades estatais quando esta ganha forma pela ins‑ titucionalização de categorias oriundas de ms. O autor propõe uma abordagem simultaneamente cognitiva e vi‑ sual das relações e das tensões entre atores políticos e comunidades locais, em contextos nacionais variados e historicamente distantes. Segundo ele, as formas pré‑modernas de Estado eram “em parte ce‑ gas” às formas locais de conhecimento das populações sujeitas ao con‑ junto de forças que impeliu à construção de Estados modernos (Scott, 1998, p. 2). Dito de outro modo: as populações, suas dinâmicas de vida e de reprodução, bem como suas fontes de recursos dispersas no terri‑ tório, foram por muito tempo ininteligíveis para os poderes e agentes de Estado, passando a ser enquadrados em seu marco somente após longos processos de extração, conscrição e taxação (cf. Tilly, 1996; Gi‑ ddens, 2008). Segundo esse argumento, boa parte do statecraft hoje cristalizado em instituições políticas modernas consistiu no desen‑ volvimento da capacidade de ler as dinâmicas do mundo. A legibilidade da sociedade, ou seja, a visibilidade da ordenação e reprodução de suas dinâmicas, é uma construção que não opera como compreensão da complexidade, mas, antes, como redução e simplificação. Ao lado de processos sociopolíticos como taxação e extração, Scott situou o que chama de formas de simplificação estatal, tais como a padronização de medidas, de mapas, de sobrenomes e da língua Novos estud. ❙❙ CEBRAP ❙❙ SÃO PAULO ❙❙ V38n02 ❙❙ 411-434 ❙❙ MAI.–AGO. 2019 419 nacional.11 Tais formas de simplificação, ou “esquemas” (schemes), foram indispensáveis para tornar as práticas e as realidades locais le‑ gíveis e traduzíveis para a intervenção dos poderes centrais — nota‑ damente nos casos nacionais europeus. [11] Para a construção e imposição de medidas comuns, ver: Anderson (2008) e Kula (1980). Legibilidade do mundo social e ação do Estado Já quando se detém em casos de construção de Estados no século xx, Scott observa como planos modernistas e autoritários de desenvolvimento, urbanização e agri‑ cultura produziram efeitos ambiental e socialmente destrutivos so‑ bre populações, cidades e áreas atingidas por eles. Afim a outras obras também dedicadas a formas populares de resistência,12 Seeing Like a State argumenta que, nesses casos nefastos, o autoritarismo político é o responsável por “aviltar”13, em nome de uma razão cientificista ou modernista, a contribuição dos recursos prático‑cognitivos de grupos e populações locais por ele denominado “metis”: “formas de conheci‑ mento enraizadas na experiência local” (Scott, 1998, p. 311). [13] Ver, em especial: Scott (1998), pp. 323 ss. Em sua conclusão, ressoando Peter Evans, Scott propõe uma deon tologia das instituições e políticas metis‑friendly, ou seja, abertas aos saberes práticos: “Uma instituição […] que tire parte de sua forma do metis em evolução das pessoas nela envolvidas enriquecerá sua gama de experiência e habilidades. […] [A] instituição metis‑friendly tanto usa como renova um bem público valioso” (1998, p. 352). Não por acaso, um dos interlocutores elogiados ao longo da obra é o incremen‑ talista Charles Lindblom: em ambos, há um laço umbilical entre fon‑ tes limitadas de conhecimento, dilemas políticos disso decorrentes e o caráter prático das alternativas disponíveis e adequadas para tomada de decisão — também ela prática — numa determinada política ou setor de política pública. Repare‑se que a legibilidade do mundo social por parte do Estado e de seus agentes, mediante esquemas, não é, por decreto da teoria, defi‑ nida como negativa ou como fonte irredimível de dominação. Em Scott, a salvaguarda dos direitos fundamentais ou a produção de bens pú‑ blicos depende de esquemas e da visibilidade que eles viabilizam, mas esquemas também animam intervenções do Estado de consequên cias catastróficas. A diferença reside na possibilidade de correção que regimes democráticos e instituições amigáveis aos saberes práticos propiciam. Assim, essa definição contrasta de maneira evidente com boa parte das metáforas visuais utilizadas pelo pensamento francês ao longo do século xx (e ainda hoje muito influentes) para designar Es‑ tado e política moderna, cuja conotação normativa era pejorativa (cf. Jay, 1993) — “panóptico” e “vigilância” em Foucault e “sociedade do espetáculo” à la Debord são dois exemplos bem conhecidos. [13] Ver, em especial: Scott (1998), pp. 323 ss. [12] Para uma crítica, ver: Monsma (2000). 20 “Seeing like a social movement”: Institucionalização simbólica e capacidades estatais cognitivas ❙❙ José Szwako e Adrian Gurza Lavalle [14] Interessantíssimo notar que, mesmo no âmbito da virada ideacio‑ nal, o aspecto simbólico das ideias foi por vezes negligenciado. Ver: Rueschemeyer (2006), p. 227. Legibilidade do mundo social e ação do Estado Utilizando, então, os insights da sociologia histórica, propomos que uma analítica da visualidade é heuristicamente proveitosa para a compreensão de dinâmicas de transformação dos esquemas esta‑ tais de ação e ordenação do mundo, apreendidas a partir de trajetórias 420 “Seeing like a social movement”: Institucionalização simbólica e capacidades estatais cognitivas ❙❙ José Szwako e Adrian Gurza Lavalle 0 “Seeing like a social movement”: Institucionalização simbólica e capacidades estatais cognitivas ❙❙ José Szwako e Adria al movement”: Institucionalização simbólica e capacidades estatais cognitivas ❙❙ José Szwako e Adrian Gurza Lavalle 420 socioestatais de interação. Nesse sentido, até mesmo uma metáfora visual largamente utilizada (em registro normativo negativo) como a de vigilância de Estado pode ter alcance explicativo renovado. “Mo‑ vimentos [sociais] dedicados ao alargamento da participação demo‑ crática devem [ou melhor, podem] ser vistos como voltados […] em direção à correção dos desequilíbrios de poder envolvidos em vigilân‑ cia” (Giddens, 2008, p. 325). Se, concordando com Scott, uma das fontes de poder do Estado reside em sua capacidade de produzir e acu‑ mular informação e conhecimento sobre grupos e recursos num dado território, essa fonte de poder é, em regimes não totalitários, passível de ser contestada, disputada e conquistada para que o Estado “fique de olho” também em realidades e dinâmicas populacionais que são objeto de interesse e valorização pelos ms. Assim, aquilo que o senso comum acadêmico chama pejorativamente de vigilância institui, com efeito, uma “dialética do controle” (idem, p. 30), na qual não apenas o Estado com seus recursos cognitivos pretende ou consegue vigiar popula‑ ções, mas, recíproca e reiteradamente, estas resistem e transformam os esquemas estatais, valendo‑se da própria capacidade estatal de vigiar e até do termo “vigilância”, agora “cidadã” (cde, Cladem; cmp, 2005). Em suma, Seeing Like a State informa nossa proposta de leitura da institucionalização de demandas dos ms a partir do exame tanto da trajetória de algumas de suas categorias e ideias centrais como de suas interações com representantes institucionais. Entendidas como re‑ cursos cognitivos, as capacidades inscritas nos saberes e no conheci‑ mento de organizações e redes civis podem e, não raro, logram interpelar e mudar as capacidades estatais. Essa afirmação pode não soar novidade para quem, como nós, tem argumentado que Estado e sc se constituem mutuamente e que as capacidades estatais, em especial as das políticas sociais, pedem o escrutínio de sua gênese sociopolítica (Gurza Lavalle et al., 2019b). Legibilidade do mundo social e ação do Estado A ênfase nas capacidades cognitivas e na dimensão simbólica constitui a face inovadora do argumento aqui apresentado, pois onde ocorre interação entre atores sociais e políticos — com graus diversos de colaboração, agonismo e antagonismo — circulam também ideais, valo‑ res e símbolos.14 Eis, esperamos, a vantagem analítica de nossa proposta: acessar o mundo das hierarquizações e das disputas e oposições simbó‑ licas dos grupos e coalizões ao redor das políticas públicas pela via das categorias dos ms e de sua institucionalização em esquemas estatais. Novos estud. ❙❙ CEBRAP ❙❙ SÃO PAULO ❙❙ V38n02 ❙❙ 411-434 ❙❙ MAI.–AGO. 2019 4 Novos estud. ❙❙ CEBRAP ❙❙ SÃO PAULO ❙❙ V38n02 ❙❙ 411-434 ❙❙ MAI.–AGO. 2019 Interações socioestatais e classificações como categorias estruturantes de políticas públicas O primeiro caso aqui examinado remete à incorporação da cate‑ goria “gênero” na estruturação das políticas públicas do Estado para‑ guaio ao longo de um processo cuidadosamente caracterizado alhures Novos estud. ❙❙ CEBRAP ❙❙ SÃO PAULO ❙❙ V38n02 ❙❙ 411-434 ❙❙ MAI.–AGO. 2019 421 como generificação do Estado. Tal processo pode ser entendido como a estruturação cognitiva de políticas, programas e instrumentos oficiais a partir da ideia de gênero (Szwako, 2012). As dinâmicas de interação mediante as quais partes do Executivo paraguaio foram permeadas pela “perspectiva de gênero” transcorreram no contexto de democra‑ tização do Paraguai que se seguiu à deposição do general Alfredo Stroessner em 1989. Esse processo de generificação (Walby, 2005)15 passou, grosso modo, por três momentos‑chave na incorporação e difusão da “pers‑ pectiva de gênero” pelo Estado paraguaio. O primeiro deles é a gênese da Secretaria da Mulher — órgão com estatuto ministerial —, criada em 1993, em boa medida como forma de articulação entre a então re‑ cém‑empossada ministra Cristina Muñoz, do Partido Colorado, e uma fração do feminismo paraguaio distinguida por sua verve partidária, o então Multisetorial de Mulheres. O segundo momento importante na generificação estatal paraguaia se deu na época da iv Conferência Mundial da Mulher ocorrida em Pequim, em 1995, que serviu como oportunidade para a projeção das demandas de organizações e ativis‑ tas da mais antiga articuladora civil feminista do país, a Coordenação de Mulheres do Paraguai (cmp).16 Já o terceiro momento abarca o pe‑ ríodo posterior a março de 1999, quando eclodiu o chamado Marzo paraguayo — ciclo de protestos deflagrado pelo assassinato do então vice‑presidente do Paraguai que resultou em violentos confrontos en‑ tre manifestantes e forças repressivas, nos quais morreram oito ma‑ nifestantes. Após 1999, uma vez pacificado o pacto no seio das elites e dos partidos paraguaios, a institucionalização de “gênero” trouxe a público um duplo processo: a incorporação dessa categoria por agên‑ cias e partes nos três poderes do Estado paraguaio, que passaram a se valer dela como forma oficial de classificação, e, ato contínuo, a con‑ tramobilização conservadora e de raiz religiosa em oposição a essa in‑ corporação (Cf. Szwako, 2014; Perissinotto; Szwako, 2017). Vejamos, então, as interações e ideias em jogo, especialmente nos dois últimos momentos dessa institucionalização. [15] Tomamos “generificação” e “gen der mainstreaming” como sinônimos, considerando especificamente o componente prático da definição de Walby (2005), p. 321. [16] “Articuladora” conforme Gurza Lavalle, Castello e Bichir (2007). Interações socioestatais e classificações como categorias estruturantes de políticas públicas A noção de “gênero” foi elaborada por parte das militantes para‑ guaias no âmbito de reivindicações formuladas desde fins do regime autoritário, quando o substantivo “mulher” dividia com o referente cognitivo “gênero” o núcleo das demandas por um órgão público voltado à “condição social das mulheres […], levando em conta a heterogeneidade de situações e as formas de discriminação por ra‑ zão de gênero” (Multisetorial, 1989, p. 7). No bojo das organiza‑ ções de pesquisa que caracterizavam uma articuladora feminista, a cmp, produzia‑se um conhecimento por meio do qual as feministas resgatavam a memória das lutas e mulheres “que muito têm feito pelo avanço da consciência de gênero” (Echauri, 1992, p. 3). Neste 422 “Seeing like a social movement”: Institucionalização simbólica e capacidades estatais cognitivas ❙❙ José Szwako e Adrian Gurza Lavalle g like a social movement”: Institucionalização simbólica e capacidades estatais cognitivas ❙❙ José Szwako e Adrian Gurz enquadramento, as mulheres e organizações com “consciência de gênero” são “feministas”, pois lutam a partir “[do] reconhecimento da discriminação social da mulher; [da] identificação da base cultural e ideológica, não natural, dessa discriminação; [e pela] elaboração de propostas e ações concretas para transformar a situação de discrimi‑ nação” (idem, 1992, p. 61, grifos nossos). p g Com base nessas e em outras reivindicações e, sobretudo, me‑ diante a aliança entre lideranças da Multisetorial e parte do Partido Colorado, houve a criação da Secretaria da Mulher em 1993. Sem recursos e orçamento robustos, o primeiro grande desafio da então ministra da Mulher seria a participação na Conferência Mundial da Mulher em Pequim. Para praticamente todos os países latino‑ame‑ ricanos, essa conferência significou uma oportunidade para as po‑ líticas estruturadas por “gênero” e para as relações travadas entre feminismos civis e autoridades (Alvarez, 2003). No caso paraguaio não foi diferente, e, do ponto de vista da mobilização das organi‑ zações ao redor da cmp, os processos preparatórios para Pequim serviram como oportunidade ímpar para mobilizar a identidade e simultaneamente fazer suas demandas pela generificação do Estado paraguaio. Na série de encontros feministas de discussão ocorri‑ dos antes da conferência internacional, a evocação à ideia de “gêne‑ ro” operou duplamente. Por um lado, a “consciência de gênero” teve ali função identitária. “[Nós] mulheres com consciência de gênero queremos construir modelos de desenvolvimento diferentes que permitam uma mudança em nossa condição sociocultural e, em consequência, na estrutura mesma da sociedade” (cmp, 1994, p. 110). Interações socioestatais e classificações como categorias estruturantes de políticas públicas Por outro, a “perspectiva de gênero” encerra a própria deman‑ da delas. “Se [nós] mulheres combinarmos o acesso ao poder com uma perspectiva de gênero, teremos capacidade para construir um modelo de sociedade diferente, sem as discriminações que hoje existem por diferentes motivos” (idem, p. 115). Em síntese: “[Nós com consciência de gênero] recomendamos que o governo tenha uma política clara de desenvolvimento econômico e social com uma perspectiva de gênero” (idem, p. 122). O legado da participação do Paraguai na capital chinesa assumiu diferentes sentidos. Se, por um lado, aproximou as lideranças da cmp e a ministra, animando quase de imediato a generificação da Secretaria da Mulher, por outro, absorveu também o peso do conservadorismo católico representado na comitiva oficial do país enviada à conferência. Na volta de Pequim, o Estado paraguaio anunciou restrições ao uso do termo “gênero”, pois “tal conceito se refere a ambos os sexos: o ho‑ mem e a mulher” (onu, 1995, p. 180). Bem entendida, essa nota marca uma oposição à lógica segundo a qual a “discriminação da mulher” tem “base cultural”, sendo “não natural”. Novos estud. ❙❙ CEBRAP ❙❙ SÃO PAULO ❙❙ V38n02 ❙❙ 411-434 ❙❙ MAI.–AGO. 2019 423 A herança direta das conferências internacionais para as políti‑ cas públicas no país ficou estampada já no nome do “Plan Nacio‑ nal de Salud Reproductiva — versión post‑Cairo” — em alusão à Conferência sobre População e Desenvolvimento, ocorrida um ano antes de Pequim, na capital egípcia. Inspirado igualmente por inte‑ rações com organizações feministas de pesquisa, este plano afirma que “[a] perspectiva de gênero nos permite identificar os papéis socialmente atribuídos a homens e mulheres que impõem estereóti‑ pos e dificultam o pleno desenvolvimento dos indivíduos” (Para‑ guai, 1997b, p. 15, grifos nossos). Embora a gênese da categoria no plano tenha sido vacilante em razão do caráter dito “polêmico” dos direitos sexuais e reprodutivos (Paraguai, 1997b, p.12), vê‑se que, na trajetória das versões posteriores e atualizações, “gênero” seguiu estruturando os principais objetivos, cujas ações deveriam assegu‑ rar “a atenção integral ao indivíduo e à comunidade com enfoque em direitos, qualidade, gênero e equidades” (Paraguai, 2003, s/p). Também nas versões mais recentes, de 2009 e de 2014, “gênero” permanece como forma classificatória do plano e, no último, chega a estruturar um capítulo de combate à “violência intrafamiliar e baseada em gênero”. Interações socioestatais e classificações como categorias estruturantes de políticas públicas Essa continuidade, contudo, não contemplava as demandas fe‑ ministas para além da incorporação do “enfoque de gênero”, pois, segundo as redes e organizações civis, não só eram alarmantes os níveis de mortalidade materna no país como também era defici‑ tário o acesso a serviços e medicamentos de saúde sexual e repro‑ dutiva (cde; Cladem; cmp, 2005). A dificuldade para assegurar medicamentos refletia uma limitação mais ampla nas capacidades estatais, especificamente na capacidade técnico‑administrativa do Ministério da Saúde paraguaio. Os graves níveis de subnotificação tornavam impossível, por exemplo, quantificar o número de óbitos no país (Martínez, 2001). Quanto à saúde sexual e reprodutiva, o déficit na capacidade instalada foi enfrentado por meio de uma coalizão de defesa chamada Daia — Disponibilidad Asegurada de Insumos Anticonceptivos [Disponibilidade Garantida de Insumos Contraceptivos], que reuniu figuras‑chave da burocracia estatal, de organizações feministas e de agências do sistema e de ajuda interna‑ cionais. Atuante entre 2003 e 2012, a Daia transformou a capacida‑ de ministerial ao racionalizar a logística de compra e distribuição de medicamentos, baixar seus custos e dotá‑los de previsão orçamen‑ tária, tornando‑se um caso modelo para a região (Narváez, 2012). De modo interessante, essa coalizão se distinguia não pelo recurso a “gênero”, mas, ecoando a conferência do Cairo, pela defesa do con‑ ceito de “planejamento familiar” — contra o qual as redes e organi‑ zações religiosas anti‑“gênero” igualmente fizeram forte oposição. 424 “Seeing like a social movement”: Institucionalização simbólica e capacidades estatais cognitivas ❙❙ José Szwako e A social movement”: Institucionalização simbólica e capacidades estatais cognitivas ❙❙ José Szwako e Adrian Gurza Laval Assim, o caso da institucionalização da categoria “gênero” e a consequente adoção da “perspectiva de gênero” em políticas de saú‑ de sexual e reprodutiva no Paraguai permite notar a diferença entre capacidades estatais de naturezas distintas. As capacidades político- ‑relacional e técnico‑administrativa estiveram encarnadas na Daia e em suas conquistas — ou, no caso das primeiras, na conexão entre ministério e cmp —, lado a lado com a capacidade cognitiva implica‑ da no “gênero”. “Gênero” se torna a lente através da qual, depois de e mediante interações com atrizes e personagens civis e internacionais, a realidade passa a ser apreendida pelo esquema formulado no Plano. [17] Cf. Decreto presidencial n. 1.946, de 28 de julho de 1996. [18] Atualmente, Confederação Na‑ cional dos Trabalhadores Rurais Agri‑ cultores e Agricultoras Familiares. Interações socioestatais e classificações como categorias estruturantes de políticas públicas Enfrentar, por vezes sem sucesso, os limites estatais no Paraguai a fim de institucionalizar, via categoria “gênero”, a noção de que homens e mulheres são construtos sociais é um avanço nada trivial, embora não garanta os resultados das políticas. A contramobilização religiosa que, de modo ininterrupto, vem disputando políticas públicas de maneira tão politizada quanto simbólica, saindo às vezes vitoriosa, está aí para mostrá‑lo (Perissinotto; Szwako, 2017). No outro caso aqui contemplado, a construção de categorias clas‑ sificatórias e sua institucionalização têm efeitos sobre uma política pública relacionada a movimentos e atores sociais do mundo rural, especificamente à categoria “agricultura familiar” e ao Programa Na‑ cional de Fortalecimento da Agricultura Familiar (Pronaf). Desde sua gênese, em 1996,17 até seus legados para políticas diversas mais recentes, passando pelas transformações na especificação de seus be‑ neficiários, o Pronaf é estruturado pelas interações com movimentos e organizações do sindicalismo rural brasileiro — sejam elas conflitivas, sejam cooperativas. De fato, o “surgimento deste programa representa o reconhecimento e a legitimação do Estado em relação às especifi‑ cidades de uma nova categoria social — os agricultores familiares” (Schneider; Mattei; Cazella, 2004, p. 21). Em âmbito associativo, a emergência da categoria “agricultura fa‑ miliar” é mais bem compreendida no bojo da reconfiguração das for‑ mas de organização e das identidades políticas do sindicalismo rural ocorrida desde fins do regime autoritário brasileiro. Os anos 1980 viram a emergência tanto de novas redes e organizações, como o Mo‑ vimento dos Trabalhadores Rurais Sem‑Terra (mst), quanto de um projeto político‑sindical dos “rurais da cut” (Central Única dos Tra‑ balhadores) (Favareto, 2006, p. 36). Com isso, a figura do “trabalha‑ dor rural”, instituída como parte do projeto modernizante da ditadura militar (Houtzager, 2004) e de representação relativamente mono‑ polizada pela Confederação Nacional dos Trabalhadores na Agricul‑ tura (Contag) durante algum tempo,18 perdeu espaço para outras identidades e suas respectivas formas de organização e manifestação, nomeadamente, “sem‑terra”, “assentados” e “agricultores familiares” Novos estud. ❙❙ CEBRAP ❙❙ SÃO PAULO ❙❙ V38n02 ❙❙ 411-434 ❙❙ MAI.–AGO. 2019 425 (Medeiros, 2001). No cenário pós‑1988, as políticas econômica e tra‑ balhista de cunho liberalizante dos governos Collor e Franco, além da conquista paulatina de protagonismo pelo mst em torno da “reforma agrária”, acabaram por colocar os sindicalismos cutista e contaguiano numa encruzilhada (Favareto, 2006, p. 40). [19] Posteriormente chamado de Projeto Alternativo de Desenvolvi‑ mento Rural Sustentável e Solidário. Interações socioestatais e classificações como categorias estruturantes de políticas públicas Isso levou essas duas en‑ tidades não só a uma aproximação até então impensável, mas também a reorientar profundamente suas pautas e estratégias, nas quais o par “agricultura familiar”/“desenvolvimento” passou a ser o cerne de um “Projeto Alternativo de Desenvolvimento Rural Sustentável”, o padrs (cut/Contag, 1998).19 Em estreita relação com essa reconfiguração sindical e mobiliza‑ ção no mundo rural, transcorreu uma mudança “paradigmática” na reflexão sobre aqueles sujeitos que, pelos modos de produção em que se inseriam, eram designados “pequenos agricultores”. Obras como Paradigmas do capitalismo agrário em questão e O desenvolvimento agrícola, para citar apenas duas numa miríade, provocaram uma ruptura episte‑ mológica, atualizando os quadros cognitivos de interpretação do rural brasileiro, de seus sujeitos e suas dinâmicas, e trazendo para primeiro plano ideias como “familiar”, “empresa familiar” e “produção fami‑ liar” (Favareto, 2010, p. 27). Contra leituras modernizantes, fossem elas marxistas ou funcionalistas, essa ruptura inovou ao enfatizar não a precariedade e os déficits dessas populações, mas “sua importân‑ cia socioeconômica, a diversidade, a capacidade produtiva e, acima de tudo, que a forma de exploração familiar foi [no Brasil e fora dele] a grande responsável pelo significativo desenvolvimento da produção de alimentos” (Picolotto, 2014, S068). Uma ruptura teórico‑acadê‑ mica com implicações político‑discursivas de peso: onde antes só se via “pequenos agricultores” passa a ser inteligível a “agricultura fami‑ liar”, desde então considerada “responsável por mais de 70% dos es‑ tabelecimentos agrícolas no Brasil” (Wanderley, 1996, s/p).li Toda essa reflexão e sua inovação categorial não ficaram restritas à academia, repercutindo “sobre o[s] discurso[s] de movimentos so‑ ciais e da burocracia governamental ligados à agricultura” (Favareto, 2010, p. 27). Mais ainda: ao lado de outras publicações elaboradas em parceria por organismos internacionais e órgãos do Executivo brasi‑ leiro, o relatório Diretrizes de política agrária e desenvolvimento sustentável para a pequena produção familiar (fao/Incra, 1994) reforçou esse con‑ junto de insights ao propor a “agricultura familiar” como conceito (Gri‑ sa, 2012, pp. 125 ss.) simultaneamente singularizado e plural, dado o modo familiar de (re)produção comum aos diversos tipos de família nele implicados. Interações socioestatais e classificações como categorias estruturantes de políticas públicas A criação do Pronaf e as transformações em seu desenho estão atravessadas por esse conjunto de forças sindicais e inovações teórico‑ -discursivas (inter alia, Medeiros, 2001; Schneider; Mattei; Cazella, 6 “Seeing like a social movement”: Institucionalização simbólica e capacidades estatais cognitivas ❙❙ José Szwako e Adria social movement”: Institucionalização simbólica e capacidades estatais cognitivas ❙❙ José Szwako e Adrian Gurza Laval 2004) por meio seja da negociação delas com gestores e órgãos pú‑ blicos (Abramovay; Veiga, 1999), seja da intensa circulação e troca de ideias entre ambas as partes (Picolotto, 2011; Grisa, 2012). Para sua gênese concorreram, também, mobilizações como o anual “Grito da Terra” e interações marcadas por altas doses de violência e repressão, como os episódios de Corumbiara e Eldorado dos Carajás. Tendo como objetivo geral fortalecer a “agricultura familiar” mediante apoio técnico‑financeiro que favoreça o desenvolvimento rural, o Pronaf copiou inicialmente do relatório fao/Incra o “modelo familiar”, bem como a categorização dos tipos de famílias aptas a ter acesso a crédito: “agricultores familiares consolidados”, “em transição” e “periféricos”. Tal categorização não esteve livre de críticas (cf. Carneiro, 1997), tanto de acadêmicos como dos movimentos rurais. Os repertórios de intera‑ ção destes últimos, tais como greves de fome e marchas, tiveram como efeito tanto alterar a tipificação das condições e linhas de financiamen‑ to quanto tornar mais plural a especificação de grupos (segmentados por faixa de capitalização) e públicos‑alvo (jovens e mulheres, por exemplo) correlatos a novas linhas de crédito reivindicadas por esses movimentos (Grisa, 2012; Mattei, 2014; Brasil, 2015; Flexor; Grisa, 2016).20 Embora seja impossível resumir aqui a gama de atores e ins‑ tituições ao redor do Pronaf ou o rico universo bibliográfico sobre o programa, cumpre destacar dois pontos. Em primeiro lugar, e de um ponto de vista mais amplo, o peso das interações socioestatais nas políticas para o âmbito rural. Protesto, proposição e cogestão fazem parte das formas de ação coletiva dos movimentos sociais e sindicais da agricultura familiar na contempora‑ neidade, ainda que com diferentes ardores, estratégias e “formatações” de outrora. [21] A crescente ampliação dos re‑ cursos em jogo não deixou de ter efei‑ tos ambíguos. Ver: Wesz (2017). [20] Tratamos aqui apenas da insti‑ tucionalização e dos desdobramen tos da categoria “agricultura fami‑ liar”. Para o escrutínio de outros processos, ver Flexor e Grisa (2016). Interações socioestatais e classificações como categorias estruturantes de políticas públicas Os movimentos sociais e sindicais da agricultura continuam acio‑ nando estratégias de protesto e de reivindicação (a exemplo dos Gritos da Terra, da Marcha das Margaridas, do Abril Vermelho, das ocupações de prédios públicos, das Jornadas de Luta da Agricultura Familiar etc.), se instrumentalizam cada vez mais para propor formatos institucionais e políticos para as políticas públicas, e reivindicam a cogestão e a execução partilhada das políticas públicas, seja visando aproximá‑las e adaptá‑las às distintas realidades sociais, seja para o empoderamento das próprias organizações sociais. (Grisa; Schneider, 2014, S142) Em segundo lugar, especificamente quanto a seus desdobramen‑ tos e legados, o Pronaf conseguiu, como anteviram Abramovay e Vei‑ ga (1999, p. 48), tornar‑se um “ambiente institucional” favorável à ampliação das bases da política nacional de crédito e desenvolvi‑ mento rural. Tal ampliação ocorreu em vários sentidos, no volume de recursos e na multiplicação de suas linhas do crédito,21 notadamente Novos estud. ❙❙ CEBRAP ❙❙ SÃO PAULO ❙❙ V38n02 ❙❙ 411-434 ❙❙ MAI.–AGO. 2019 427 com a chegada do Partido dos Trabalhadores (pt) à Presidência da República, em 2003. A partir de então, o programa foi englobado no “macroenquadramento” (masterframe) socioassistencial (cf. Ben‑ ford e Snow, 2000) característico dos primeiros governos petistas. Os desdobramentos incidiram também sobre outras políticas ino‑ vadoras, como as de segurança alimentar, aquisição de alimentos e construção de mercados alimentares alternativos (Flexor; Grisa, 2016; Schmitt; Grisa, 2013; Grisa, 2012). Por outro lado, do ponto de vista de outros modos de institucionalização, aquela ampliação foi acompanhada de uma complexificação na divisão interna do Mi‑ nistério do Desenvolvimento Agrário. Às já existentes secretarias da Agricultura Familiar e de Reordenamento Agrário juntou‑se, em 2003, a Secretaria de Desenvolvimento Territorial. A criação des‑ sa secretaria ecoava demandas de setores dos movimentos e orga‑ nizações rurais descontentes com o Pronaf e com o Ministério do Desenvolvimento Agrário (mda) e respondia a críticas acadêmicas que apontavam desigualdades inter‑regionais intocadas ou mesmo reproduzidas pelo programa (cf. Mattei, 2014). Sob a influência de organismos internacionais, a “abordagem territorial” emergiu como referente e passou ao centro de duas novas políticas (cf. Sabourin, 2014): o Programa de Desenvolvimento Sustentável de Territórios Rurais, que, em 2003, substituiu uma linha do Pronaf, e o Programa Territórios da Cidadania (ptc), de 2008. À parte as críticas passíveis a ambos os programas (cf. 428 “Seeing like a social movement”: Institucionalização simbólica e capacidades estatais cognitivas ❙❙ José Szwako e Adrian Gurza Lavalle Institucionalização simbólica: questões e agenda de pesquisa Inserido no debate sobre institucionalização de reivindicações de ms e osc desenvolvido entre os campos de estudos de partici‑ pação, ms e políticas públicas, este artigo se propôs a examinar o modo de institucionalização simbólica, o qual tem recebido menos atenção na literatura. Aproveitando as metáforas e os insights cogni‑ tivo‑visuais de J. Scott, tencionamos observar as formas pelas quais o Estado opera por meio de categorias de classificação ou, para res‑ peitar a embocadura do autor referido, de “esquemas” estatais. Ob‑ servamos dois casos em que esses esquemas foram construídos no processo de interação entre autoridades e agentes públicos, de um lado, e movimentos, redes e osc, de outro. Interessa aqui atentar para as implicações analíticas dos traços comuns neles encontrados, e não examinar suas divergências ou explicar suas características. Focamos em duas ordens de implicação extraídas de nossa observa‑ ção: uma empírico‑comparativa, pertinente para o estudo de políti‑ cas públicas, e outra teórico‑conceitual, relativa à crítica de alguns pressupostos da obra de Scott, mas não restrita a ela. Primeiro, e mais evidente quanto aos traços comuns nos casos analisados, reafirma‑se uma máxima cara à literatura especializada: as políticas públicas não são afeitas a análises simples e etapistas. Seus processos, atores e ritmos não são reciprocamente exteriores nem estão cronologicamente dispostos, como se seguissem — nos casos que envolvem ms — de um momento inicial, no qual a reivindica‑ ção dos movimentos seria pura e autonomamente elaborada, para um momento posterior, em que esta seria incorporada por gestores vir‑ tuosos ou aliados. Interações com outros atores e discursos estatais e sociais atravessam a formulação das categorias pelas quais militantes e organizações civis concebem suas reivindicações. A produção das categorias “agricultura familiar” e “gênero” passou pela interlocução com setores acadêmicos e pela apropriação das formas de cognição gestadas em pesquisas, ou em organizações civis de pesquisa, com relação àquelas personagens e redes da sc. Essas interações e trocas discursivas, por sua vez, também não são alheias à aproximação com autoridades e à circulação internacional, como se nota pelos exemplos das conferências de Cairo e Pequim ou pela influência de organismos como a Organização das Nações Unidas para Alimentação e Agri‑ cultura (fao). Assim, há uma sociogênese nos esquemas estatais, o que sugere cautela perante divisões que separam as políticas de sua disputa sociopolítica, confiantes em abordagens setoriais endógenas. Interações socioestatais e classificações como categorias estruturantes de políticas públicas Favareto, 2010; Sabourin, 2014), importa‑nos destacar que também neles os efeitos da “agricultura familiar” como esquema de priorização do Estado se fizeram imprimir. No caso do ptc, a “agricultura familiar” conquistou o estatuto de crivo estruturador não só normativamen‑ te previsto, mas efetivo como critério relevante de elegibilidade para os “territórios da cidadania” (Andrade et al., 2016). Porém, para que pudesse operar como capacidade cognitiva mediante a qual o Estado seleciona e hierarquiza municípios e territórios, essa categoria devia antes ser inteligível àqueles agentes públicos que haveriam de empre‑ gá‑la. Tal inteligibilidade foi, como tentamos demonstrar, construída por processos de interação conflitivos e cooperativos que propiciaram trocas e circulação de ideias entre Estado e atores sociais e sindicais do mundo rural — quer desde o “ambiente institucional” produzido pelo Pronaf, quer antes dele. Foi, então, por meio dessas trajetórias de interações socioestatais que a “agricultura familiar” ganhou a con‑ dição de esquema de classificação estatal, tornando um grupo social de agricultores legível e visível ao público e ao Estado na forma insti‑ tucionalizada e impessoal de categoria “responsável por garantir boa parte da segurança alimentar do país, como importante fornecedora de alimentos para o mercado interno” (ibge, 2009, s/p). 8 “Seeing like a social movement”: Institucionalização simbólica e capacidades estatais cognitivas ❙❙ José Szwako e Adria al movement”: Institucionalização simbólica e capacidades estatais cognitivas ❙❙ José Szwako e Adrian Gurza Lavalle 428 Novos estud. ❙❙ CEBRAP ❙❙ SÃO PAULO ❙❙ V38n02 ❙❙ 411-434 ❙❙ MAI.–AGO. 2019 Institucionalização simbólica: questões e agenda de pesquisa Decerto alguns setores são mais afeitos a tais abordagens; porém, em âmbitos como os das sexualidades, das etnicidades, das ruralidades e das biodiversidades, o diálogo e a mútua influência entre agentes Novos estud. ❙❙ CEBRAP ❙❙ SÃO PAULO ❙❙ V38n02 ❙❙ 411-434 ❙❙ MAI.–AGO. 2019 429 públicos, saber científico (em especial, o antropológico) e saberes tra‑ dicionais têm resultado na produção e na defesa de direitos. Outra implicação extraída do cotejo reside naquilo que o neoinsti‑ tucionalismo histórico chama de “dependência da trajetória” (cf. Grisa; Schneider, 2014; Flexor; Grisa, 2016). Do ponto de vista da análise de políticas públicas, isso significa que opções institucionais anteriores circunscrevem caminhos e elevam custos de mudanças posteriores, dificultando determinadas decisões e facilitando outras. Do ponto de vista da análise das interações entre políticas e movimentos, significa que aquilo que é tomado como “início” de determinada política pú‑ blica contém uma sociogênese prévia que também pesa nos rumos da política. Assim, à diferença do que se aponta em boa parte das pesqui‑ sas desenvolvidas no Brasil sobre repertórios e Estado, as interações socioestatais aqui analisadas não aguardaram até o ano propício de 2003 para conquistar e produzir encaixes e efeitos institucionais na administração federal. No caso paraguaio não é diferente: as militan‑ tes e organizações feministas não esperaram que uma legenda ou coa lizão partidária “aliada” chegasse ao topo do Poder Executivo, como ocorreu com Lugo em 2008, para lutar por seus valores e ideais de sociedade e de relações de gênero. Esse deslocamento temporal é me‑ todologicamente salutar para apreender, por contraste, a porosidade do Estado. Em outras palavras, iluminar quanto determinadas políti‑ cas sociais devem às interações socioestatais do passado é, a nosso ver, fundamental para melhor compreender e qualificar a chamada per‑ meabilidade estatal. Institucionalização simbólica: questões e agenda de pesquisa O Pronaf é, nesse sentido, exemplar, pois serviu como o “ambiente institucional” prévio fundamental, no diagnóstico profético de Abramovay e Veiga (1999), para o incremento do volume de recursos e linhas de financiamento nele disponíveis e também para o desenvolvimento de outras políticas, com novos programas, instru‑ mentos e encaixes institucionais.22 [22] Se tomássemos a trajetória das interações socioestatais de militantes e organizações do movimento negro brasileiro pré e pós‑1988, os traços elencados anteriormente (a saber, interlocução com outros discursos, especialmente o acadêmico, e depen‑ dência da trajetória iterativa prévia à chegada ao governo federal de um partido visto como aliado) também apareciam na incorporação estatal da categoria “negros” no Brasil. As interações entre agentes estatais, entidades internacionais e movimentos sociais sugerem desconfiança, a nosso ver, ante oposições como aquela em que Scott (1998, pp. 323 ss.) contrapõe “saber local” a “explicação científica” ou “saber prático” a “tékhn-e”. Centrado em “pro‑ jetos autoritários” de modernização, Scott tende a reificar a distância entre metis e esquemas estatais, de modo que uma suposta impossibi‑ lidade de mediações entre ambos carrega consequências funestas para grupos e territórios atingidos. Mesmo se e quando afirma que esque‑ mas são “insustentáveis sem o saber prático”, aqueles tenderiam a “des‑ cartar” este último, tal como em sua relação com a tékhn-e, da qual esse saber difere radicalmente (Scott, 1998, pp. 7 ss.; pp. 319 ss.). Ora, nos‑ sos casos infirmam tal imaginação teórica na medida em que interações socioestatais prenhes de saberes, ideias e categorias entre universida‑ de, Estado, organismos internacionais, ms e sc tendem a operar como 430 “Seeing like a social movement”: Institucionalização simbólica e capacidades estatais cognitivas ❙❙ José Szwako e A social movement”: Institucionalização simbólica e capacidades estatais cognitivas ❙❙ José Szwako e Adrian Gurza Laval 430 mediadores sociopolíticos à raiz das políticas públicas. Enquanto nas análises (ou nos casos) de Scott faltam tais mediadores, uma concep‑ ção homogeneizante e reificada de “Estado” como fonte irredimível de dominação, captura e governamentalidade — concepção corriqueira nas análises movimentalistas — tende a turvar a compreensão daquelas mediações sociopolíticas que tornaram possível a transformação dos esquemas e suas consequências para a estruturação de políticas. Institucionalização simbólica: questões e agenda de pesquisa Se é ver‑ dade que as instituições do Estado produzem categorias e “problemas sociais” sobre os quais intervêm (Bourdieu; Wacquant; Farage, 1994), não é menos verdade que organizações e movimentos da sc disputam e também fabricam categorias pelas quais reivindicam representar gru‑ pos ligados a injustiças e “problemas sociais” e, por vezes, deslocam as primeiras pelas segundas. A adesão normativa a posturas deontológicas anti‑Estado não leva apenas a uma incompreensão de como o Estado muda interativa e parcialmente sua cognição de partes da população e do território, pode levar também à subestimação da presença e do papel desempenhado por atores e atrizes civis na estruturação das categorias oficiais de classificação e das disputas em torno delas. As disputas e mudanças ocorridas nas últimas décadas nas formas oficiais de classificação ainda não tiveram, a nosso ver, tratamento ade‑ quado, à altura da complexidade das circulações e interações socioes‑ tatais e de suas implicações cognitivo‑simbólicas. Em sua dimensão cognitiva, a estruturação de uma política orientada pelo argumento do caráter de construto social das diferenças entre homens e mulheres, ou da “alimentação da nação” propiciada pela “agricultura familiar”, depende de uma lógica específica de raciocínio, em cuja raiz há trajetórias e inte‑ rações socioestatais. É a operação de tal sociogênese no plano simbólico e a construção de capacidades cognitivas estatais a partir dela que es‑ peramos ter demonstrado. Em sua dimensão simbólica, as categorias que estruturaram políticas inovadoras tocam em valores e ideais sobe‑ jamente caros à autoimagem nacional. Daí, então, o pânico do público cívico e do contramovimento paraguaios diante de valores igualitários potencialmente implicados na noção de gênero. Bastante reais, tais pâ‑ nicos e disputas são eminentemente simbólicos, são disputas ao redor da fabricação sociopolítica não apenas das categorias pelas quais o Esta‑ do lê e reconhece a sociedade e suas partes, mas, simultaneamente, das formas pelas quais a sociedade é capaz de se conhecer e se reconhecer. Recebido para publicação em 11 de setembro de 2018. Aprovado para publicação em 5 de maio de 2019. NOVOS ESTUDOS CEBRAP 114, mai.–ago. 2019 pp. 411‑434 José Szwako [https://orcid.org/0000‑0002‑4764‑6533] é professor do programa de pós‑gradua ção em sociologia do Instituto de Estudos Sociais e Políticos da Universidade do Estado do Rio de Janeiro (Iesp‑Uerj) e bolsista Prociência (Uerj). Foi corresponsável pelos argumentos centrais e pela redação do artigo. Institucionalização simbólica: questões e agenda de pesquisa Adrian Gurza Lavalle [https://orcid.org/0000‑0002‑8998‑9833] é professor do departamento de ciência política da Universidade de São Paulo (usp) e pesquisador do Centro de Estudos da Metró‑ pole (cem) e do Cebrap. Foi corresponsável pelos argumentos centrais e pela redação do artigo. Novos estud. ❙❙ CEBRAP ❙❙ SÃO PAULO ❙❙ V38n02 ❙❙ 411-434 ❙❙ MAI.–AGO. 2019 431 Referências bibliográficas Sociological Theory, v. 12, n. 1, 1994, pp. 1‑18. g y pp Carlos, Euzeneia. Movimentos sociais e instituições participativas: efeitos organizacionais, relacionais e discursivos. Tese (Doutorado em Ciência Política) — usp, São Paulo, 2012. g Carlos, Euzeneia. Movimentos sociais e instituições participativas: efeitos organizacionais, relacionais e discursivos. Te Carneiro, Maria J. “Política pública e agricultura familiar: uma leitura do Pronaf”. Estudos Sociedade e Agricultura, 1997, pp. 70‑83. Carneiro, Maria J.; Danton, Thais. “Agricultura e biodiversidade nas ciências sociais brasileiras: alimentando a Carneiro, Maria J.; Danton, Thais. “Agricultura e biodiversidade nas ciências sociais brasileiras: alimentando a comunicação entre ciência e políticas públicas”. Sociologias, n. 30, 2012, pp. 252‑89. cde; Cladem; cmp. Informe Sombra cedaw: vigilancia ciudadana sobre los derechos humanos de las mujeres en Paraguay. Assunção, 2005. cde; Cladem; cmp. Informe Sombra cedaw: vigilancia ciudadana sobre los derechos humanos de las mujeres en Paraguay. Assunção, 2005. Chaves, Christine A. A marcha nacional dos sem‑terra: um estudo sobre a fabricação do social. Rio de Janeiro: Relume Dumará 2000 Chaves, Christine A. A marcha nacional dos sem‑terra: um estudo sobre a fabricação do social. Rio de Janeiro: Relume Dumará, 2000. Cingolani, Luciana. “The State of State Capacity”. Unumerit Working Paper Series, n. 53, Universidade de Maastricht, Maastricht, 2013, pp. 1‑52. pp Clemens, Elizabeth. The People’s Lobby. Organizational Innovation and the Rise of Interest Group Politics in the United States, pp Clemens, Elizabeth. The People’s Lobby. Organizational Innovation and the Rise of Interest Group Politics in the United States, 1890‑1925. Chicago: Chicago University Press, 1997. g g y ordinación de Mujeres del Paraguay (cmp). “Mujeres paraguayas camin Coordinación de Mujeres del Paraguay (cmp). “Mujeres paraguayas camino a Beijing, Kuña Paraguay Beijing Rapere — Documento No Gubernamental del Paraguay para el Foro Internacional sobre la Mujer de Beijing 95”. Mimeo. Assunção, 1994. cut/contag. Projeto Alternativo de Desenvolvimento Rural Sustentável. Mimeo, 1998. Disponível em: http://www.contag.org.br/imagens/f2215padrss.pdf. Acesso em: 15/7/2019. cut/contag. Projeto Alternativo de Desenvolvimento Rural Sustentável. Mimeo, 1998. Disponível em: contag. Projeto Alternativo de Desenvolvimento Rural Sustentável http://www.contag.org.br/imagens/f2215padrss.pdf. Acesso em: 15/7/2019. Dagnino, E. “Civil Society in Latin America”. In: Edwards, Michael (org.). The Oxford Handbook of Civil Society. Oxford: Oxford University Press, 2011. Dagnino, E.; Olvera, A.; Panfichi, A. “Para uma outra leitura da disputa pela construção democrática na América Latina”. In: A disputa pela construção democrática na América Latina. São Paulo/Campinas: Paz e Terra/Editora da Unicamp, 2006. p Douglas, Mary. Cómo piensan las instituciones. Referências bibliográficas Abers, Rebecca; Oliveira, Marília. “Nomeações políticas no Ministério do Meio Ambiente (2003‑2013): interco‑ nexões entre ongs, partidos e governos”. Opinião Pública, v. 21, 2015, pp. 336‑64. Abramovay, Ricardo; Veiga, José Eli. “Novas instituições para o desenvolvimento rural: o caso do Programa a ovay, ca do;Ve ga,Jo é ova t tu çõe pa a o de e vo v e to u a o ca o do og a a Nacional de Fortalecimento da Agricultura Familiar (Pronaf)”. Texto para discussão n. 641. Brasília: Ipea, 1999. Albuquerque, Maria do Carmo. “O movimento pelos direitos da criança e do adolescente e o controle social sobre a política socioeducativa: repertórios de interação socioestatal”. Política & Sociedade, v. 14, n. 29, 2015, pp. 31‑66. Albuquerque, Maria do Carmo. “O movimento pelos direitos da criança e do adolescente e o controle social sobre a política socioeducativa: repertórios de interação socioestatal”. Política & Sociedade, v. 14, n. 29, 2015, pp. 31‑66. Alonso, Angela. As teorias dos movimentos sociais: um balanço do debate . Lua Nova, n. 76, 2009, pp. 49‑86. Alvarez, Sonia et al. “Encontrando os feminismos latino‑americanos e caribenhos”. Revista Estudos Feministas, v. 11, n. 2, 2003, pp. 541‑75. Anderson, Benedict. Comunidades imaginadas: reflexão sobre a origem e a difusão do nacionalismo. São Paulo: Companhia das Letras, 2008. Andrade, Pedro G. et al. “Avaliação dos critérios de elegibilidade do Programa Territórios da Cidadania”. Revista Andrade, Pedro G. et al. “Avaliação dos critérios de elegibilidade do Programa Territórios da Cidadania”. Revis de Economia e Sociologia Rural, v. 54, n. 4, 2016, pp. 599‑614. de Economia e Sociologia Rural, v. 54, n. 4, 2016, pp. 599‑614. Benford, Robert; Snow, David. “Framing Processes and Social Movements: An Overview and Assessment”. Annual Review of Sociology, n. 26, 2000, pp. 611‑39. Berger, Peter; Luckmann, Thomas. A construção social da realidade. Petrópolis: Vozes, 2004. Berger, Peter; Luckmann, Thomas. A construção social da realidade. Petrópolis: Vozes, 2004. Berger, Peter; Luckmann, Thomas. A construção social da r Bichir, Renata; Brettas, Gabriela; Canato, Pamella. “Multi‑level Governance in Federal Contexts: the Social Assis‑ Bichir, Renata; Brettas, Gabriela; Canato, Pamella. “Multi‑level Governance in Federal Cont tance Policy in the City of São Paulo”. Brazilian Political Science Review, v. 11, n. 2, 2017. tance Policy in the City of São Paulo”. Brazilian Political Science Review, v. 11, n. 2, 2017. Bourdieu, Pierre; Wacquant, Loïc; Farage, Samar. “Rethinking the State: Genesis and Structure of the Bureaucratic Field”. Referências bibliográficas Grisa, Catia; Schneider, Sergio. “Três gerações de políticas públicas para a agricultura familiar e formas de intera‑ ção entre sociedade e Estado no Brasil”. Revista de Economia e Sociologia Rural, v. 52, 2014, pp. 125‑46. Gurza Lavalle, Adrian; Carlos, Euzeneia, Dowbor, Monika; Szwako, José (orgs.). Movimentos sociais e institucionaliza‑ Gurza Lavalle, Adrian; Carlos, Euzeneia, Dowbor, Monika; Szwako, José (orgs.). Movimentos sociais e institucionaliza‑ as sociais, raça e gênero no Brasil pós‑transição. Rio de Janeiro: Eduerj, 2019a. . “Movimentos sociais, institucionalização e domínios de agência”. In: Movimentos sociais e institucionalização: políticassociais raçaegêneronoBrasilpós transição RiodeJaneiro:Eduerj 2019b pp 21 86 . “Movimentos sociais, institucionalização e domínios de agência”. In: Movimentos sociais e institucionalização: políticas sociais, raça e gênero no Brasil pós‑transição. Rio de Janeiro: Eduerj, 2019b, pp. 21‑86. . “Movimentos sociais, institucionalização e domínios de agência”. In: Movimentos sociais e institucionalização: políticas sociais, raça e gênero no Brasil pós‑transição. Rio de Janeiro: Eduerj, 2019b, pp. 21‑86. Gurza Lavalle, Adrian; Castello, Graziella; Bichir, Renata M. “Protagonistas na sociedade civil: redes e centrali‑ dades de organizações civis em São Paulo”. Dados — Revista de Ciências Sociais, v. 50, n. 3, 2007, pp. 465‑98. Gurza Lavalle, Adrian; Szwako, José. “Sociedade civil, Estado e autonomia: argumentos, contra‑argumentos e avanços no debate”. Opinião Pública, v. 21, n. 1, 2015, pp. 157‑87. Gutierres, Kellen Alves. Projetos políticos, trajetórias e estratégias: a política de assistência social entre o partido e o Estado. Tese Gutierres, Kellen Alves. Projetos políticos, trajetórias e estratégias: a política de assistência social entre o partido e o Estado. Te Gutierres, Kellen Alves. Projetos políticos, trajetórias e estratégias: a política de (Doutorado em Ciências Sociais) — Unicamp, Campinas, 2015. (Doutorado em Ciências Sociais) — Unicamp, Campinas, 2015. Houtzager, Peter. Os últimos cidadãos: conflito e modernização no Brasil rural (1964‑1995). São Paulo: Globo, 2004. Houtzager, Peter. Os últimos cidadãos: conflito e modernização no Brasil rural (1964‑1995). São Paulo: Globo, 2004. Hoyler,Telma.“Produção habitacional via mercado:quem produz,como e onde?”.NovosEstudosCebrap,v.35,n Houtzager, Peter. Os últimos cidadãos: conflito e modernização no Brasil rural (1964‑1995). São Paulo: Globo, 2004. Hoyler, Telma. “Produção habitacional via mercado: quem produz, como e onde?”. Novos Estudos Cebrap, v. 35, n. 1, 2016, pp. 139‑57. Hoyler, Telma. “Produção habitacional via mercado: quem produz, como e onde?”. Novos Estudos Cebrap, v. 35, n. 1, 2016, pp. 139‑57. ibge. Censo Agropecuário 2006. Agricultura familiar: primeiros resultados — Brasil, grandes regiões e unidades da federação. Rio de Janeiro: ibge, 2009. ibge. Referências bibliográficas Censo Agropecuário 2006. Agricultura familiar: primeiros resultados — Brasil, grandes regiões e unidades da federação. Rio de Janeiro: ibge, 2009. Jay, Martin. Downcast Eyes: The Denigration of Vision in Twentieth‑Century French Thought. Berkeley: University of Ca‑ lifornia Press, 1993. Kula, Witold. Las medidas y los hombres. Cidade do México: Siglo xxi, 1980. Lindvall, Johannes; Teorel, Jan. State Capacity as Power: A Conceptual Framework. Lund: Lund University Publi‑ cations, 2017. Marques, Eduardo. “Redes sociais e poder no Estado brasileiro: aprendizados a partir de políticas urbanas”. Revista Brasileira de Ciências Sociais, v. 21, n. 60, 2006, pp. 15‑41. . “Government, Political Actors and Governance in Urban Policies in Brazil and São Paulo: concepts for a FutureResearchAgenda” BrazilianPoliticalScienceReview v 7 n 3 2013 pp 8‑35 , p Future Research Agenda”. Brazilian Political Science Review, v. 7, n. 3, 2013, pp. 8‑35. re Research Agenda”. Brazilian Political Science Review, v. 7, n. 3, 2013, pp. 8‑35 nez, Esperanza. “Derecho a Salud”. In: Codehupy. Informe de Derechos Humanos Mattei, Lauro. “Evolução do crédito do Pronaf para as categorias de agricultores familiares A e A/C entre 2000 e Mattei, Lauro. “Evolução do crédito do Pronaf para as categorias de ag 2010”. Revista Econômica do Nordeste, v. 45, n. 3, 2014, pp. 58‑69. McAdam, Doug. “The Framing Function of movement Tactics: Strategic Dramaturgy in the American Civil Rights Movement”. In: McAdam, Doug; McCarthy, John D.; Zald, Mayer N. (orgs.). Comparative Perspectives on Social Movements. Political Opportunities, Mobilizing Structures, and Cultural Framings. Cambridge: Cambridge Univer‑ sity Press, 1996, pp. 338‑55. Medeiros, Leonildes. “‘Sem terra’, ‘assentados’, ‘agricultores familiares’: considerações sobre os conflitos sociais e as formas de organização dos trabalhadores rurais brasileiros”. In: Giarracca, Norma (org.). ¿Una nueva ruralidad en América Latina?. Buenos Aires: Clacso, 2001, pp. 103‑28. g g ruralidad en América Latina?. Buenos Aires: Clacso, 2001, pp. 103‑28. ruralidad en América Latina?. Buenos Aires: Clacso, 2001, pp. 103‑28. Minarelli, Guilherme. “Planejamento urbano sob a ótica dos registros municipais de propriedade imobiliária: inefetividade, instrumentos e capacidades no caso de São Paulo”. Revista Brasileira de Ciências Sociais, no prelo. Monsma, Karl. “James C. Scott e a resistência cotidiana no campo: uma avaliação crítica”. Revista Brasileira de Infor‑ mação Bibliográfica em Ciências Sociais, Rio de Janeiro, n. 49, 2000, pp. 95‑121. Monsma, Karl. “James C. Scott e a resistência cotidiana no campo: uma avaliação crítica”. Revista Brasileira de Infor‑ mação Bibliográfica em Ciências Sociais, Rio de Janeiro, n. Referências bibliográficas Madri: Alianza, 1986. Dowbor, Monika. A arte da institucionalização: estratégias de mobilização dos sanitaristas (1974‑2006). Tese (Doutorado em Ciência Política) — usp, São Paulo, 2012. Durkheim, Émile; Mauss, Marcel. Primitive Classification. Londres: Cohen & West, 2009. auri, Carmen et al. Hacia una presencia diferente. Mujeres, organización y feminis fao/incra. Diretrizes de política agrária e desenvolvimento sustentável para a pequena produção familiar. Brasília: fao/incra, 1994. Favareto, Arilson. “Agricultores, trabalhadores: os trinta anos do novo sindicalismo rural no Brasil”. Revista Brasi‑ leira de Ciências Sociais, n. 62, out. 2006, pp. 27‑44. . “Tendências contemporâneas dos estudos e políticas sobre o desenvolvimento territorial”. In: Instituto . “Tendências contemporâneas dos estudos e políticas sobre o desenvolvimento territorial”. In: Instituto Interamericano de Cooperação para a Agricultura (org.). Políticas de desenvolvimento territorial rural no Brasil: avanços e desafios. Brasília: iica, 2010. Interamericano de Cooperação para a Agricultura (org.). Políticas de desenvolvimento territorial rural no Brasil: avanços e desafios. Brasília: iica, 2010. Flexor, Georges; Grisa, Catia. “Políticas de seguridad alimentaria y agricultura familiar en Brasil: actores, ideas e instituciones”. América Latina Hoy, v. 74, 2016, pp. 39‑53. instituciones”. América Latina Hoy, v. 74, 2016, pp. 39‑53. Giddens, Anthony. O Estado‑nação e a violência: segundo volume de uma crítica contemporânea ao materialismo histórico. São Paulo: Edusp, 2008. Goldman, Marcio. Como funciona a democracia. Rio de Janeiro: 7 Letras, 2006. Goldman, Marcio. Como funciona a democracia. Rio de Janeiro: 7 Letras, 2006. Goldman, Marcio. Como funciona a democracia. Rio de Janeiro: 7 Letras, 2006. 432 “Seeing like a social movement”: Institucionalização simbólica e capacidades estatais cognitivas ❙❙ José Szwako e Adrian Gurza Lavalle 432 Gomide, Alexandre; Boschi, Renato. Capacidades estatais em países emergentes: o Brasil em perspectiva comparada. Rio de Janeiro: Ipea, 2016. Gomide, Alexandre; Pereira, Karine; Machado, Raphael. “Apresentação: o conceito de capacidade estatal e a pes‑ i i ífi ” R i t S i d d C lt Gomide, Alexandre; Pereira, Karine; Machado, Raphael. “Apresentação: o conceito de capacidade estatal e a pes‑ quisa científica”. Revista Sociedade e Cultura, v. 20, n. 1, 2017, pp. 3‑12. quisa científica”. Revista Sociedade e Cultura, v. 20, n. 1, 2017, pp. 3‑12. Gomide, Alexandre; Pires, Roberto. Capacidades estatais e democracia: arranjos institucionais de políticas públicas. Rio de Janeiro: Ipea, 2014. Grisa, Catia. Políticas públicas para a agricultura familiar no Brasil: produção e institucionalização das ideias. Tese (Dou‑ torado em Ciências) — ufrrj, Rio de Janeiro, 2012. Referências bibliográficas 49, 2000, pp. 95‑121. mação Bibliográfica em Ciências Sociais, Rio de Janeiro, n. 49, 2000, pp. 95‑12 Multisetorial de Mulheres. “Críticas y sugestiones al capítulo 9: ‘promoción de la mujer’ del Plan Nacional de desarrollo económico y social 89/90”. Mimeo, 1989. Multisetorial de Mulheres. “Críticas y sugestiones al capítulo 9: ‘promoción de la mujer’ del Plan Nacional de desarrollo económico y social 89/90”. Mimeo, 1989. Narváez, Carmen. Sistematización de la experiencia del Comité Daia. Paraguay. 2012. Disponível em: <www.healthpo‑ licyproject.com/pubs/130_ParaguayDAIA.pdf>. Acesso em: 23/12/2017. Narváez, Carmen. Sistematización de la experiencia del Comité Daia. Paraguay. 2012. Disponível em: <www.healthpo‑ licyproject.com/pubs/130_ParaguayDAIA.pdf>. Acesso em: 23/12/2017. Narváez, Carmen. Sistematización de la experiencia del Comité Daia. Paraguay. 2012. Disponível em: <www.healthpo‑ licyproject.com/pubs/130_ParaguayDAIA.pdf>. Acesso em: 23/12/2017. onu. Informe de la Cuarta Conferencia Mundial sobre la Mujer, Beijing. A/CONF.177/20/Rev.1. Nova York: onu, 1995. Disponível em: <www.un.org/womenwatch/daw/beijing/pdf/Beijing%20full%20report%20S.pdf> Aces‑ so em: 23/12/2017. onu. Informe de la Cuarta Conferencia Mundial sobre la Mujer, Beijing. A/CONF.177/20/Rev.1. Nova York: onu, 1995. Disponível em: <www.un.org/womenwatch/daw/beijing/pdf/Beijing%20full%20report%20S.pdf> Aces‑ so em: 23/12/2017. Paraguai, República do. Plan Nacional de Igualdad de Oportunidades para las Mujeres 1997‑2001. Assunção: Secretaría de la Mujer, 1997a. Paraguai, República do. Plan Nacional de Igualdad de Oportunidades para las Mujeres 1997‑2001. Assunção: Secretaría de la Mujer, 1997a. . Plan Nacional de Salud Reproductiva — versión post‑Cairo. Assunção: Ministerio de Salud Pública, 1997b. . Plan Nacional de Salud Sexual y Reproductiva, 2003‑2008. Mimeo, 2003. Perissinotto, Renato; Szwako, José. “Movimentos sociais como teóricos políticos: Wolin, ideias e políticas públi‑ cas”. Lua Nova, n. 102, 2017, pp. 231‑63. Picolotto, Everton. As mãos que alimentam a nação: agricultura familiar, sindicalismo e política. Tese (Doutorado em Ciências) — ufrrj. Rio de Janeiro, 2011. Picolotto, Everton. As mãos que alimentam a nação: agricultura familiar, sindicalismo e política. Tese (Doutorado em Ciências) — ufrrj. Rio de Janeiro, 2011. . “Os atores da construção da categoria agricultura familiar no Brasil”. Revista de Economia e Sociologia Rural, v. 52, 2014, pp. 63‑84. . “Os atores da construção da categoria agricultura familiar no Brasil”. Revista de Economia e Sociologia Rural, v. 52, 2014, pp. 63‑84. Novos estud. ❙❙ CEBRAP ❙❙ SÃO PAULO ❙❙ V38n02 ❙❙ 411-434 ❙❙ MAI.–AGO. 2019 433 Romão Netto, José Veríssimo; Assumpção‑Rodrigues, Marta Maria. “Skill Formation, Cultural Policies, and Ins‑ titutional Hybridity: Bridging the Gap between Politics and Policies at Federal and State Levels in Brazil”. Cogent Social Sciences, v. 3, n. 3, 2017, pp. 1‑19. Disponível em: http://doi.org/10.1080/23311886.2017.1361601. Referências bibliográficas A / / Romão Netto, José Veríssimo; Assumpção‑Rodrigues, Marta Maria. “Skill Formation, Cultural Policies, and Ins‑ titutional Hybridity: Bridging the Gap between Politics and Policies at Federal and State Levels in Brazil”. Cogent Social Sciences, v. 3, n. 3, 2017, pp. 1‑19. Disponível em: http://doi.org/10.1080/23311886.2017.1361601. Acesso em: 23/12/2017. Romão Netto, José Veríssimo; Assumpção‑Rodrigues, Marta Maria. “Skill Formation, Cultural Policies, and Ins‑ titutional Hybridity: Bridging the Gap between Politics and Policies at Federal and State Levels in Brazil”. titutional Hybridity: Bridging the Gap between Politics and Policies at Federal and State Levels in Brazil . Cogent Social Sciences, v. 3, n. 3, 2017, pp. 1‑19. Disponível em: http://doi.org/10.1080/23311886.2017.1361601. Acesso em: 23/12/2017. Cogent Social Sciences, v. 3, n. 3, 2017, pp. 1‑19. Disponível em: http://doi.org/10.1080/23311886.2017.1361601. Acesso em: 23/12/2017. Rueschemeyer, Dietrich. “How and Why Ideas Matter”. In: Goodin, Robert E.; Tilly, Charles (orgs.). The Oxford Handbook of Contextual Political Analysis. Oxford: Oxford University Press, 2006, pp. 227‑50. Rueschemeyer, Dietrich. “How and Why Ideas Matter”. In: Goodin, Robert E.; Tilly, Charles (orgs.). The Oxford H db k f C t t lP liti lA l i O f d O f dU i it P 2006 227 50 Sabourin, Eric. “Evolução da política federal de desenvolvimento territorial no Brasil”. Novos Cadernos NAEA, v. 18, n. 1, 2014, pp. 123‑43. Schmidt, Vivian. “Taking Ideas and Discourse Seriously: Explaining Change through Discursive Institutionalism Schmidt, Vivian. “Taking Ideas and Discourse Seriously: Explaining Change through Discursive Inst as the Fourth ‘New Institutionalism’”. European Political Science Review, v. 2, n. 1, 2010, pp. 1‑25. h ‘New Institutionalism’”. European Political Science Review, v. 2, n. 1, 2010, p Schmitt, Claudia; Grisa, Catia. “Agroecologia, mercados e políticas públicas: uma análise a partir dos instrumen‑ Schmitt, Claudia; Grisa, Catia. Agroecologia, mercados e políticas públicas: uma análise a partir dos instrumen‑ tos de ação governamental”. In: Niederle, Paulo André; Almeida, Luciano de; Vezzani, Fabiane Machado (orgs.). Agroecologia: práticas, mercados e políticas para uma nova agricultura. Curitiba: Kairós, 2013, pp. 215‑65. Schneider Sergio;Mattei Lauro;Cazella Ademir “Histórico caracterização e dinâmica recente do Pronaf tos de ação governamental . In: Niederle, Paulo André; Almeida, Luciano de; Vezzani, Fabiane Machado (orgs.). Agroecologia: práticas, mercados e políticas para uma nova agricultura. Curitiba: Kairós, 2013, pp. 215‑65. Schneider,Sergio;Mattei,Lauro;Cazella,Ademir.“Histórico,caracterização e dinâmica recente do Pronaf — g (orgs.). Agroecologia: práticas, mercados e políticas para uma nova agricultura. Curitiba: Kairós, 2013, pp. 215‑65. Referências bibliográficas S h id S i M i L C ll Ad i “Hi ó i i ã di â i d P f (orgs.). Agroecologia: práticas, mercados e políticas para uma nova agricultura. Curitiba: Kairós, 2013, pp. 215‑65. Schneider, Sergio; Mattei, Lauro; Cazella, Ademir. “Histórico, caracterização e dinâmica recente do Pronaf — Programa Nacional de Fortalecimento da Agricultura Familiar (1995‑2003)”. In: Morizzi, Paulo Eduardo; Schneider, Sergio; Silva, Marcelo Kunrath. Políticas públicas e participação social no Brasil rural. Porto Alegre: Editora da ufrgs, 2004, pp. 21‑49. Programa Nacional de Fortalecimento da Agricultura Familiar (1995‑2003)”. In: Morizzi, Paulo Eduardo; Schneider, Sergio; Silva, Marcelo Kunrath. Políticas públicas e participação social no Brasil rural. Porto Alegre: Editora da ufrgs, 2004, pp. 21‑49. Scott, James. Seeing Like a State. Why Certain Schemes to Improve Human Condition Have Failed. New Haven/Lon‑ dres: Yale University Press, 1998. Skocpol, Theda. Protecting Soldiers and Mothers: The Political Origins of Social Policy in the United States. Cambridge, MA: Harvard University Press, 1992. Skocpol, Theda. Protecting Soldiers and Mothers: The Political Origins of Social Policy in the United States. Cambridge, MA: Harvard University Press, 1992. Souza Lima, Antônio C.; Castro, João. “Notas para uma abordagem antropológica da(s) política(s) pública(s)”. Anthropológicas, v. 26, n. 2, 2015, pp. 17‑54. Souza Lima, Antônio C.; Castro, João. “Notas para uma abordagem antropológica da(s) política(s) pública(s)”. Anthropológicas, v. 26, n. 2, 2015, pp. 17‑54. Szwako, José. “Del otro lado de la vereda: luta feminista e construção democrática no Paraguai pós‑ditatorial”. Tese ( ) (Doutorado em Ciências Sociais) — Unicamp, Campinas, 2012. . “O ‘mau desempenho’ de Lugo: gênero, religião e contramovimento na última destituição presidencial paraguaia”. Opinião Pública, v. 20, n. 1, 2014, pp. 132‑55. Teixeira, Carla; Souza Lima, Antonio C. “A antropologia da administração e da governança no Brasil: área temática ou ponto de dispersão?”. In: Duarte, Luiz F. D.; Martins, Carlos B. (orgs.): Horizontes das Ciências Sociais no Brasil: antropologia. São Paulo: Anpocs, 2010, pp. 51‑95. ou ponto de dispersão?”. In: Duarte, Luiz F. D.; Martins, Carlos B. (orgs.): Horizontes das Ciências Sociais no Brasil: antropologia. São Paulo: Anpocs, 2010, pp. 51‑95. Tilly, Charles. Coerção, capital e Estados europeus. São Paulo: Edusp, 1996. Tilly, Charles. Coerção, capital e Estados europeus. São Paulo: Edusp, 1996. Tilly, Charles; Tarrow, Sidney. Contentious Politics. Nova York: Oxford University Press, 2006. Walby, Sylvia. “Gender Mainstreaming: Productive Tensions in Theory and Practice”. Social Politics, v. 12, n. 3, 2005, pp. Referências bibliográficas 321‑43. Walby, Sylvia. “Gender Mainstreaming: Productive Tensions in Theory and Practice”. Social Politics, v. 12, n. 3, 2005, pp. 321‑43. Wanderley, Maria de Nazareth. “Raízes históricas do campesinato brasileiro”. xx Encontro Anual da Anpocs. Caxambu, 1996. Wesz, Valdemar. “A trajetória do Pronaf Agroindústria no Brasil (1998/99‑2016/17): um olhar sobre as condições de financiamento e o público beneficiado”. In: Maluf, Renato S.; Flexor, Georges (orgs.). Questões agrárias, agrícolas e rurais: conjunturas e políticas públicas. 1. ed. Rio de Janeiro: E‑Papers, 2017. Wesz, Valdemar. “A trajetória do Pronaf Agroindústria no Brasil (1998/99‑2016/17): um olhar sobre as condições de financiamento e o público beneficiado”. In: Maluf, Renato S.; Flexor, Georges (orgs.). Questões agrárias, agrícolas e rurais: conjunturas e políticas públicas. 1. ed. Rio de Janeiro: E‑Papers, 2017. i pi , ; , g ( g ) Q g , agrícolas e rurais: conjunturas e políticas públicas. 1. ed. Rio de Janeiro: E‑Papers, 2017. ícolas e rurais: conjunturas e políticas públicas. 1. ed. Rio de Janeiro: E‑Papers, 20
https://openalex.org/W3006111520	https://jmir.org/api/download?alt_name=mhealth_v8i4e14969_app3.pdf&filename=2e6693c8eb7d0e7c14d2928f3f890ae4.pdf	English	null	A Social Group-Based Information-Motivation-Behavior Skill Intervention to Promote Acceptability and Adoption of Wearable Activity Trackers Among Middle-Aged and Older Adults: Cluster Randomized Controlled Trial	JMIR mhealth and uhealth	2,020	cc-by	16,758	CONSORT-EHEALTH (V 1.6.1) - Submission/Publication Form The CONSORT-EHEALTH checklist is intended for authors of randomized trials evaluating web-based and Internet-based applications/interventions, including mobile interventions, electronic games (incl multiplayer games), social media, certain telehealth applications, and other interactive and/or networked electronic applications. Some of the items (e.g. all subitems under item 5 - description of the intervention) may also be applicable for other study designs. The goal of the CONSORT EHEALTH checklist and guideline is to be The goal of the CONSORT EHEALTH checklist and guideline is to be a) a guide for reporting for authors of RCTs, The goal of the CONSORT EHEALTH checklist and a) a guide for reporting for authors of RCTs, The goal of the CONSORT EHEALTH checklist and guideline is a) a guide for reporting for authors of RCTs, b) to form a basis for appraisal of an ehealth trial (in terms of validity) CONSORT-EHEALTH items/subitems are MANDATORY reporting items for studies published in the Journal of Medical Internet Research and other journals / scientific societies endorsing the checklist. Items numbered 1., 2., 3., 4a., 4b etc are original CONSORT or CONSORT-NPT (non- pharmacologic treatment) items. Items with Roman numerals (i., ii, iii, iv etc.) are CONSORT-EHEALTH i / l ifi i Items with Roman numerals (i., ii, iii, iv etc.) are CONSORT-EHEALTH extensions/clarifications. As the CONSORT-EHEALTH checklist is still considered in a formative stage, we would ask that you also RATE ON A SCALE OF 1-5 how important/useful you feel each item is FOR THE PURPOSE OF THE CHECKLIST and reporting guideline (optional). Mandatory reporting items are marked with a red . In the textboxes, either copy & paste the relevant sections from your manuscript into this form - please include any quotes from your manuscript in QUOTATION MARKS, or answer directly by providing additional information not in the manuscript, or elaborating on why the item was not relevant for this study. YOUR ANSWERS WILL BE PUBLISHED AS A SUPPLEMENTARY FILE TO YOUR PUBLICATION IN JMIR AND ARE CONSIDERED PART OF YOUR PUBLICATION (IF ACCEPTED). i Please fill in these questions diligently. Information will not be copyedited, so please use proper spelling and grammar, use correct capitalization, and avoid abbreviations. DO NOT FORGET TO SAVE AS PDF _AND_ CLICK THE SUBMIT BUTTON SO YOUR ANSWERS ARE IN OUR DATABASE !!! Citation Suggestion (if you append the pdf as Appendix we suggest to cite this paper in the caption): Mobile Health Interventions J Med Internet Res 2011;13(4):e126 URL: http://www.jmir.org/2011/4/e126/ doi: 10.2196/jmir.1923 PMID: 22209829 必填 Your name * First Last Jing Liao Primary Affiliation (short), City, Country * University of Toronto, Toronto, Canada Sun Yat-sen University, Guangzhou, China Your e-mail address * abc@gmail.com liaojing5@mail.sysu.edu.cn Title of your manuscript * Provide the (draft) title of your manuscript. Social-Group Based Deployment Intervention to Promote Acceptability and Adoption of Wearable Activity Trackers among Mid- and Older- Aged Adults: A Cluster Randomized Controlled Trial Name of your App/Software/Intervention * If there is a short and a long/alternate name, write the short name first and add the long name in brackets. Mobile Health Interventions J Med Internet Res 2011;13(4):e126 URL: http://www.jmir.org/2011/4/e126/ doi: 10.2196/jmir.1923 PMID: 22209829 Evaluated Version (if any) e.g. "V1", "Release 2017-03-01", "Version 2.0.27913" Evaluated Version (if any) e.g. "V1", "Release 2017-03-01", "Version 2.0.27913" Evaluated Version (if any) e.g. "V1", "Release 2017-03-01", "Version 2.0.27913" Accessibility * Approximately Daily Approximately Weekly Approximately Monthly Approximately Yearly "as needed" 其他: Primary Medical Indication/Disease/Condition * e.g. "Stress", "Diabetes", or define the target group in brackets after the condition, e.g. "Autism (Parents of children with)", "Alzheimers (Informal Caregivers of)" Not applicable. Primary Outcomes measured in trial * comma-separated list of primary outcomes reported in the trial The current study focused on the process Secondary/other outcomes Are there any other outcomes the intervention is expected to affect? 您的回答 Recommended "Dose" * What do the instructions for users say on how often the app should be used? Primary Medical Indication/Disease/Condition * e.g. "Stress", "Diabetes", or define the target group in brackets after the condition, e.g "Autism (Parents of children with)", "Alzheimers (Informal Caregivers of)" Primary Medical Indication/Disease/Condition * e.g. "Stress", "Diabetes", or define the target group in brackets after the condition, e.g. "Autism (Parents of children with)", "Alzheimers (Informal Caregivers of)" Not applicable. unknown / not evaluated 0-10% 11-20% 21-30% 31-40% 41-50% 51-60% 61-70% 71%-80% 81-90% 91-100% 其他: Approx. Percentage of Users (starters) still using the app as recommended after 3 months * Approx. Percentage of Users (starters) still using the app as recommended after 3 months * yes: all primary outcomes were significantly better in intervention group vs control partly: SOME primary outcomes were significantly better in intervention group vs control no statistically significant difference between control and intervention potentially harmful: control was significantly better than intervention in one or more outcomes inconclusive: more research is needed 其他: not submitted yet - in early draft status not submitted yet - in late draft status, just before submission submitted to a journal but not reviewed yet submitted to a journal and after receiving initial reviewer comments submitted to a journal and accepted, but not published yet published 其他: Overall, was the app/intervention effective? * Article Preparation Status/Stage * At which stage in your article preparation are you currently (at the time you fill in this form) TITLE AND ABSTRACT 1a) TITLE: Identification as a randomized trial in the title yes 其他: 1a) Does your paper address CONSORT item 1a? * I.e does the title contain the phrase "Randomized Controlled Trial"? (if not, explain the reason under "other") 1a-i) Identify the mode of delivery in the title Identify the mode of delivery. Preferably use “web-based” and/or “mobile” and/or “electronic game” in the title. Avoid ambiguous terms like “online”, “virtual”, “interactive”. Use “Internet-based” only if Intervention includes non-web-based Internet components (e.g. email), use “computer-based” or “electronic” only if offline products are used. Use “virtual” only in the context of “virtual reality” (3-D worlds). Use “online” only in the context of “online support groups”. Complement or substitute product names with broader terms for the class of products (such as “mobile” or “smart phone” instead of “iphone”), especially if the application runs on different platforms. subitem not at all important 1 2 3 4 5 essential subitem not at all important 1 2 3 4 5 essential subitem not at all important 1 2 3 4 5 essential Journal * If you already know where you will submit this paper (or if it is already submitted), please provide the journal name (if it is not JMIR, provide the journal name under "other") not submitted yet / unclear where I will submit this Journal of Medical Internet Research (JMIR) p g If this is a JMIR submission, please provide the manuscript tracking number under "other" (The ms tracking number can be found in the submission acknowledgement email, or when you login as author in JMIR. If the paper is already published in JMIR, then the ms tracking number is the four-digit number at the end of the DOI, to be found at the bottom of each published article in JMIR) no ms number (yet) / not (yet) submitted to / published in JMIR 其他: JMU ms#14969 Does your paper address subitem 1a-ii? Does your paper address subitem 1a-ii? Copy and paste relevant sections from manuscript title (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study Yes, our title includes non-web-based components as "Social-Group Based Deployment Intervention". subitem not at all important 1 2 3 4 5 essential 1a-ii) Non-web-based components or important co- interventions in title Mention non-web-based components or important co-interventions in title, if any (e.g., “with telephone support”). Mention non-web-based components or important co-interventions in title, if any (e.g., “with telephone support”). Does your paper address subitem 1a-i? * Copy and paste relevant sections from manuscript title (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study Yes, we specify in our title the mode of delivery as “Wearable Activity Trackers”. 1b-i) Key features/functionalities/components of the intervention and comparator in the METHODS section of the ABSTRACT Mention key features/functionalities/components of the intervention and comparator in the abstract. If possible, also mention theories and principles used for designing the site. Keep in mind the needs of systematic reviewers and indexers by including important synonyms. (Note: Only report in the abstract what the main paper is reporting. If this information is missing from the main body of text, consider adding it) subitem not at all important 1 2 3 4 5 essential subitem not at all important 1 2 3 4 5 essential Does your paper address subitem 1a-iii? * y p p Copy and paste relevant sections from manuscript title (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study Yes, we highlight our target group as "Mid and older- Aged Adults" in the title. 1b) ABSTRACT: Structured summary of trial design, methods, results, and conclusions NPT extension: Description of experimental treatment, comparator, care providers, centers, and blinding status. Does your paper address subitem 1b-i? * y p p Copy and paste relevant sections from the manuscript abstract (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study Yes, we state in the methods section of the abstract as " These dancing groups were randomized 1:1 into two arms, and both received wrist-worn activity trackers and instructions at the baseline face-to-face assessment. Based on the Information-Motivation-Behavior Skill framework, the intervention arm was in addition given a tutorial on the purpose of exercise monitoring (Information), encouraged to compete in exercise and share exercise records with their dancing peers (Motivation), and further assisted with the use of activity tracker (Behavior Skill)." 1b-ii) Level of human involvement in the METHODS section of the ABSTRACT Clarify the level of human involvement in the abstract, e.g., use phrases like “fully automated” vs. “therapist/nurse/care provider/physician-assisted” (mention number and expertise of providers involved, if any). (Note: Only report in the abstract what the main paper is reporting. If this information is missing from the main body of text, consider adding it) subitem not at all important 1 2 3 4 5 essential Does your paper address subitem 1b-ii? Does your paper address subitem 1b ii? Copy and paste relevant sections from the manuscript abstract (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study Yes, we specify the level of human involvement in the methods section of the abstract as “Based on the Information-Motivation-Behavior Skill framework, the intervention arm was in addition given a tutorial on the purpose of exercise monitoring (Information), encouraged to compete in exercise and share exercise records with their dancing peers (Motivation), and further assisted with the use of activity tracker (Behavior Skill)." 1b-iii) Open vs. closed, web-based (self-assessment) vs. face-to-face assessments in the METHODS section of the ABSTRACT Mention how participants were recruited (online vs. offline), e.g., from an open access website or from a clinic or a closed online user group (closed usergroup trial), and clarify if this was a purely web-based trial, or there were face-to-face components (as part of the intervention or for assessment). Clearly say if outcomes were self-assessed through questionnaires (as common in web-based trials). Note: In traditional offline trials, an open trial (open-label trial) is a type of clinical trial in which both the researchers and participants know which treatment is being administered. To avoid confusion, use “blinded” or “unblinded” to indicated the level of blinding instead of “open”, as “open” in web-based trials usually refers to “open access” (i.e. participants can self-enrol). (Note: Only report in the abstract what the main paper is reporting. If this information is missing from the main body of text, consider adding it) subitem not at all important 1 2 3 4 5 essential subitem not at all important 1 2 3 4 5 essential subitem not at all important 1 2 3 4 5 essential Does your paper address subitem 1b-iii? y p p Copy and paste relevant sections from the manuscript abstract (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study We state our methods part as “These dancing groups were randomized 1:1 into two arms, which both received wrist-worn activity trackers and instructions at the baseline face-to-face assessment.” 1b-iv) RESULTS section in abstract must contain use data 1b-iv) RESULTS section in abstract must contain use data Report number of participants enrolled/assessed in each group, the use/uptake of the intervention (e.g., attrition/adherence metrics, use over time, number of logins etc.), in addition to primary/secondary outcomes. (Note: Only report in the abstract what the main paper is reporting. If this information is missing from the main body of text, consider adding it) Does your paper address subitem 1b-iv? Copy and paste relevant sections from the manuscript abstract (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study We address this point in the result section of the abstract as "All dancing groups were followed up for the post-intervention reassessment, with 61 out of 69 participants (88%) of the intervention arm (seven groups) and 56 out of 80 participants (70%) of the control arm (six group). Participants’ sociodemographic characteristics (mean age 62 years, retired) and health status were comparable between two arms, except for the intervention arm had fewer female participants and lower cognitive test scores. Our intervention significantly increased participants’ overall acceptability by 6.8 points (95% CI 2.2 to 11.4), mainly driven by promoted motivation (adjusted group difference 2.0, 95% CI: 0.5 to 3.6), increased usefulness (adjusted group difference 2.5, 95% CI: 0.9 to 4.1) and better perceived ease-of-use (adjusted group difference 1.2, 95%CI 0.1 to 2.4); but not enjoyment and comfort (adjusted group difference 0.9, 95% CI -0.4 to 2.3). Higher adoption was also showed among participants of the intervention arm, who were twice more likely to have valid daily step account data than their controlled counterparts (adjusted Incidence Relative Risk (IRR) 2.0, 95% CI: 1.2 to 3.3). The average daily step counts, 7803 vs 5653 steps/day for the intervention and the control respectively, were similar between two arms (adjusted IRR=1.4, 95% CI: 0.7 to 2.5)." 1b-v) CONCLUSIONS/DISCUSSION in abstract for negative trials Conclusions/Discussions in abstract for negative trials: Discuss the primary outcome - if the trial is negative (primary outcome not changed), and the intervention was not used, discuss whether negative results are attributable to lack of uptake and discuss reasons. (Note: Only report in the abstract what the main paper is reporting. If this information is missing from the main body of text, consider adding it) subitem not at all important 1 2 3 4 5 essential INTRODUCTION 2a) In INTRODUCTION: Scientific background and explanation of rationale 2a) In INTRODUCTION: Scientific background and explanation of rationale Does your paper address subitem 1b-v? Copy and paste relevant sections from the manuscript abstract (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study 您的回答 2a-i) Problem and the type of system/solution ) y y Describe the problem and the type of system/solution that is object of the study: intended as stand-alone intervention vs. incorporated in broader health care program? Intended for a particular patient population? Goals of the intervention, e.g., being more cost-effective to other interventions, replace or complement other solutions? (Note: Details about the intervention are provided in “Methods” under 5) subitem not at all important 1 2 3 4 5 essential subitem not at all important 1 2 3 4 5 essential 2a-ii) Scientific background, rationale: What is known about the (type of) system Scientific background, rationale: What is known about the (type of) system that is the object of the study (be sure to discuss the use of similar systems for other conditions/diagnoses, if appropiate), motivation for the study, i.e. what are the reasons for and what is the context for this specific study, from which stakeholder viewpoint is the study performed, potential impact of findings [2]. Briefly justify the choice of the comparator. Does your paper address subitem 2a-ii? * y p p Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study We reviewed what have done previously in Paragraph 3 (Intro). We reviewed what have done previously in Paragraph 3 (Intro). 2b) In INTRODUCTION: Specific objectives or hypotheses Does your paper address CONSORT subitem 2b? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study Does your paper address subitem 2a-i? * Does your paper address subitem 2a-i? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study We outlined in the first two paragraphs of the Introduction the ever- increasing use of mobile technology in health care, and the digital divide considering the limited use of mobile technology among older adults. Through reviewing previous literature on improving technology acceptability and adoption among this population (Paragraph 3, Intro), we proposed our theory-based intervention (Paragraph 4, Intro). METHODS 3a) Description of trial design (such as parallel, factorial) including allocation ratio subitem not at all important 1 2 3 4 5 essential 4a) Eligibility criteria for participants 3b-i) Bug fixes, Downtimes, Content Changes Bug fixes, Downtimes, Content Changes: ehealth systems are often dynamic systems. A description of changes to methods therefore also includes important changes made on the intervention or comparator during the trial (e.g., major bug fixes or changes in the functionality or content) (5-iii) and other “unexpected events” that may have influenced study design such as staff changes, system failures/downtimes, etc. [2]. Does your paper address subitem 3b-i? Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study 您的回答 3b-i) Bug fixes, Downtimes, Content Changes Bug fixes, Downtimes, Content Changes: ehealth systems are often dynamic systems. A description of changes to methods therefore also includes important changes made on the intervention or comparator during the trial (e.g., major bug fixes or changes in the functionality or content) (5-iii) and other “unexpected events” that may have influenced study design such as staff changes, system failures/downtimes, etc. [2]. subitem not at all important 1 2 3 4 5 essential Does your paper address CONSORT subitem 3a? * Does your paper address CONSORT subitem 3a? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study We introduced our trial design as “This phase 1 report covers the trial conducted between November 2017 and October 2018, consisting of seven-month recruitment and baseline assessment, three-month intervention, and the post-intervention reassessment. While phase 2 focuses on health outcomes, this interim study prioritizes implementation outcomes. The study was carried out in Guangzhou, the capital city of Guangdong province, China. The trial was developed guided by the CONSORT-EHEALTH Checklist and CONSORT-Checklist for reporting a cluster randomized controlled trial” The allocation ratio was stated in “Random Allocation” section as " All random allocation was done at the cluster level, namely, by the square-dancing groups. After the recruitment of all eligible participants, a statistician otherwise not associated with the project allocated participants by their square- dancing groups equally into two arms (1:1), following a simple randomization process. Although the participants were aware of the interventional nature of the study, they were blinded to their allocation status. Outcome assessors were blinded to the group assignments and were different from researchers who conducted and monitored the interventions." 3b) Important changes to methods after trial commencement (such as eligibility criteria), with reasons Does your paper address CONSORT subitem 3b? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study There was no important changes to the methods after trial There was no important changes to the methods after trial commencement. subitem not at all important 1 2 3 4 5 essential 4a-i) Computer / Internet literacy Computer / Internet literacy is often an implicit “de facto” eligibility criterion - this should be explicitly clarified. Does your paper address subitem 4a-i? Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study 您的回答 subitem not at all important 1 2 3 4 5 essential 4a-i) Computer / Internet literacy Computer / Internet literacy is often an implicit “de facto” eligibility criterion - this should be explicitly clarified. Does your paper address subitem 4a-i? Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study 您的回答 subitem not at all important 1 2 3 4 5 essential 4a-i) Computer / Internet literacy Computer / Internet literacy is often an implicit “de facto” eligibility criterion - this should be explicitly clarified. 1 2 3 4 5 4a-i) Computer / Internet literacy Computer / Internet literacy is often an implicit “de facto” eligibility criterion - this should be explicitly clarified. 4a-i) Computer / Internet literacy Computer / Internet literacy is often an implicit “de facto” eligibility criterion - this should be explicitly clarified. subitem not at all important 1 2 3 4 5 essential Does your paper address CONSORT subitem 4a? * Does your paper address CONSORT subitem 4a? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study Participants’ eligibility was described as " Square-dancing groups regularly practicing in those selected places were used as the sampling frame. Dancing groups were eligible for the current study if the dancing style was not ballroom dance, and the group total size was no less than twenty with more than half of the dancers aged 45 years and older. Square dancers of the eligible dancing group, who were 1) community- residents of Guangzhou; 2) regularly practiced square dancing at least once per week in the past 12 months; 3) aged 45 years and older; and 4) agreed to participate in our study if recruited. Participants were excluded if they 1) had serious and uncontrolled diseases related to heart, brain, lung, liver and kidney or any acute complications; and 2) had no smart- phone devices (as the data recorded by the wearable activity trackers can only be uploaded to the cloud via the paired smart-phone devices)". Does your paper address subitem 3b-i? Does your paper address subitem 3b-i? Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study 您的回答 4a) Eligibility criteria for participants Does your paper address subitem 4a-i? Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study 您的回答 4a-ii) Open vs. closed, web-based vs. face-to-face assessments: Open vs. closed, web-based vs. face-to-face assessments: Mention how participants were recruited (online vs. offline), e.g., from an open access website or from a clinic, and clarify if this was a purely web-based trial, or there were face-to-face components (as part of the intervention or for assessment), i.e., to what degree got the study team to know the participant. In online-only trials, clarify if participants were quasi-anonymous and whether having multiple identities was possible or whether technical or logistical measures (e.g., cookies, email confirmation, phone calls) were used to detect/prevent these. subitem not at all important 1 2 3 4 5 essential subitem not at all important 1 2 3 4 5 essential subitem not at all important 1 2 3 4 5 essential Does your paper address subitem 4a-ii? * y p p Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study We described our recruitment and assessments strategies as “Major public squares and parks of three old districts of Guangzhou, namely Yuexiu, Haizhu and Liwan, were identified via online map (i.e. Baidu map), considering their land area and visitor flow, and sit in residential areas. Using a restricted randomizing sampling approach, eight squares and parks per district were chosen at random. We recruited participant in the selected square and parks, using advertisements and flyers." and " Participants were initially screened for eligibility via onsite interviews, and eligible participants were invited for health checkups at the local community health centers on a scheduled date." 4a-iii) Information giving during recruitment 4a-iii) Information giving during recruitment Information given during recruitment. Specify how participants were briefed for recruitment and in the informed consent procedures (e.g., publish the informed consent documentation as appendix, see also item X26), as this information may have an effect on user self-selection, user expectation and may also bias results. subitem not at all important 1 2 3 4 5 essential subitem not at all important subitem not at all important Does your paper address subitem 4b-i? * Does your paper address subitem 4b-i? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study Data collection methods were described as the answer above. Data collection methods were described as the answer above. 4b-ii) Report how institutional affiliations are displayed Report how institutional affiliations are displayed to potential participants [on ehealth media], as affiliations with prestigious hospitals or universities may affect volunteer rates, use, and reactions with regards to an intervention.(Not a required item – describe only if this may bias results) Does your paper address subitem 4a-iii? Does your paper address subitem 4a-iii? Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study Informed consent was obtained from our participants, as stated in the article "All participants read and signed the written informed consent approved by the Institutional Review Board (No. L2016-004) of the School of Public Health of Sun Yat-sen University." 4b) Settings and locations where the data were collected 4b) Settings and locations where the data were collected Does your paper address CONSORT subitem 4b? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study We address the data collection method as “Control variables were collected during the recruitment and baseline health checkup prior to the intervention by investigators and clinical staff who were blinded to the intervention assignment. During the intervention, data on participants’ daily physical activity level (e.g. step counts) were automatically recorded and uploaded by the wearable activity trackers and their paired smart phones, as indicators for adoption. At the post-intervention assessment, participants evaluated their satisfaction with the activity trackers by the 14-item questionnaire, as an indicator for acceptability.” subitem not at all important 1 2 3 4 5 essential subitem not at all important 1 2 3 4 5 essential 5) The interventions for each group with sufficient details to allow replication, including how and when they were actually administered 4b-i) Report if outcomes were (self-)assessed through online questionnaires Clearly report if outcomes were (self-)assessed through online questionnaires (as common in web-based trials) or otherwise. Does your paper address subitem 4b-i? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study Data collection methods were described as the answer above. 4b-ii) Report how institutional affiliations are displayed Report how institutional affiliations are displayed to potential participants [on ehealth media], as affiliations with prestigious hospitals or universities may affect volunteer rates, use, and reactions with regards to an intervention.(Not a required item – describe only if this may bias results) Does your paper address subitem 4b-ii? Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study 您的回答 Clearly report if outcomes were (self-)assessed through online questionnaires (as common in web-based trials) or otherwise. Does your paper address subitem 4b-ii? Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study 您的回答 5-i) Mention names, credential, affiliations of the developers, sponsors, and owners Mention names, credential, affiliations of the developers, sponsors, and owners [6] (if authors/evaluators are owners or developer of the software, this needs to be declared in a “Conflict of interest” section or mentioned elsewhere in the manuscript). subitem not at all important 1 2 3 4 5 essential Does your paper address subitem 5-i? Does your paper address subitem 5-i? Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study We provided detailed information on our intervention components in the Intervention & Procedures section of the Methods. 5-ii) Describe the history/development process Describe the history/development process of the application and previous formative evaluations (e.g., focus groups, usability testing), as these will have an impact on adoption/use rates and help with interpreting results. Does your paper address subitem 5-ii? y p p Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study 您的回答 Does your paper address subitem 5-iii? Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study 5 iv) Quality assurance methods Provide information on quality assurance methods to ensure accuracy and quality of information provided [1], if applicable. subitem not at all important 1 2 3 4 5 essential Does your paper address subitem 5-iii? Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study 您的回答 5-iv) Quality assurance methods Provide information on quality assurance methods to ensure accuracy and quality of information provided [1], if applicable. Does your paper address subitem 5-iv? Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study 您的回答 Does your paper address subitem 5-iii? 5-iii) Revisions and updating Revisions and updating. Clearly mention the date and/or version number of the application/intervention (and comparator, if applicable) evaluated, or describe whether the intervention underwent major changes during the evaluation process, or whether the development and/or content was “frozen” during the trial. Describe dynamic components such as news feeds or changing content which may have an impact on the replicability of the intervention (for unexpected events see item 3b). subitem not at all important 1 2 3 4 5 essential subitem not at all important 1 2 3 4 5 essential 5-vi) Digital preservation Digital preservation: Provide the URL of the application, but as the intervention is likely to change or disappear over the course of the years; also make sure the intervention is archived (Internet Archive, webcitation.org, and/or publishing the source code or screenshots/videos alongside the article). As pages behind login screens cannot be archived, consider creating demo pages which are accessible without login. subitem not at all important 1 2 3 4 5 essential subitem not at all important 1 2 3 4 5 essential Does your paper address subitem 5-v? Does your paper address subitem 5-v? Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study subitem not at all important 1 2 3 4 5 essential subitem not at all important 1 2 3 4 5 essential 5-v) Ensure replicability by publishing the source code, and/or providing screenshots/screen-capture video, and/or providing flowcharts of the algorithms used Ensure replicability by publishing the source code, and/or providing screenshots/screen- capture video, and/or providing flowcharts of the algorithms used. Replicability (i.e., other researchers should in principle be able to replicate the study) is a hallmark of scientific reporting. Does your paper address subitem 5-v? Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study 您的回答 5-vi) Digital preservation Digital preservation: Provide the URL of the application, but as the intervention is likely to change or disappear over the course of the years; also make sure the intervention is archived (Internet Archive, webcitation.org, and/or publishing the source code or screenshots/videos alongside the article). As pages behind login screens cannot be archived, consider creating demo pages which are accessible without login. Does your paper address subitem 5-vi? Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study 您的回答 5-v) Ensure replicability by publishing the source code, and/or providing screenshots/screen-capture video, and/or providing flowcharts of the algorithms used Ensure replicability by publishing the source code, and/or providing screenshots/screen- capture video, and/or providing flowcharts of the algorithms used. Replicability (i.e., other researchers should in principle be able to replicate the study) is a hallmark of scientific reporting. Ensure replicability by publishing the source code, and/or providing screenshots/screen- capture video, and/or providing flowcharts of the algorithms used. Replicability (i.e., other researchers should in principle be able to replicate the study) is a hallmark of scientific reporting. subitem not at all important 1 2 3 4 5 essential subitem not at all important 1 2 3 4 5 essential Does your paper address subitem 5-iv? y p p Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study 您的回答 Does your paper address subitem 5-vi? Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study 您的回答 Does your paper address subitem 5-vii? * We described the use of the wearable activity trackers in our study as "Both the intervention and control arms were equipped with wrist-worn activity trackers free of charge at the baseline assessment." 5-vii) Access Access: Describe how participants accessed the application, in what setting/context, if they had to pay (or were paid) or not, whether they had to be a member of specific group. If known, describe how participants obtained “access to the platform and Internet” [1]. To ensure access for editors/reviewers/readers, consider to provide a “backdoor” login account or demo mode for reviewers/readers to explore the application (also important for archiving purposes, see vi). subitem not at all important 1 2 3 4 5 essential Does your paper address subitem 5-vii? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study Does your paper address subitem 5-viii? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study We provided detailed information on the mode of delivery, the theory- based intervention components in the Intervention & Procedures section of the Method, and in Figure 1. 5-ix) Describe use parameters Describe use parameters (e.g., intended “doses” and optimal timing for use). Clarify what instructions or recommendations were given to the user, e.g., regarding timing, frequency, heaviness of use, if any, or was the intervention used ad libitum. subitem not at all important 1 2 3 4 5 essential 5-viii) Mode of delivery, p Describe mode of delivery, features/functionalities/components of the intervention and comparator, and the theoretical framework [6] used to design them (instructional strategy [1], behaviour change techniques, persuasive features, etc., see e.g., [7, 8] for terminology). This includes an in-depth description of the content (including where it is coming from and who developed it) [1],” whether [and how] it is tailored to individual circumstances and allows users to track their progress and receive feedback” [6]. This also includes a description of communication delivery channels and – if computer- mediated communication is a component – whether communication was synchronous or asynchronous [6]. It also includes information on presentation strategies [1], including page design principles, average amount of text on pages, presence of hyperlinks to other resources, etc. [1]. subitem not at all important 1 2 3 4 5 essential subitem not at all important subitem not at all important Does your paper address subitem 5-ix? y p p Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study Information on use parameters were given in the Intervention & Procedures section of Methods, for instance as " Participants at both arms received 30-minute demonstration on the core functions of these activity trackers (e.g. how to wear, read the displays and charge the trackers), and were instructed to wear them over the entire day until going to bed." Does your paper address subitem 5-x? We specified the level of human involvement for each component we used in our intervention. For instance, for the set-up and troubleshooting of wearable activity trackers we stated "One project facilitator was assigned to assist one dancing group, who conducted the initial set-ups, provided troubleshooting, and monitored participants’ uploaded PA data via the managerial accounts." 5-x) Clarify the level of human involvement ) Clarify the level of human involvement (care providers or health professionals, also technical assistance) in the e-intervention or as co-intervention (detail number and expertise of professionals involved, if any, as well as “type of assistance offered, the timing and frequency of the support, how it is initiated, and the medium by which the assistance is delivered”. It may be necessary to distinguish between the level of human involvement required for the trial, and the level of human involvement required for a routine application outside of a RCT setting (discuss under item 21 – generalizability). subitem not at all important 1 2 3 4 5 essential subitem not at all important 1 2 3 4 5 essential subitem not at all important Does your paper address subitem 5-x? Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study 5-xi) Report any prompts/reminders used 5-xi) Report any prompts/reminders used Report any prompts/reminders used: Clarify if there were prompts (letters, emails, phone calls, SMS) to use the application, what triggered them, frequency etc. It may be necessary to distinguish between the level of prompts/reminders required for the trial, and the level of prompts/reminders for a routine application outside of a RCT setting (discuss under item 21 – generalizability). Does your paper address subitem 5-xi? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study For the intervention arm, we specified our reminders as “Six booster educational voice-messages on PA related topics such as warming up & relaxing exercises were delivered twice a month via participants’ WeChat groups (defined below). These messages consisted of a one-minute voice message, along with the corresponding transcript and illustrations." 5-xii) Describe any co-interventions (incl. training/support) Describe any co-interventions (incl. training/support): Clearly state any interventions that are provided in addition to the targeted eHealth intervention, as ehealth intervention may not be designed as stand-alone intervention. This includes training sessions and support [1]. It may be necessary to distinguish between the level of training required for the trial, and the level of training for a routine application outside of a RCT setting (discuss under item 21 – generalizability. subitem not at all important 1 2 3 4 5 essential subitem not at all important 1 2 3 4 5 essential subitem not at all important 1 2 3 4 5 essential Does your paper address subitem 5-xii? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study We described co-intervention for both arms as “Participants at both arms received 30-minute demonstration on the core functions of these activity trackers (e.g. how to wear, read the displays and charge the trackers), and were instructed to wear them over the entire day until going to bed.” Additional co-intervention was only provided to the intervention arm based on the information-motivation-behavior skill components. 6a) Completely defined pre-specified primary and secondary outcome measures, including how and when they were assessed 6a) Completely defined pre-specified primary and secondary outcome measures, including how and when they were assessed Does your paper address CONSORT subitem 6a? * Does your paper address CONSORT subitem 6a? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study We defined our outcomes as "The current interim study focused on the process outcomes, namely, the acceptability and adoption of wearable activity trackers. Acceptability was defined as users’ subjective perception and experiences, and was evaluated by a 14-item users’ feedback questionnaire (Multimedia Appendix 1), adapted from previous usability and acceptability studies. Rated on a five-point Likert scale from 1‘strongly disagree’ to 5 ‘strongly agree’, this questionnaire assessed users’ acceptability in four main domains: enjoyment & comfort (three items, range 3~15; Cronbach alpha=.85), motivation to use (four items, range 4~20; Cronbach alpha=.83), usefulness (four items, range 4~20; Cronbach alpha=.89), and perceived ease-of-use (three items, range 3~15; Cronbach alpha=.76). A total score was calculated to indicate users’ overall experience (Cronbach alpha=.93, range 14~70). Adoption was defined as users’ interaction and usage behavior, and was evaluated objectively via the uploaded step count data in two ways: 1) the percentage of days having valid step records over individuals’ follow-up days (average 90.7 days), and 2) the average daily step counts per person of these valid step records. Daily step counts less than the 5th percentile of the study sample’s daily step counts (i.e. 1,311 steps per day) were treated as invalid records and were removed, as those steps might represent non-wear and inappropriate use of the activity trackers." 6b) Any changes to trial outcomes after the trial commenced, with reasons 7a) How sample size was determined NPT: When applicable, details of whether and how the clustering by care provides or centers was addressed Does your paper address CONSORT subitem 6b? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study NOT applicable. 6b) Any changes to trial outcomes after the trial commenced, with reasons subitem not at all important 1 2 3 4 5 essential subitem not at all important 1 2 3 4 5 essential 6a-i) Online questionnaires: describe if they were validated for online use and apply CHERRIES items to describe how the questionnaires were designed/deployed If outcomes were obtained through online questionnaires, describe if they were validated for online use and apply CHERRIES items to describe how the questionnaires were designed/deployed [9]. Does your paper address subitem 6a-i? Copy and paste relevant sections from manuscript text 您的回答 6a-ii) Describe whether and how “use” (including intensity of use/dosage) was defined/measured/monitored Describe whether and how “use” (including intensity of use/dosage) was defined/measured/monitored (logins, logfile analysis, etc.). Use/adoption metrics are important process outcomes that should be reported in any ehealth trial. Does your paper address subitem 6a-ii? Copy and paste relevant sections from manuscript text We described the use monitoring as “Adoption was defined as users’ interaction and usage behavior, and was evaluated objectively via the uploaded step count data in two ways: 1) the percentage of days having valid step records over individuals’ follow-up days (average 90.7 days), and 2) the average daily step counts per person of these valid step records. Daily step counts less than the 5th percentile of the study sample’s daily step counts (i.e. 1,311 steps per day) were treated as invalid records and were removed, as those steps might represent non- wear and inappropriate use of the activity trackers.” subitem not at all important 1 2 3 4 5 essential subitem not at all important 1 2 3 4 5 essential 6a-ii) Describe whether and how “use” (including intensity of use/dosage) was defined/measured/monitored Describe whether and how “use” (including intensity of use/dosage) was defined/measured/monitored (logins, logfile analysis, etc.). Use/adoption metrics are important process outcomes that should be reported in any ehealth trial. Does your paper address subitem 6a-ii? Copy and paste relevant sections from manuscript text We described the use monitoring as “Adoption was defined as users’ interaction and usage behavior, and was evaluated objectively via the uploaded step count data in two ways: 1) the percentage of days having valid step records over individuals’ follow-up days (average 90.7 days), and 2) the average daily step counts per person of these valid step records. Daily step counts less than the 5th percentile of the study sample’s daily step counts (i.e. 1,311 steps per day) were treated as invalid records and were removed, as those steps might represent non- wear and inappropriate use of the activity trackers.” 6a-iii) Describe whether, how, and when qualitative feedback from participants was obtained Describe whether, how, and when qualitative feedback from participants was obtained (e.g., through emails, feedback forms, interviews, focus groups). 6a-iii) Describe whether, how, and when qualitative feedback from participants was obtained Describe whether, how, and when qualitative feedback from participants was obtained (e.g., through emails, feedback forms, interviews, focus groups). p p Describe whether, how, and when qualitative feedback from participants was obtained (e.g., through emails, feedback forms, interviews, focus groups). subitem not at all important 1 2 3 4 5 essential Does your paper address subitem 6a-iii? Copy and paste relevant sections from manuscript text We stated in the Data collection section: “To further explore users’ experiences with the activity trackers, participants’ qualitative feedbacks were also collected by the group facilitators. In reference to the acceptability questionnaire, participants were encouraged to elaborate their self-monitoring experiences regarding enjoyment & comfort, motivation to use, usefulness, and ease-of-use. Their feedbacks were analyzed in a deductive manner to extract information concerning the barriers and facilitators of each acceptability domain. A formal coding process was not applied.” Does your paper address subitem 7a-i? Does your paper address subitem 7a-i? Copy and paste relevant sections from manuscript title (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study The sample size calculation was described as "The main study’s sample size was calculated based on changes in physical activity levels. It was calculated that 12 square dancing groups with an average of 15 participants per arm would have 85% power to detect an increase in physical activity from 1302 to 1500 MET-min per week, assuming an intracluster correlation of 0.05 and a 5% type I error. To further account for a 20% attrition ratio, 24 dancing groups across three districts were needed to fulfill a total sample size of 440 individual participants." 7b) When applicable, explanation of any interim analyses and stopping guidelines Does your paper address CONSORT subitem 6b? * y p p Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study NOT applicable. NPT: When applicable, details of whether and how the clustering by care provides or centers was addressed 7a-i) Describe whether and how expected attrition was taken into account when calculating the sample size Describe whether and how expected attrition was taken into account when calculating the sample size. 7a-i) Describe whether and how expected attrition was taken into account when calculating the sample size Describe whether and how expected attrition was taken into account when calculating the sample size. g p Describe whether and how expected attrition was taken into account when calculating the sample size. subitem not at all important 1 2 3 4 5 essential 8a) Method used to generate the random allocation sequence NPT: When applicable, how care providers were allocated to each trial group Does your paper address CONSORT subitem 7b? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study Not applicable. Does your paper address CONSORT subitem 8a? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study We used a simple randomization process to generate the random allocation sequence, as described in the Random Allocation section “All random allocation was done at the cluster level, namely, by the square- dancing groups. After the recruitment of all eligible participants, a statistician otherwise not associated with the project allocated participants by their square-dancing groups equally into two arms (1:1), following a simple randomization process.” 8b) Type of randomisation; details of any restriction (such as blocking and block size) Does your paper address CONSORT subitem 8b? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study Does your paper address CONSORT subitem 8b? * We used a simple randomization process to generate the random allocation sequence, without restriction. We used a simple randomization process to generate the random allocation sequence, without restriction. 9) Mechanism used to implement the random allocation sequence (such as sequentially numbered containers), describing any steps taken to conceal the sequence until interventions were assigned 9) Mechanism used to implement the random allocation sequence (such as sequentially numbered containers), describing any steps taken to conceal the sequence until interventions were assigned Does your paper address subitem 11a-i? * We described the blinding as "After the recruitment of all eligible participants, a statistician otherwise not associated with the project allocated participants by their square-dancing groups equally into two arms (1:1), following a simple randomization process. Although the participants were aware of the interventional nature of the study, they were blinded to their allocation status. Outcome assessors were blinded to the group assignments and were different from researchers who conducted and monitored the interventions." Does your paper address CONSORT subitem 9? * Does your paper address CONSORT subitem 9? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study We described the allocation concealment as “Although the participants were aware of the interventional nature of the study, they were blinded to their allocation status. Outcome assessors were blinded to the group assignments and were different from researchers who conducted and monitored the interventions.” 10) Who generated the random allocation sequence, who enrolled participants, and who assigned participants to interventions Does your paper address CONSORT subitem 10? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study We stated in the Methods: “After the recruitment of all eligible participants, a statistician otherwise not associated with the project allocated participants by their square-dancing groups equally into two arms (1:1), following a simple randomization process." 11a) If done, who was blinded after assignment to interventions (for example, participants, care providers, those assessing outcomes) and how NPT: Whether or not administering co-interventions were blinded to group assignment 11a-i) Specify who was blinded, and who wasn’t Specify who was blinded, and who wasn’t. Usually, in web-based trials it is not possible to blind the participants [1, 3] (this should be clearly acknowledged), but it may be possible to blind outcome assessors, those doing data analysis or those administering co- interventions (if any). Does your paper address subitem 11a-i? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study 11b) If relevant, description of the similarity of interventions (this item is usually not relevant for ehealth trials as it refers to similarity of a placebo or sham intervention to a active medication/intervention) 11a-ii) Discuss e.g., whether participants knew which intervention was the “intervention of interest” and which one was the “comparator” Informed consent procedures (4a-ii) can create biases and certain expectations - discuss e.g., whether participants knew which intervention was the “intervention of interest” and which one was the “comparator”. subitem not at all important 1 2 3 4 5 essential Does your paper address subitem 11a-ii? Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study We described this as "Although the participants were aware of the interventional nature of the study, they were blinded to their allocation status." Does your paper address CONSORT subitem 11b? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study Not applicable. NPT: When applicable, details of whether and how the clustering by care providers or centers was addressed Does your paper address CONSORT subitem 12a? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study We addressed the clustering as “The intent-to-treat (ITT) analysis was adopted to examine the treatment effects, to minimize the selection bias and keep the original randomization design” and “The intention-to-treat (ITT) analysis was adopted to examine the treatment effects, to minimize the selection bias and keep the original randomization design” and " As participants clustered within dance groups, multilevel linear regression models were used to test for the intervention effect on continuous outcomes (i.e. acceptability) at individual participant level, while taking cluster level variation due to dancing groups into account. Similarly, multilevel negative binomial models were fitted to count outcomes (i.e. adoption), which followed an over-dispersed Poisson-like distribution. All models were adjusted for baseline covariates that were empirically suggested strong predictors for the adoption of wearable trackers. Length of individual follow-up days was further adjusted in the multilevel negative binomial model for daily step counts." 12a-i) Imputation techniques to deal with attrition / missing values Imputation techniques to deal with attrition / missing values: Not all participants will use the intervention/comparator as intended and attrition is typically high in ehealth trials. Specify how participants who did not use the application or dropped out from the trial were treated in the statistical analysis (a complete case analysis is strongly discouraged, and simple imputation techniques such as LOCF may also be problematic [4]). subitem not at all important 1 2 3 4 5 essential subitem not at all important 1 2 3 4 5 essential subitem not at all important x26-ii) Outline informed consent procedures Outline informed consent procedures e.g., if consent was obtained offline or online (how? Checkbox, etc.?), and what information was provided (see 4a-ii). See [6] for some items to be included in informed consent documents. Does your paper address subitem 12a-i? * y p p Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study We described the imputation method as “Missing baseline covariates and missing outcomes were multiply imputed under the missing at random assumption, using individual demographic information, health status, other outcomes and cluster identifiers, separately by randomized arms to avoid biasing treatment effects towards the null. Altogether, 20 sets of complete datasets were imputed based on the chained equations. Primary analyses were then performed on each complete dataset and combined results were obtained according to the Rubin’s combination rules." 12b) Methods for additional analyses, such as subgroup analyses and adjusted analyses Does your paper address CONSORT subitem 12b? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study We only conducted sensitivity analysis with complete cases. Does your paper address subitem X26-i? Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study We stated the IRB approval as "All participants read and signed the written informed consent approved by the Institutional Review Board (No. L2016-004) of the School of Public Health of Sun Yat-sen University." Does your paper address subitem X26-ii? Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study X26-iii) Safety and security procedures Safety and security procedures, incl. privacy considerations, and any steps taken to reduce the likelihood or detection of harm (e.g., education and training, availability of a hotline) Does your paper address subitem X26-iii? Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study 13b) For each group, losses and exclusions after randomisation, together with reasons 13b-i) Attrition diagram Strongly recommended: An attrition diagram (e.g., proportion of participants still logging in or using the intervention/comparator in each group plotted over time, similar to a survival curve) or other figures or tables demonstrating usage/dose/engagement. subitem not at all important 1 2 3 4 5 essential subitem not at all important 1 2 3 4 5 essential RESULTS 13a) For each group, the numbers of participants who were randomly assigned, received intended treatment, and were analysed for the primary outcome NPT: The number of care providers or centers performing the intervention in each group and the number of patients treated by each care provider in each center Does your paper address CONSORT subitem 13a? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study We specified the number of participants for each group as “Of the 88 dancing groups initially assessed for eligibility, 26 groups did not meet the cluster inclusion criteria mainly because of dancing style and group size. Nearly half of these groups (38/88, 43%) assessed declined to participate due to lack of trust or time. The rest 11 groups were also not eligible, as their group members were not local residents (n=2), hadn’t been regularly practicing square dancing in the past 12 months (n=5), or less than two participants of the given group were willing to participate (n=4). The remaining 13 dancing groups were 1:1 randomized into the intervention arm (n=7) and the control arm (n=6). Among these eligible groups, 69 out of 82 participants (84 %) of the intervention arm and 80 out of 98 participants (82%) of the control arm received the allocated treatment. During the follow-up, no dancing groups withdrew; while five participants of the intervention arm and 19 of the control arm were lost to follow up, two participants of each arm had discontinued intervention due to technical problem, and one participant of the intervention arm and three participants of the control arm withdrew from the study. The final ITT analysis sample was based on the 149 participants of 13 dancing groups, out of whom 117 (78.5%) filled the users’ feedback questionnaire of the wearable activity trackers.” 13b) For each group, losses and exclusions after randomisation, together with reasons Does your paper address CONSORT subitem 13b? RESULTS (NOTE: Preferably, this is shown in a CONSORT flow diagram) * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study Does your paper address CONSORT subitem 13b? (NOTE: Preferably, this is shown in a CONSORT flow diagram) * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study Loss and exclusion was presented in the flowchat (Figure 2), and we also stated in the Results as “During the follow-up, no dancing groups withdrew; while five participants of the intervention arm and 19 of the control arm were lost to follow up, two participants of each arm had discontinued intervention due to technical problem, and one participant of the intervention arm and three participants of the control arm withdrew from the study. The final ITT analysis sample was based on the 149 participants of 13 dancing groups, out of whom 117 (78.5%) filled the users’ feedback questionnaire of the wearable activity trackers." Does your paper address subitem 14a-i? Does your paper address subitem 14a i? Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study 14b) Why the trial ended or was stopped (early) Does your paper address subitem 13b-i? y Copy and paste relevant sections from the manuscript or cite the figure number if applicable (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study 14a) Dates defining the periods of recruitment and follow-up subitem not at all important 1 2 3 4 5 essential 14b) Why the trial ended or was stopped (early) Does your paper address CONSORT subitem 14a? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study We addressed the period of recruitment and follow-up in the Method part as “This interim report covers the trial conducted between November 2017 and October 2018, consisting of seven-month recruitment and baseline assessment, three-month intervention, and the post-intervention reassessment.” 14a-i) Indicate if critical “secular events” fell into the study period Indicate if critical “secular events” fell into the study period, e.g., significant changes in Internet resources available or “changes in computer hardware or Internet delivery resources” Does your paper address subitem 14a-i? Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study 您的回答 Does your paper address CONSORT subitem 14b? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study Not applicable. Does your paper address CONSORT subitem 14a? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study We addressed the period of recruitment and follow-up in the Method part as “This interim report covers the trial conducted between November 2017 and October 2018, consisting of seven-month recruitment and baseline assessment, three-month intervention, and the post-intervention reassessment.” 14a-i) Indicate if critical “secular events” fell into the study period Indicate if critical “secular events” fell into the study period, e.g., significant changes in Internet resources available or “changes in computer hardware or Internet delivery resources” 15) A table showing baseline demographic and clinical characteristics for each group NPT: When applicable, a description of care providers (case volume, qualification, expertise, etc.) and centers (volume) in each group Does your paper address CONSORT subitem 15? * y Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study We provided participants’ baseline demographic and clinical characteristics in the Table 1. Does your paper address CONSORT subitem 14b? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study Does your paper address subitem 16-i? * Does your paper address subitem 16-i? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study We clearly indicated the number of participant in the result and in the flowchart (Figure 2). As we stated, “The final ITT analysis sample was based on the 149 participants of 13 dancing groups, out of whom 117 (78.5%) filled the users’ feedback questionnaire of the wearable activity trackers.” 16) For each group, number of participants (denominator) included in each analysis and whether the analysis was by original assigned groups subitem not at all important 1 2 3 4 5 essential subitem not at all important 1 2 3 4 5 essential 16-i) Report multiple “denominators” and provide definitions Report multiple “denominators” and provide definitions: Report N’s (and effect sizes) “across a range of study participation [and use] thresholds” [1], e.g., N exposed, N consented, N used more than x times, N used more than y weeks, N participants “used” the intervention/comparator at specific pre-defined time points of interest (in absolute and relative numbers per group). Always clearly define “use” of the intervention. Does your paper address subitem 16-i? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study We clearly indicated the number of participant in the result and in the flowchart (Figure 2). As we stated, “The final ITT analysis sample was based on the 149 participants of 13 dancing groups, out of whom 117 (78.5%) filled the users’ feedback questionnaire of the wearable activity trackers.” 16-ii) Primary analysis should be intent-to-treat Primary analysis should be intent-to-treat, secondary analyses could include comparing only “users”, with the appropriate caveats that this is no longer a randomized sample (see 18-i). 16) For each group, number of participants (denominator) included in each analysis and whether the analysis was by original assigned groups 16-i) Report multiple “denominators” and provide definitions i Report multiple “denominators” and provide definitions: Report N’s (and effect sizes) “across a range of study participation [and use] thresholds” [1], e.g., N exposed, N consented, N used more than x times, N used more than y weeks, N participants “used” the intervention/comparator at specific pre-defined time points of interest (in absolute and relative numbers per group). Always clearly define “use” of the intervention. subitem not at all important 1 2 3 4 5 essential subitem not at all important 1 2 3 4 5 essential subitem not at all important 1 2 3 4 5 essential 15-i) Report demographics associated with digital divide issues In ehealth trials it is particularly important to report demographics associated with digital divide issues, such as age, education, gender, social-economic status, computer/Internet/ehealth literacy of the participants, if known. Does your paper address subitem 15-i? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study y p p Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study We reported "Sociodemographic characteristics and health status were similar between the two arms. Most of the participants were married older women (mean age 62 years), retired, with an educational degree of senior high school, had diagnosed chronic diseases but were physically and mentally sound. The intervention arm had fewer female participants and lower mean TICS scores than the control arm. The average group size was 9.9 and 13.3 participants for the intervention arm and the control arm respectively." Does your paper address subitem 16-ii? Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study We applied ITT for the primary outcome analysis, which was mentioned in the statistical analysis section and results section. 17a) For each primary and secondary outcome, results for each group, and the estimated effect size and its precision (such as 95% confidence interval) Does your paper address CONSORT subitem 17a? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study For each primary outcome we provided estimated effects with its 95% confidence interval, for instance, “After adjusting for clustering effect, baseline unbalanced covariates and predictors for adoption, the absolute group difference in the overall acceptability score was estimated to be 6.8 points (95% CI 2.2 to 11.4) higher in the intervention arm than the control arm.” 16-ii) Primary analysis should be intent-to-treat ) y y Primary analysis should be intent-to-treat, secondary analyses could include comparing only “users”, with the appropriate caveats that this is no longer a randomized sample (see 18-i). subitem not at all important 1 2 3 4 5 essential subitem not at all important 17a-i) Presentation of process outcomes such as metrics of use and intensity of use In addition to primary/secondary (clinical) outcomes, the presentation of process outcomes such as metrics of use and intensity of use (dose, exposure) and their operational definitions is critical. This does not only refer to metrics of attrition (13-b) (often a binary variable), but also to more continuous exposure metrics such as “average session length”. These must be accompanied by a technical description how a metric like a “session” is defined (e.g., timeout after idle time) [1] (report under item 6a). subitem not at all important 1 2 3 4 5 essential subitem not at all important subitem not at all important Does your paper address subitem 17a-i? Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study As our study was aimed to explore the acceptability and adoption of wearable activity trackers by mid- and older- aged adults, we specifically reported measures regarding these two process outcomes, for instance, “Regarding adoption outcomes measured by step count data, the medium of the percentage of days that participants had valid step count record were 44.1% vs 11.4% for the intervention and control arms, with 10% and 25% of each arm having invalid step counts records.” 17b) For binary outcomes, presentation of both absolute and relative effect sizes is recommended Does your paper address CONSORT subitem 17b? * Does your paper address CONSORT subitem 17b? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study We reported both absolute and relative effect size for each outcome measures by intervention arms with (Table 2) and without (Appendix table 1) multiple imputation and covariates adjustments. 18) Results of any other analyses performed, including subgroup analyses and adjusted analyses, distinguishing pre-specified from exploratory Does your paper address CONSORT subitem 18? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study We reported results among complete cases analyses as sensitivity analysis in Appendix table 2. 18-i) Subgroup analysis of comparing only users A subgroup analysis of comparing only users is not uncommon in ehealth trials done, it must be stressed that this is a self-selected sample and no longer an u sample from a randomized trial (see 16-iii). 18-i) Subgroup analysis of comparing only users A subgroup analysis of comparing only users is not uncommon in ehealth trials, but if done, it must be stressed that this is a self-selected sample and no longer an unbiased sample from a randomized trial (see 16-iii). Does your paper address CONSORT subitem 19? * Does your paper address CONSORT subitem 19? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study 19-i) Include privacy breaches, technical problems subitem not at all important 1 2 3 4 5 essential subitem not at all important 1 2 3 4 5 essential Does your paper address subitem 19-i? Does your paper address subitem 19-i? Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study Does your paper address subitem 18-i? 您的回答 (for specific guidance see CONSORT for harms) subitem not at all important 1 2 3 4 5 essential subitem not at all important 1 2 3 4 5 essential Does your paper address CONSORT subitem 19? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study Our study had no harms for our participants, and any potential unintended effects or use of the wearable activity trackers were reported in the qualitative feedback from participant section, such as “some participants commented that they preferred the sleep monitoring function to the PA monitoring function.” 19-i) Include privacy breaches, technical problems Include privacy breaches, technical problems. This does not only include physical “harm” to participants, but also incidents such as perceived or real privacy breaches [1], technical problems, and other unexpected/unintended incidents. “Unintended effects” also includes unintended positive effects [2]. Does your paper address subitem 19-i? Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study 您的回答 19-ii) Include qualitative feedback from participants or observations from staff/researchers Include qualitative feedback from participants or observations from staff/researchers, if available, on strengths and shortcomings of the application, especially if they point to unintended/unexpected effects or uses. This includes (if available) reasons for why people did or did not use the application as intended by the developers. Our study had no harms for our participants, and any potential unintended effects or use of the wearable activity trackers were reported in the qualitative feedback from participant section, such as “some participants commented that they preferred the sleep monitoring function to the PA monitoring function.” 19-i) Include privacy breaches, technical problems Include privacy breaches, technical problems. This does not only include physical “harm” to participants, but also incidents such as perceived or real privacy breaches [1], technical problems, and other unexpected/unintended incidents. “Unintended effects” also includes unintended positive effects [2]. 19-ii) Include qualitative feedback from participants or observations from staff/researchers Include qualitative feedback from participants or observations from staff/researchers, if available, on strengths and shortcomings of the application, especially if they point to unintended/unexpected effects or uses. This includes (if available) reasons for why people did or did not use the application as intended by the developers. Does your paper address subitem 19-ii? Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study We reported the qualitative feedback from our participants in the Qualitative feedback from participants and researchers section, as “Qualitative feedbacks from participants and researchers’ observations indicated some common problems encountered by both arms. In terms of enjoyment and comfort of wearing, participants noted that activity trackers were extremely uncomfortable to wear when the weather was hot and humid, let alone during dancing if they sweat heavily. Some participants reflected that the figures displayed on the trackers were too small to read, such that they had to wear glasses to read them. Regarding usefulness, a few participants thought that the function of the wearable activity trackers was quite limited; while other functions, such as blood pressure monitoring, were more useful and relevant from their point of view. For instance, some participants commented that they preferred the sleep monitoring function to the PA monitoring function. As for ease-of-use, our researchers found that they received most common inquiries about how to charge the trackers and how to synchronize data with the paired smart phone.” 22) Interpretation consistent with results, balancing benefits and harms, and considering other relevant evidence NPT: In addition, take into account the choice of the comparator, lack of or partial blinding, and unequal expertise of care providers or centers in each group 22) Interpretation consistent with results, balancing benefits and harms, and considering other relevant evidence 22) Interpretation consistent with results, balancing benefits and harms, and considering other relevant evidence 22) Interpretation consistent with results, balancing benefits and harms, and considering other relevant evidence NPT: In addition, take into account the choice of the comparator, lack of or partial blinding, and unequal expertise of care providers or centers in each group 22-i) Restate study questions and summarize the answers suggested by the data, starting with primary outcomes and process outcomes (use) Restate study questions and summarize the answers suggested by the data, starting with primary outcomes and process outcomes (use). subitem not at all important 1 2 3 4 5 essential Does your paper address subitem 22-i? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study Does your paper address subitem 22-i? * We summarized our main findings in line with the study questions in the first paragraph of the Discussion, as "We assessed the effectiveness of a social group-based deployment intervention informed by the IMBS framework to promote wearable activity trackers’ acceptability and adoption among community-dwelling mid- and older-aged adults. In line with our hypotheses, the results revealed that our intervention significantly increased participants’ acceptability, mainly driven by improvements in the perceived motivation, usefulness and ease-of-use. The intervention also effectively promoted participants’ adoption, quantified as twice the amount of valid step count data of the intervention arm than the control arm.” subitem not at all important 1 2 3 4 5 essential 22-ii) Highlight unanswered new questions, suggest future research Highlight unanswered new questions, suggest future research. subitem not at all important 1 2 3 4 5 essential 22-ii) Highlight unanswered new questions, suggest future research Highlight unanswered new questions, suggest future research. Highlight unanswered new questions, suggest future research. Does your paper address subitem 22-ii? Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study We concluded our study by highlighting its limitations alongside directions for future studies, such as “Second, as our intervention package addressed the three main barriers of behavior changes jointly according to the IMBS framework, we were not able to distinguish the unique contribution of each intervention component to the program effect. Additional research design may be considered in future studies, such as the factorial experiment that may allow the evaluation of sole and joint effects of such an intervention. "and “Lastly, as a phase 1 study, we have yet to capture and report long-term acceptability, adoption and health-related outcomes, which are needed to establish the intervention’s long-term behavior maintenance and effectiveness." 20) Trial limitations, addressing sources of potential bias, imprecision, and, if relevant, multiplicity of analyses 20-i) Typical limitations in ehealth trials ) yp Typical limitations in ehealth trials: Participants in ehealth trials are rarely blinded. Ehealth trials often look at a multiplicity of outcomes, increasing risk for a Type I error. Discuss biases due to non-use of the intervention/usability issues, biases through informed consent procedures, unexpected events. subitem not at all important Does your paper address subitem 21-i? Does your paper address subitem 21-i? Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study We addressed our generalizability as “The participants recruited thus were likely to be more prone to technology than the general mid- and older- adults. However, this self-selection of the participants should not affect the internal validity of our program effects as the intervention and control arms shared similar characteristics and were randomly assigned. The self-selection, however, limited our program implication to the broader mid- and older- aged population.” 21-i) Generalizability to other populations Generalizability to other populations: In particular, discuss generalizability to a general Internet population, outside of a RCT setting, and general patient population, including applicability of the study results for other organizations subitem not at all important 1 2 3 4 5 essential Does your paper address subitem 20-i? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study Limitations of the current analysis were outlined as “Several limitations of our study are worth noting. First, significantly underestimating the challenges in the recruitment, we recruited only 13 dancing groups rather than the 24 groups as expected. The participants recruited thus were likely to be more prone to technology than the general mid- and older- adults. However, this self-selection of the participants should not affect the internal validity of our program effects as the intervention and control arms shared similar characteristics and were randomly assigned. The self-selection, however, limited our program implication to the broader mid- and older- aged population. Second, as our intervention package addressed the three main barriers of behavior changes jointly according to the IMBS framework, we were not able to distinguish the unique contribution of each intervention component to the program effect. Additional research design may be considered in future studies, such as the factorial experiment that may allow the evaluation of sole and joint effects of such an intervention. Third, our intervention relied on social interactions and required regular assistance from the research staff, particularly in the initial phase. Although social support and comparison functions have been integrated into many activity trackers nowadays, our assistance level is likely to be higher than what commercially available activity trackers typically provide. Lastly, as an interim study, we have yet to capture and report long-term acceptability, adoption and health-related outcomes, which are needed to establish the intervention’s long-term behavior maintenance and effectiveness." 21) Generalisability (external validity, applicability) of the trial findings NPT: External validity of the trial findings according to the intervention, comparators, patients, and care providers or centers involved in the trial 21-i) Generalizability to other populations Generalizability to other populations: In particular, discuss generalizability to a general Internet population, outside of a RCT setting, and general patient population, including applicability of the study results for other organizations 21-ii) Discuss if there were elements in the RCT that would be different in a routine application setting Discuss if there were elements in the RCT that would be different in a routine application setting (e.g., prompts/reminders, more human involvement, training sessions or other co- interventions) and what impact the omission of these elements could have on use, adoption, or outcomes if the intervention is applied outside of a RCT setting. subitem not at all important 1 2 3 4 5 essential Does your paper address subitem 21-ii? Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study We discussed the RCT in comparison to a routine application as “Although social support and comparison functions have been integrated into many activity trackers nowadays, our assistance level is likely to be higher than what commercially available activity trackers typically provide.” OTHER INFORMATION 23) Registration number and name of trial registry Does your paper address CONSORT subitem 23? * Does your paper address CONSORT subitem 23? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study X27) Conflicts of Interest (not a CONSORT item) subitem not at all important 1 2 3 4 5 essential About the CONSORT EHEALTH checklist Guangdong Province Natural Science Foundation (2017A030310346, 2018A0303130046) and Sun Yat-sen Scientific Research Foundation for Early Career Researchers (17ykpy15). The founders play no role in the study design, data collection and analysis, or result interpretation. X27-i) State the relation of the study team towards the system being evaluated In addition to the usual declaration of interests (financial or otherwise), also state the relation of the study team towards the system being evaluated, i.e., state if the authors/evaluators are distinct from or identical with the developers/sponsors of the intervention. Does your paper address subitem X27-i? Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study This is no conficts of interest to be declared. X27) Conflicts of Interest (not a CONSORT item) X27-i) State the relation of the study team towards the system being evaluated In addition to the usual declaration of interests (financial or otherwise), also state the relation of the study team towards the system being evaluated, i.e., state if the authors/evaluators are distinct from or identical with the developers/sponsors of the intervention. ChiCTR-IOC-17013185 Does your paper address CONSORT subitem 24? * Cite a Multimedia Appendix, other reference, or copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study The trial protocol can be acquired from the lead author. Does your paper address CONSORT subitem 25? * Does your paper address CONSORT subitem 25? * Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study As mentioned in Acknowledgement, our study was supported the Guangdong Province Natural Science Foundation (2017A030310346, 2018A0303130046) and Sun Yat-sen Scientific Research Foundation for Early Career Researchers (17ykpy15). The founders play no role in the study design, data collection and analysis, or result interpretation. Does your paper address subitem X27-i? y Copy and paste relevant sections from the manuscript (include quotes in quotation marks "like this" to indicate direct quotes from your manuscript), or elaborate on this item by providing additional information not in the ms, or briefly explain why the item is not applicable/relevant for your study This is no conficts of interest to be declared. About the CONSORT EHEALTH checklist yes, major changes yes, minor changes no yes no 其他: As a result of using this checklist, did you make changes in your manuscript? * What were the most important changes you made as a result of using this checklist? We provided more detailed description regarding the study methods in accordance with the checklist. How much time did you spend on going through the checklist INCLUDING making changes in your manuscript * Roughly 2-3 days. As a result of using this checklist, do you think your manuscript has improved? * Any other comments or questions on CONSORT EHEALTH t Would you like to become involved in the CONSORT EHEALTH group? g p This would involve for example becoming involved in participating in a workshop and writing an "Explanation and Elaboration" document yes no 其他: As a result of using this checklist, did you make changes in your manuscript? * We provided more detailed description regarding the study methods in accordance with the checklist. As a result of using this checklist, do you think your manuscript has improved? * yes no 其他: STOP - Save this form as PDF before you click submit To generate a record that you filled in this form, we recommend to generate a PDF of this page (on a Mac, simply select "print" and then select "print as PDF") before you submit it. To generate a record that you filled in this form, we recommend to generate a PDF of this page (on a Mac, simply select "print" and then select "print as PDF") before you submit it. When you submit your (revised) paper to JMIR, please upload the PDF as supplementary file. Don't worry if some text in the textboxes is cut off, as we still have the complete information in our database. Thank you! Click submit so we have your answers in our database! 此内容不是由Google 所创建，Google 不对其作任何担保。举报滥用行为 - 服务条款 - 隐私权政策
https://openalex.org/W2713713727	https://pure.rug.nl/ws/files/44243677/The_association_between_various_smoking_behaviors_cotinine_biomarkers_and_skin_autofluorescence_a_marker_for_advanced_glycation_end_product_accumulation.pdf	English	null	The association between various smoking behaviors, cotinine biomarkers and skin autofluorescence, a marker for advanced glycation end product accumulation	PloS one	2,017	cc-by	9,540	University of Groningen The association between various smoking behaviors, cotinine biomarkers and skin autofluorescence, a marker for advanced glycation end product accumulation van Waateringe, Robert P.; Mook-Kanamori, Marjonneke J.; Slagter, Sandra N.; van der Klauw, Melanie M.; van Vliet-Ostaptchouk, Jana V.; Graaff, Reindert; Lutgers, Helen L.; Suhre, Karsten; Selim, Mohammed M. El-Din; Mook-Kanamori, Dennis O. Published in: PLoS ONE E.-D., Mook-Kanamori, D. O., & Wolffenbuttel, B. H. R. (2017). The association between various smoking behaviors, cotinine biomarkers and skin autofluorescence, a marker for advanced glycation end product accumulation. PLoS ONE, 12(6), Article 0179330. https://doi.org/10.1371/journal.pone.0179330 Citation for published version (APA): van Waateringe, R. P., Mook-Kanamori, M. J., Slagter, S. N., van der Klauw, M. M., van Vliet-Ostaptchouk, J. V., Graaff, R., Lutgers, H. L., Suhre, K., Selim, M. M. E.-D., Mook-Kanamori, D. O., & Wolffenbuttel, B. H. R. (2017). The association between various smoking behaviors, cotinine biomarkers and skin autofluorescence, a marker for advanced glycation end product accumulation. PLoS ONE, 12(6), Article 0179330. https://doi.org/10.1371/journal.pone.0179330 Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license. More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne- amendment. Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Citation for published version (APA): van Waateringe, R. P., Mook-Kanamori, M. J., Slagter, S. N., van der Klauw, M. M., van Vliet-Ostaptchouk, J. V., Graaff, R., Lutgers, H. L., Suhre, K., Selim, M. M. E.-D., Mook-Kanamori, D. O., & Wolffenbuttel, B. H. R. (2017). The association between various smoking behaviors, cotinine biomarkers and skin autofluorescence, a marker for advanced glycation end product accumulation. PLoS ONE, 12(6), Article 0179330. https://doi.org/10.1371/journal.pone.0179330 University of Groningen The association between various smoking behaviors, cotinine biomarkers and skin autofluorescence, a marker for advanced glycation end product accumulation van Waateringe, Robert P.; Mook-Kanamori, Marjonneke J.; Slagter, Sandra N.; van der Klauw, Melanie M.; van Vliet-Ostaptchouk, Jana V.; Graaff, Reindert; Lutgers, Helen L.; Suhre, Karsten; Selim, Mohammed M. El-Din; Mook-Kanamori, Dennis O. Published in: PLoS ONE University of Groningen The association between various smoking behaviors, cotinine biomarkers and skin autofluorescence, a marker for advanced glycation end product accumulation van Waateringe, Robert P.; Mook-Kanamori, Marjonneke J.; Slagter, Sandra N.; van der Klauw, Melanie M.; van Vliet-Ostaptchouk, Jana V.; Graaff, Reindert; Lutgers, Helen L.; Suhre, Karsten; Selim, Mohammed M. El-Din; Mook-Kanamori, Dennis O. Published in: PLoS ONE The association between various smoking behaviors, cotinine biomarkers and skin autofluorescence, a marker for advanced glycation end product accumulation van Waateringe, Robert P.; Mook-Kanamori, Marjonneke J.; Slagter, Sandra N.; van der Klauw, Melanie M.; van Vliet-Ostaptchouk, Jana V.; Graaff, Reindert; Lutgers, Helen L.; Suhre, Karsten; Selim, Mohammed M. El-Din; Mook-Kanamori, Dennis O. Published in: PLoS ONE The association between various smoking behaviors, cotinine biomarkers and skin autofluorescence, a marker for advanced glycation end product accumulation van Waateringe, Robert P.; Mook-Kanamori, Marjonneke J.; Slagter, Sandra N.; van der Kl M l i M Vli t O t t h k J V G ff R i d t L t H l L The association between various smoking behaviors, cotinine biomarkers and skin autofluorescence, a marker for advanced glycation end product accumulation van Waateringe, Robert P.; Mook-Kanamori, Marjonneke J.; Slagter, Sandra N.; van der Klauw, Melanie M.; van Vliet-Ostaptchouk, Jana V.; Graaff, Reindert; Lutgers, Helen L.; Suhre, Karsten; Selim, Mohammed M. El-Din; Mook-Kanamori, Dennis O. Published in: PLoS ONE van Waateringe, Robert P.; Mook-Kanamori, Marjonneke J.; Slagter, Sandra N.; van der Klauw, Melanie M.; van Vliet-Ostaptchouk, Jana V.; Graaff, Reindert; Lutgers, Helen L.; Suhre, Karsten; Selim, Mohammed M. El-Din; Mook-Kanamori, Dennis O. Published in: PLoS ONE van Waateringe, Robert P.; Mook-Kanamori, Marjonneke J.; Slagter, Sandra N.; van der Klauw, Melanie M.; van Vliet-Ostaptchouk, Jana V.; Graaff, Reindert; Lutgers, Helen L.; Suhre, Karsten; Selim, Mohammed M. El-Din; Mook-Kanamori, Dennis O. Published in: PL S ONE DOI: 10.1371/journal.pone.0179330 DOI: 10.1371/journal.pone.0179330 IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Document Version Publisher's PDF, also known as Version of record Publication date: 2017 Publication date: 2017 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): van Waateringe, R. P., Mook-Kanamori, M. J., Slagter, S. N., van der Klauw, M. M., van Vliet-Ostaptchouk, J. V., Graaff, R., Lutgers, H. L., Suhre, K., Selim, M. M. Download date: 24-10-2024 Take-down policy Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. RESEARCH ARTICLE OPEN ACCESS * r.p.van.waateringe@umcg.nl Citation: van Waateringe RP, Mook-Kanamori MJ, Slagter SN, van der Klauw MM, van Vliet- Ostaptchouk JV, Graaff R, et al. (2017) The association between various smoking behaviors, cotinine biomarkers and skin autofluorescence, a marker for advanced glycation end product accumulation. PLoS ONE 12(6): e0179330. https:// doi.org/10.1371/journal.pone.0179330 Methods Copyright: © 2017 van Waateringe 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. In a cross-sectional study, we evaluated participants from the LifeLines Cohort Study and the Qatar Metabolomics Study on Diabetes (QMDiab). In the LifeLines Cohort Study smok- ing behavior and secondhand smoking were assessed in 8,905 individuals including 309 individuals (3.5%) with type 2 diabetes. In QMDiab, cotinine biomarkers were measured in saliva, plasma and urine in 364 individuals of whom 188 (51%) had type 2 diabetes. Skin autofluorescence was measured non-invasively in all participants using the AGE Reader. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: The manuscript is based on data from the LifeLines cohort study. LifeLines adheres to standards for open data availability. The data catalogue of LifeLines is publicly accessible on www.lifelines.net. All international researchers can apply for data at the LifeLines research office Background Skin autofluorescence, a biomarker for advanced glycation end products (AGEs) accumula- tion, has been shown to predict diabetes-related cardiovascular complications and is associ- ated with several environmental and lifestyle factors. In the present study, we examined the association between various smoking behaviors and skin autofluorescence, as well as the association between several cotinine biomarkers and skin autofluorescence, using both epi- demiological and metabolomics data. Editor: Ram Nagaraj, University of Colorado Denver School of Medicine, UNITED STATES Editor: Ram Nagaraj, University of Colorado Denver School of Medicine, UNITED STATES Received: December 8, 2016 Accepted: May 26, 2017 Published: June 20, 2017 Copyright: © 2017 van Waateringe 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: December 8, 2016 Accepted: May 26, 2017 Published: June 20, 2017 The association between various smoking behaviors, cotinine biomarkers and skin autofluorescence, a marker for advanced glycation end product accumulation Robert P. van Waateringe1, Marjonneke J. Mook-Kanamori2,3, Sandra N. Slagter1, Melanie M. van der Klauw1, Jana V. van Vliet-Ostaptchouk1, Reindert Graaff1, Helen L. Lutgers4, Karsten Suhre5,6, Mohammed M. El-Din Selim7, Dennis O. Mook-Kanamori2,8, Bruce H. R. Wolffenbuttel1 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Department of Endocrinology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands, 2 Department of Biostatistics, Epidemiology and Scientific Computing, Epidemiology Section, King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia, 3 Department of Physiology and Biophysics, Weill Cornell Medical College, Doha, Qatar, 4 Department of Internal Medicine, Medical Center Leeuwarden, Leeuwarden, The Netherlands, 5 Bioinformatics Core, Weill Cornell Medical College, Doha, Qatar, 6 Research Centre for Environmental Health, Helmholtz Zentrum Munchen, Neuherberg, Germany, 7 Department of Dermatology, Hamad Medical Corporation, Doha, Qatar, 8 Department of Clinical Epidemiology, Leiden University Medical Center, Leiden, the Netherlands r.p.van.waateringe@umcg.nl Introduction Competing interests: RG is founder and shareholder of DiagnOptics BV, Groningen, the Netherlands, manufacturing autofluorescence readers (http://www.diagnoptics.com) which has been used in the present study. This does not alter our adherence to PLOS ONE policies on sharing data and materials. Advanced glycation end products (AGEs) are the final products of non-enzymatic glycation and oxidative reactions [1] and comprise a group of irreversibly modified proteins, lipids and nucleic acids [2]. AGEs form stable structures with—and accumulate in—tissues as a result of aging [3,4]. The formation and accumulation of AGEs is increased in conditions such as diabe- tes and renal insufficiency [5,6]. AGE accumulation can be measured non-invasively in the skin with a device known as the AGE Reader (Diagnoptics Technologies, Groningen, The Netherlands) [7]. Previous studies have demonstrated that higher skin autofluorescence (SAF) is associated with, and a good predictor of the development of cardiovascular morbidity and mortality in patients with diabetes and (end-stage) renal failure [8–10]. In addition, recent studies have shown that SAF levels are increased in patients with chronic obstructive pulmo- nary disease and peripheral artery disease, independent of diabetes status [11,12]. Several studies have shown elevated SAF levels in smokers compared to non-smokers [9,13]. Moreover, we have recently found both current smoking and the number of pack-years to be strongly associated with higher SAF levels in a large-scale general population [14]. Active and passive tobacco smoking increase the risk of cardiovascular disease and type 2 diabetes, while higher SAF levels are associated with both conditions. As tobacco smoke has been reported to be an exogenous source of AGEs, its accumulation might be considered as an underlying mechanism leading to cardiovascular disease and type 2 diabetes [15]. In studies examining the risk of tobacco smoking on disease outcome, smoking behavior is assessed by general questionnaires. Questionnaires regarding tobacco use are prone to under- estimation and reporting biases [16]. Furthermore, the majority of these questionnaires do not include questions regarding secondhand smoking. An alternative way to examine exposure to tobacco smoke is through assessment of biomarkers for tobacco smoke, measured in either urine, saliva or blood [17]. Cotinine, the main metabolite of nicotine, is considered to be a valid biomarker for envi- ronmental tobacco smoke exposure due to its high sensitivity, specificity and long half-life time [18,19]. Conclusions In the present study, we have demonstrated that secondhand smoking is associated with higher skin autofluorescence levels whereas smoking cessation has a beneficial effect on skin autofluorescence. Finally, urinary cotinine N-oxide might be used as an alternative way for questionnaires to examine the effect of (environmental) tobacco smoking on skin autofluorescence. Smoking behavior and skin autofluorescence biomarker of nicotine exposure, was found to be positively associated with skin autofluores- cence in the QMDiab study (p = 0.03). biomarker of nicotine exposure, was found to be positively associated with skin autofluores- cence in the QMDiab study (p = 0.03). (LLscience@umcg.nl). The LifeLines system allows access for reproducibility of the study results. The QMDiab study was supported by ‘Biomedical Research Program’ funds at Weill Cornell Medical College in Qatar, a program funded by the Qatar Foundation. Support for some of the experiments was provided by the Weill Cornell Medical College in Qatar (WCMC-Q) bioinformatics and virtual metabolomics core which is funded by the Qatar Foundation. Dennis Mook-Kanamori is supported by Dutch Science Organization (ZonMW-VENI Grant 916.14.023). This work was supported by Netherlands Consortium for Healthy Ageing (NCHA), Bio-SHaRE-EU, Biobank Standardisation and Harmonisation for research excellence in the European Union. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Results Skin autofluorescence levels increased with a higher number of hours being exposed to sec- ondhand smoking. Skin autofluorescence levels of former smokers approached levels of never smokers after around 15 years of smoking cessation. Urinary cotinine N-oxide, a 1 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0179330 June 20, 2017 PLOS ONE \| https://doi.org/10.1371/journal.pone.0179330 June 20, 2017 Introduction Since secondhand smoking might be underestimated using questionnaires, it would be interesting whether SAF is a more accurate and reliable measure to detect—long- term—exposure to secondhand smoking. The aim of the present study was to examine the effect of both active and secondhand smoking as well as smoking cessation on SAF. In addition, using metabolomics data we also assessed the association between several cotinine biomarkers and SAF. PLOS ONE \| https://doi.org/10.1371/journal.pone.0179330 June 20, 2017 2 / 15 Smoking behavior and skin autofluorescence Materials and methods Study design For this cross-sectional study, we used data from two different populations, the Dutch Life- Lines Cohort Study and Qatar Metabolomics Study on Diabetes (QMDiab). The LifeLines Cohort Study is a multidisciplinary prospective population-based cohort study with a unique three-generation design that examines the health and health-related behaviors of 8,905 partici- pants in the northern part of The Netherlands [20]. For the current study, we included all par- ticipants from Western European origin between 18 and 80 years old with both SAF measurements and information regarding smoking behavior, who participated in our previous studies regarding (genetic) determinants of SAF [14,21]. Exclusion criteria were missing data for smoking status (n = 104) and severely impaired renal function (serum creatinine >140 micromol/L (n = 14) leaving 8,905 individuals, of whom 309 (3.5%) with type 2 diabetes, avail- able for analysis. Before participating in the study, all participants provided written informed consent. The study protocol was approved by the medical ethical review committee of the Uni- versity Medical Center Groningen, The Netherlands. The Qatar Metabolomics Study on Diabetes (QMDiab) [22] is a large collaborative effort between the Dermatology Department of Hamad Medical Center (HMC) in Doha, Qatar and Weill Cornell Medical College—Qatar (WCMC-Q). Subjects were enrolled between February 2012 and June 2012. Before participating in the study, all participants provided written informed consent. Ethical approval was obtained from the Institutional Review Board from both HMC and WCMC-Q. In total, 374 subjects above the age of 18 participated in the study. Smoking data were missing from ten participants, leav- ing 364 participants of whom 185 (51%) with type 2 diabetes for analyses. PLOS ONE \| https://doi.org/10.1371/journal.pone.0179330 June 20, 2017 Data collection Information regarding ethnicity and smoking behavior was obtained from questionnaires. In QMDiab, ethnicity was determined based on the birthplace of the participant, both parents and four grandparents as described previously [23]. Data on smoking behavior was collected by a detailed self-administered questionnaire. Subjects were classified accord- ing to their smoking status as never smoker, former smoker or current smoker (types of tobacco: cigarette, cigarillo, cigar, pipe tobacco). Never smokers were those who had not smoked during the last month and had never smoked for longer than a year. Former smok- ers were defined as those who had reported smoking for more than a whole year and who had not smoked during the last month and had stopped smoking. Those who had smoked for longer than a year and had not stopped smoking were classified as current smoker. Esti- mation of total tobacco use of current smokers and their classification into light, moderate and heavy smokers were estimated based on the following quantities: one cigarette = 1 gram tobacco. Light smoking was defined as 10 gram/day or less, moderate as 11 to 20 gram/day and heavy as more than 20 gram/day. Pack-years of smoking was calculated as the number of packs of cigarettes smoked per day multiplied by the number of years a subject has smoked. Finally, information about the age at start and quitting smoking as well as whether a subject have been exposed to secondhand smoking at home was obtained by the questionnaire. Diagnosis of diabetes mellitus was established at their LifeLines baseline visit either by a sin- gle fasting blood plasma glucose level 7.0 mmol/L, or when participants reported to have dia- betes which was checked with their medication use (i.e. use of oral blood-glucose lowering agents and/or insulin). 3 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0179330 June 20, 2017 Smoking behavior and skin autofluorescence AGE reader For both study groups, SAF was measured with the AGE Reader (Diagnoptics Technologies, the Netherlands). This method has been described in detail previously [7,13]. SAF measures the accumulation of AGEs in the skin which relationship has been demonstrated by measuring AGEs from skin biopsies [7]. The AGE Reader illuminates a skin surface of approximately 4 cm2, guarded against surrounding light, with an excitation light source whose wavelength is between 300 and 420nm (peak intensity at ~ 370nm). Emission light and reflected excitation light from the skin are measured with an internal spectrometer in the range 300 to 600nm. Measurements were performed on the volar side of the forearm, 10cm below the elbow, at room temperature. SAF was calculated by dividing the average emitted light intensity per nanometer in the range of 420–600 nm by the average exci- tated light intensity per nanometer in the range 300–420 nm and multiplying by 100, and is expressed in arbitrary units (AU). Previous studies have shown an error percentage of around 5–6% when repeated SAF measurement were taken over a single day in control subjects and diabetic patients [7]. The AGE Reader measures SAF also in individuals with a pigmented skin, but only when the UV reflection is above 6%. With a UV reflection below 6% SAF is not given when using the current AGE Reader software. Biochemical measures Blood was collected in the fasting state between 8.00 and 10.00 a.m. and transported to the LifeLines laboratory facility at room temperature or at 4˚C, depending on the sample require- ments. On the day of collection, fasting blood glucose was measured using a hexokinase method. HbA1c (EDTA-anticoagulated) was analyzed using a turbidimetric inhibition immu- noassay on a Cobas Integra 800 CTS analyzer (Roche Diagnostics Nederland BV, Almere, The Netherlands). Creatinine clearance was calculated using the Cockcroft-Gault formula [24]. Cotinine was used as a biomarker for environmental tobacco smoke exposure. Cotinine was measured in non-fasting saliva, plasma and urine specimens which were collected and processed using standardized protocols [22]. In brief, saliva was obtained using the Salivette system following the manufacturer’s recommendations (Sarstedt, Germany). After collection, samples were stored on ice for transportation. Within six hours after sample collection, all samples were centrifuged at 2,500g for 10 min- utes, aliquoted, and stored at -80˚C. Metabolic profiling was achieved using ultra-high-performance liquid-phase chromatogra- phy (UHPLC) and gas-chromatography separation, coupled with tandem mass spectrometry (GCMS) at Metabolon Inc. using established procedures (Durham, NC, USA) [25]. The units represent ion counts as measured by the mass spectrometer, which represent semi-quantitative values. In total, 1,568 different metabolites were detected. Osmolality in saliva and urine was measured for normalization purposes. Anthropometry Using standardized protocols, trained technicians measured body weight with the participant wearing light clothing and without shoes with a 0.1 kg precision. Height was measured without shoes to the nearest 0.5 cm. Body Mass Index (BMI) was calculated as weight (kg) divided by height squared (m2). Results The clinical characteristics of the LifeLines participants according to their smoking status are shown in Table 1. Mean (±SD) age was 48 ± 12 years in never smokers, 53 ± 11 years in former smokers and 46 ± 10 years for current smokers (p<0.0001). BMI was significantly higher among former smokers compared to never and current smokers (p<0.0001). Current smokers had significantly more pack-years smoked compared to former smokers (16.5 vs 7.7 pack- years, p<0.0001). In former smokers, median time since smoking cessation was 16.7 years (interquartile range 8.0–26.4). Mean SAF levels were significantly higher among current smok- ers compared to former and never smokers (in non-diabetic subjects, p<0.0001 and in type 2 diabetic individuals, p<0.001). Moreover, in all smoking groups, subjects with type 2 diabetes had significantly higher SAF levels compared to individuals without diabetes (within never smokers, p = 0.001 and within former- and current smokers, p<0.0001). Fig 1 shows SAF levels for different smoking classes according to the amount of tobacco smoked per day. SAF levels were highest among heavy smokers compared to light smokers, former and never smokers (p<0.0001). Significantly higher SAF levels were found in moderate vs. light smokers (p<0.01) but not in heavy vs. moderate smokers (p = 0.08). The effect of smoking cessation on SAF is shown in Fig 2. SAF Z-scores of former smokers approached levels of never smokers after 15 years of smoking cessation, also after adjusting for age and BMI. At last, we examined the effect of secondhand smoking on SAF (adjusted for age and diabe- tes status) in never smokers (as well as former smokers who have stopped smoking for more than 15 years, which is based on the previous analysis). A gradual increase in SAF with the number of hours reportedly being exposed to secondhand was observed (Fig 3). Individuals who had been exposed to secondhand smoking for more than 11 hours per day had significantly higher SAF levels compared to subjects who had been exposed 1–5 hours per day, but not compared to individuals exposed for 6–10 hours or 0 hours per day. The characteristics of the QMDiab Study participants are shown in Table 1. Mean (±SD) age was 46 ± 13 years in never smokers, 51 ± 12 years among former smokers and 47 ± 11 years in current smokers (p = 0.02). The prevalence of type 2 diabetes was highest among for- mer smokers (57%). Statistical analysis Data are shown as mean ± standard deviation (SD) or median and interquartile range (IQR) in case of non-normally distributed data. SAF Z-scores were calculated based on the total 4 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0179330 June 20, 2017 Smoking behavior and skin autofluorescence population. Univariate Analysis of Variance (ANOVA) with a post-hoc Bonferroni test was used to determine differences between the smoking groups. The effect of secondhand smoking (hours per day) on SAF Z scores (adjusted for age, creatinine clearance and diabetes status) was assessed in never smokers and former smokers. We investigated the effect of smoking ces- sation (in years after smoking abstinence) on SAF (Z-scores) and adjusted in the analysis for age, BMI, creatinine clearance and diabetes status. Multivariable linear regression analysis was performed to examine the association between cotinine biomarkers measured in different specimens and SAF. SPSS (version 22, IBM, Armonk, NY, USA) was used for statistical analy- sis. A P-value <0.05 was considered statistically significant. PLOS ONE \| https://doi.org/10.1371/journal.pone.0179330 June 20, 2017 Smoking behavior and skin autofluorescence Table 1. Subjects characteristics of the study populations. Characteristics LifeLines cohort Never smokers Former smokers Current smokers N 3614 3321 1970 Type 2 diabetes/non-diabetes 106 (3) / 3508 (97) 147 (4) / 3174 (96) 56 (3) / 1914 (97) Age (years) 48 ± 12 53 ± 11* 46 ± 10 Gender (male/female) n (%) 1361 (38) / 2253 (62) 1423 (43) / 1898 (57) 913 (46) / 1057 (54) Body mass index (kg/m2) 26.4 ± 4.4 27.0 ± 4.2 * 26.1 ± 4.2 Creatinine clearance (ml/min) 113 ± 31 110 ± 31 119 ± 32 * Fasting blood glucose type 2 diabetes 7.7 ± 1.9 8.2 ± 2.6 7.8 ± 2.4 non-diabetes 5.0 ± 0.5 5.1 ± 0.5 * 5.0 ± 0.5 HbA1c (%) type 2 diabetes 6.7 ± 1.1 7.0 ± 1.2 6.7 ± 1.2 non-diabetes 5.5 ± 0.3 5.6 ± 0.3 * 5.6 ± 0.3 HbA1c (mmol/mol) type 2 diabetes 50.0 ± 12.3 52.7 ± 12.7 50.2 ± 12.9 non-diabetes 36.9 ± 3.3 37.3 ± 3.5 * 37.3 ± 3.4 Estimated diabetes duration (years) 6.5 (2.9–11.2) 6.6 (3.1–12.3) 4.3 (3.7–7.3) Duration since stop smoking (years) n.a. 16.7 (8.0–26.4) n.a. Pack-years n.a. Results There were no significant differences regarding both salivary cotinine and plasma cotinine among the smoking groups. Both urinary cotinine (p<0.05) as well as cotinine N-oxide (p<0.0001) levels were highest for current smokers compared to former and never- smokers. Five markers for tobacco smoke were evaluated: cotinine in saliva, plasma and urine, and cotinine N-oxide and hydroxy-cotinine in urine. The sensitivity, specificity and the predictive value of the five markers for current smoking are shown in S1 Table. Measured in 148 subjects, cotinine N-oxide had the highest sensitivity (100%) but a relatively low specificity (63.8%) for current smoking. 5 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0179330 June 20, 2017 (Continued) 7.7 (3.2–15.0) 16.5 (9.6–25.0) * SAF (AU) type 2 diabetes 2.27 ± 0.49 2.48 ± 0.53 2.65 ± 0.61 non-diabetes 1.94 ± 0.40 2.09 ± 0.43 2.14 ± 0.48 * Age-adjusted SAF Z-scores type 2 diabetes 0.05 ± 0.16 0.65 ± 0.16 1.98 ± 0.26 * non-diabetes -0.34 ± 0.02 -0.15 ± 0.03 0.57 ± 0.04 *** Characteristics QMDiab cohort Never Smokers Former Smokers Current smokers N 269 61 34 Type 2 diabetes/non-diabetes 133 (49) / 136 (51) 35 (57) / 26 (43) 17 (50) / 17 (50) Age (years) 46 ± 13 51 ± 12 47 ± 11* Gender (male/female) n (%) 101 (38) / 168 (62) 56 (92) / 5 (8) 29 (85) / 5 (15) * Body mass index (kg/m2) 29.9 ± 6.3 28.4 ± 4.2 28.1 ± 4.8 Ethnicity Arab (%) 157 (58) 27 (44) 18 (53) South Asian (%) 75 (28) 25 (41) 12 (35) Filipino (%) 27 (10) 6 (10) 3 (9) Other or mix (%) 10 (4) 3 (5) 1 (3) Serum creatinine (umol/L) 70 ± 17 89 ± 23 * 77 ± 16 HbA1c (%) type 2 diabetes 8.0 ± 1.8 8.3 ± 1.6 8.2 ± 2.4 non-diabetes 5.5 ± 0.4 5.7 ± 0.4 5.7 ± 0.4 HbA1c (mmol/mol) type 2 diabetes 64.3 ± 19.4 67.2 ± 17.5 66.5 ± 26.2 non-diabetes 36.6 ± 4.6 38.5 ± 0.3 38.0 ± 4.7 Estimated diabetes duration (years) 10.5 ± 10.0 11.4 ± 10.6 8.7 ± 10.0 Pack-years n.a. 10.8 ± 14.3 14.0 ± 13.9 Cotinine (saliva, ion counts) 51.4 x 104 ± 45.2 x 104 (n = 23) 34.6 x 104 ± 21.2 x 104 (n = 12) 71.4 x 104 ± 83.8 x 104 (n = 27) Cotinine (plasma, ion counts) 28.1 x 104 ± 14.4 x 104 (n = 17) 22.3 x 104 ± 13.4 x 104 (n = 14) 35.0 x 104 ± 19.8 x 104 (n = 30) Cotinine (urine, ion counts) 30.6 x 104 ± 67.8 x 104 (n = 37) 43.6 x 104 ± 42.2 x 104 (n = 20) 81.3 x 104 ± 87.1 x 104 (n = 32) * (Continued) Table 1. Subjects characteristics of the study populations. (Continued) PLOS ONE \| https://doi.org/10.1371/journal.pone.0179330 June 20, 2017 6 / 15 Smoking behavior and skin autofluorescence Table 1. (Continued) Cotinine N oxide (urine, ion counts) 3.0 x 104 ± 4.8 x 104 (n = 88) 4.1 x 104 ± 3.8 x 104 (n = 28) 12.9 x 104 ± 10.1 x 104 (n = 33) *** Hydroxy-cotinine (urine, ion counts) 191.8 x 104 ± 148.0 x 104 (n = 10) 158.6 x 104 ± 68.1 x 104 (n = 5) 277.8 x 104 ± 275.9 x 104 (n = 14) SAF (AU) type 2 diabetes 2.43 ± 0.72 2.29 ± 0.58 2.49 ± 0.62 non-diabetes 2.10 ± 0.51 2.04 ± 0.58 2.23 ± 0.74 Age-adjusted SAF Z-scores type 2 diabetes 0.24 ± 0.25 0.66 ±0.38 0.50 ± 0.67 non-diabetes -0.10 ± 0.18 -0.72 ± 0.41 -0.04 ± 0.59 Data are presented as means ± standard deviation, or median (interquartile range) and number (%).SAF, Skin autoﬂuorescence; AU, Arbitrary Units. * p<0.05 Data are presented as means ± standard deviation, or median (interquartile range) and number (%).SAF, Skin autoﬂuorescence; AU, Arbitrary Units. * 0 05 https://doi.org/10.1371/journal.pone.0179330.t001 Cotinine N-oxide was detected in 115 out of 316 individuals (36.4%) who reported to be former or never smoker. However, cotinine in plasma (detected in 61 subjects) had the highest predictive value for current smoking (AUC = 0.94, 95% CI: 0.89, 0.98). Table 2 shows the results from the multivariable linear regression analyses between smok- ing variables (smoking status and cotinine biomarkers) and SAF. Current smoking status was significantly associated with higher SAF in the total population. After adjusting for confound- ers, urinary cotinine N-oxide was found positively and significantly (p = 0.03) associated with SAF in the total population and in individuals without diabetes. PLOS ONE \| https://doi.org/10.1371/journal.pone.0179330 June 20, 2017 Discussion In the present study, we combined data from a large population-based cohort study in the Netherlands with metabolomics data from a smaller cohort study in Qatar to examine the effect of smoking intensity, secondhand smoking and smoking cessation on SAF, as well as the association between cotinine biomarkers and SAF. In our previous study, we have already reported have higher SAF levels in smokers com- pared to non-smokers [14]. The present study shows that smoking intensity influences SAF levels as well. SAF levels were highest in heavy smokers compared to light smokers, whereas no significant difference was found between heavy and moderate smokers. Hoonhorst et. al. found a positive association between the amount of pack-years smoked and higher SAF levels among patients with chronic obstructive pulmonary disease indicating a dose-dependent effect [12]. Ohkuma et. al. examined the association between tobacco smoking and glycaemic control in type 2 diabetes patients and reported a significant increase in HbA1c levels with higher smoking intensity [26]. While Cerami et. al. have shown AGEs to be present in aqueous extracts of tobacco, Nicholl et. al. found higher AGEs levels in the lenses and blood vessels of tobacco smokers, demon- strating that tobacco smoke is an exogenous source of AGEs [15,27]. Tobacco smoke is associ- ated with increased systemic oxidative stress, which may in turn contribute to exogenous AGEs formation as well [28]. AGEs have negative effects on insulin sensitivity, which might be due to increased oxidative stress and inflammation [29,30]. In addition, several AGEs can form cross-links within the vascular wall which results in impaired protein function and as a result increased vascular stiffness [31]. Finally, interaction of circulating AGEs with the AGEs receptor (RAGE) stimulates the production of pro-inflammatory cytokines, enhances PLOS ONE \| https://doi.org/10.1371/journal.pone.0179330 June 20, 2017 7 / 15 Smoking behavior and skin autofluorescence PLOS ONE \| https://doi.org/10.1371/journal.pone.0179330 June 20, 2017 Fig 1. Skin autofluorescence stratified for smoking class (LifeLines Cohort Study). Bars represent mean SAF Z scores (adjusted for age, creatinine clearance and diabetes), whiskers reflect standard error of the mean. Never smoker (n = 3670), Former smoker (n = 3321), Light smoker (0–10 gram tobacco per day, n = 878), Moderate smoker (10–20 gram tobacco per day, n = 537), heavy smoker (>20 gram tobacco per day, n = 475). SAF, skin autofluorescence; AU, arbitrary units; NS, not significant. https://doi.org/10.1371/journal.pone.0179330.g001 https://doi.org/10.1371/journal.pone.0179330.g001 oxidative stress [32] and causes endothelial dysfunction [33]. Therefore, tobacco smoke as an exogenous source of AGEs accumulation, may play a potential role in the underlying patho- physiological mechanism of type 2 diabetes [13] and a wide range of cardiovascular diseases, including (sub)clinical atherosclerosis, [34] coronary artery [35] and peripheral artery disease [36], Using metabolomics data we have shown that cotinine N-oxide, a biomarker for environ- mental tobacco smoke exposure, is significantly associated with higher SAF levels in a group of individuals without diabetes. Therefore, urinary cotinine N-oxide might be used as an alterna- tive way for questionnaires in demonstrating the effect of tobacco smoking on SAF. Remark- ably, we found high levels of cotinine in never smokers, particularly in urine and saliva. Cotinine concentrations are four to five times higher in urine than in plasma and saliva which makes urine more eligible for detecting low-level exposure [37]. This could explain our detected sensitivity of 100% for cotinine N-oxide and supports our thoughts regarding the PLOS ONE \| https://doi.org/10.1371/journal.pone.0179330 June 20, 2017 8 / 15 Fig 2. Effect of smoking cessation on skin autofluorescence in former smokers participating in the LifeLines study. Dots show mean SAF Z-scores (adjusted for age, BMI, creatinine clearance and diabetes status). Whiskers reflect standard error of the mean. SAF, skin autofluorescence https://doi org/10 1371/journal pone 0179330 g002 Smoking behavior and skin autofluorescence Smoking behavior and skin autofluorescence PLOS ONE \| https://doi.org/10.1371/journal.pone.0179330 June 20, 2017 Fig 2. Effect of smoking cessation on skin autofluorescence in former smokers participating in the LifeLines study. Dots show mean SAF Z-scores (adjusted for age, BMI, creatinine clearance and diabetes status). Whiskers reflect standard error of the mean. SAF, skin autofluorescence Fig 2. Effect of smoking cessation on skin autofluorescence in former smokers participating in the LifeLines study. Dots show mean SAF Z-scores (adjusted for age, BMI, creatinine clearance and diabetes status). Whiskers reflect standard error of the mean. SAF, skin autofluorescence https://doi.org/10.1371/journal.pone.0179330.g002 high levels of cotinine N-oxide in never smokers caused by secondhand smoking. Moreover, data from the third National Health and Nutrition Examination Survey (NHANES) suggest that around 90% of never smokers have detectable levels of serum cotinine [38]. It has been reported that the serum cut-off point to discriminate adult active smokers from non-smokers is 3 ng/ml [39]. Several explanations might be given for the fact that we observed high levels of cotinine in non-smokers. Firstly, in Qatar and the Middle-East, many public areas (such as res- taurants) in the Qatar and the Middle-East still allow smoking, increasing the exposure to sec- ondhand smoking in non-smokers. Secondly, we cannot exclude the possibility of heavy secondhand smoke exposure for some of the individuals, as well as the fact of misclassification of subjects smoking status according to their self-reports (e.g. smokers who occasionally smoke might be in fact considered as active smokers). In the study from Benowitz et. al, some of the individuals who reported to be a non-smoker had cotinine levels similar to levels of active smokers [39]. According to the cotinine levels reported in our study, it is likely that at least a proportion of non-smokers were in fact active smokers. Thirdly, racial/ethnic differ- ences in the rate of metabolism of nicotine and cotinine have been described [40]. This PLOS ONE \| https://doi.org/10.1371/journal.pone.0179330 June 20, 2017 9 / 15 Smoking behavior and skin autofluorescence Fig 3. Effect of secondhand smoking on skin autofluorescence in never- and former smokers participating in the LifeLines study. Bars represent mean SAF Z scores (adjusted for age, creatinine clearance and diabetes status) in never smokers and former smokers who stopped smoking for more than 15 years, whiskers reflect standard error of the mean. 0 hours (n = 4213), 1–5 hours (n = 676), 6–10 hours (n = 78), >11 hours (n = 15). SAF, skin autofluorescence; Arbitrary Units, AU; NS, not significant. PLOS ONE \| https://doi.org/10.1371/journal.pone.0179330 June 20, 2017 Smoking behavior and skin autofluorescence Table 2. Smoking status and cotinine markers related to skin autofluorescence using multivariable analyses stratified by diabetes status (QMDiab Study). Coefﬁcient Beta (CI) P value Coefﬁcient Beta(CI) P value Coefﬁcient Beta (CI) P value All subjects Non-diabetes Type 2 diabetes Questionnaire Current smoker 0.24 (0.04, 0.45) 0.02 0.22 (-0.03, 0.47) 0.08 0.35 (0.00, 0.69) 0.05 Former smoker 0.05 (-0.12, 0.21) 0.59 0.17 (-0.19, 0.22) 0.87 0.08 (-0.19, 0.35) 0.56 Cotinine markers Cotinine saliva 0.01 (-0.05, 0.07) 0.85 0.02 (-0.04, 0.08) 0.53 -0.02 (-0.15, 0.11) 0.75 Cotinine plasma 0.05 (-0.01, 0.10) 0.12 0.07 (-0.01, 0.15) 0.07 0.04 (-0.05, 0.13) 0.40 Cotinine urine 0.05 (-0.00, 0.11) 0.07 0.06 (-0.01, 0.12) 0.06 0.08 (-0.05, 0.22) 0.23 Cotinine N Oxide (urine) 0.06 (0.01, 0.12) 0.03 0.08 (-0.01, 0.15) 0.03 0.06 (-0.04, 0.16) 0.21 Hydroxy Cotinine (urine) 0.04 (-0.02, 0.10) 0.17 0.04 (-0.03, 0.10) 0.29 0.05 (-0.05, 0.16) 0.32 Values represent regression coefﬁcients (95% conﬁdence interval) and their corresponding p-values. Model was adjusted for age, gender, ethnicity, body mass index, reﬂectance, creatinine clearance and the presence of type 2 diabetes. For categorical or dichotomous variables, the effect estimates represent the difference in skin AF compared to the reference group. Markers in saliva and urine were normalized by osmolality and subsequently Z-score normalized to make effect sizes comparable. Thus, effect estimates represent change in SAF per standard deviation increase of cotinine marker. Cotinine metabolites were measured in n = 330 (saliva), n = 358 (plasma), and n = 360 (urine). htt //d i /10 1371/j l 0179330 t002 markers related to skin autofluorescence using multivariable analyses stratified by diabetes status (QMDiab Table 2. Smoking status and cotinine markers related to skin autofluorescence using multivariable analyses stratified by diabetes status (QMDiab Study). Coefﬁcient Beta (CI) P value Coefﬁcient Beta(CI) P value Coefﬁcient Beta (CI) P value All subjects Non-diabetes Type 2 diabetes Questionnaire Current smoker 0.24 (0.04, 0.45) 0.02 0.22 (-0.03, 0.47) 0.08 0.35 (0.00, 0.69) 0.05 Former smoker 0.05 (-0.12, 0.21) 0.59 0.17 (-0.19, 0.22) 0.87 0.08 (-0.19, 0.35) 0.56 Cotinine markers Cotinine saliva 0.01 (-0.05, 0.07) 0.85 0.02 (-0.04, 0.08) 0.53 -0.02 (-0.15, 0.11) 0.75 Cotinine plasma 0.05 (-0.01, 0.10) 0.12 0.07 (-0.01, 0.15) 0.07 0.04 (-0.05, 0.13) 0.40 Cotinine urine 0.05 (-0.00, 0.11) 0.07 0.06 (-0.01, 0.12) 0.06 0.08 (-0.05, 0.22) 0.23 Cotinine N Oxide (urine) 0.06 (0.01, 0.12) 0.03 0.08 (-0.01, 0.15) 0.03 0.06 (-0.04, 0.16) 0.21 Hydroxy Cotinine (urine) 0.04 (-0.02, 0.10) 0.17 0.04 (-0.03, 0.10) 0.29 0.05 (-0.05, 0.16) 0.32 Table 2. Smoking status and cotinine markers related to skin autofluorescence using multivariable ana Study) Values represent regression coefﬁcients (95% conﬁdence interval) and their corresponding p-values. Model was adjusted for age, gender, ethnicity, body mass index, reﬂectance, creatinine clearance and the presence of type 2 diabetes. For categorical or dichotomous variables, the effect estimates represent the difference in skin AF compared to the reference group. Markers in saliva and urine were normalized by osmolality and subsequently Z-score normalized to make effect sizes comparable. Thus, effect estimates represent change in SAF per standard deviation increase of cotinine marker. Cotinine metabolites were measured in n = 330 (saliva), n = 358 (plasma), and n = 360 (urine). https://doi.org/10.1371/journal.pone.0179330.t002 active smokers [43]. Accumulating evidence has shown that secondhand smoking increases the risk of type 2 diabetes [44] and coronary artery disease [45,46] by increasing oxidative stress, systemic inflammation and endothelial dysfunction [28,47]. Furthermore, tobacco smoking may contribute to insulin resistance [48] and has been demonstrated to be associated with a higher risk of pancreatitis and pancreatic cancer suggesting that chemical compounds in tobacco smoke may have direct toxic effects on pancreatic β cells [49]. In a similar way as active smoking, it is highly likely that enhanced AGEs formation is involved in the pathophysi- ological pathway by which secondhand smoking increases the risk of both type 2 diabetes and cardiovascular diseases. To our knowledge, this is the first study showing that quitting smoking has a reversible effect on SAF levels. We observed that SAF Z-scores of former smokers gradually decreased with increasing years since quitting smoking, even after adjusting for BMI. https://doi org/10 1371/journal pone 0179330 g003 Fig 3. Effect of secondhand smoking on skin autofluorescence in never- and former smokers participating in the LifeLines study. Bars represent mean SAF Z scores (adjusted for age, creatinine clearance and diabetes status) in never smokers and former smokers who stopped smoking for more than 15 years, whiskers reflect Fig 3. Effect of secondhand smoking on skin autofluorescence in never- and former smokers participating in the LifeLines study. Bars represent mean SAF Z scores (adjusted for age, creatinine clearance and diabetes status) in never smokers and former smokers who stopped smoking for more than 15 years, whiskers reflect standard error of the mean. 0 hours (n = 4213), 1–5 hours (n = 676), 6–10 hours (n = 78), >11 hours (n = 15). SAF, skin autofluorescence; Arbitrary Units, AU; NS, not significant. https://doi.org/10.1371/journal.pone.0179330.g003 supports our thoughts about secondhand exposure in (non-caucasian) non-smokers having a slower nicotine metabolism, and as a result, have elevated cotinine levels. Fourthly, exposure to thirdhand smoke might also be an explanation for the high cotinine levels observed in non-smokers. Thirdhand smoke is generally considered to be residual tobacco smoke left on (indoor) surfaces such as sofas and wallpapers. Finally, several kinds of nutrition, such as vegetables and black tea, contain small amounts of nicotine but these levels are almost negligible. In addition to active smoking, we were able to demonstrate that secondhand smoking is associated with higher SAF levels as well. Although we did not find all groups to be signifi- cantly different, a marked increase in SAF levels with higher exposure to secondhand smoking was observed. From these results, we learn that secondhand smoking can be added to the other determinants associated with SAF as demonstrated in our previous study [14]. Secondhand smoke is a combination of sidestream smoke and mainstream smoke which is exhaled smoke by an active smoker [41]. Sidestream smoke contains a relatively higher con- centration of unfiltered toxic gases and small respirable particles than mainstream smoke [42] and is thus potentially more hazardous compared to the smoke which is directly inhaled by 10 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0179330 June 20, 2017 Values represent regression coefﬁcients (95% conﬁdence interval) and their corresponding p-values. Model was adjusted for age, gender, ethnicity, body mass index, reﬂectance, creatinine clearance and the presence of type 2 diabetes. For categorical or dichotomous variables, the effect estimates represent the difference in skin AF compared to the reference group. Markers in saliva and urine were normalized by osmolality and subsequently Z-score normalized to make effect sizes comparable. Thus, effect estimates represent change in SAF per standard deviation increase of cotinine marker. Cotinine metabolites were measured in n = 330 (saliva), n = 358 (plasma), and n = 360 (urine). PLOS ONE \| https://doi.org/10.1371/journal.pone.0179330 June 20, 2017 Conclusions This study clearly demonstrated that secondhand smoking is associated with higher SAF levels whereas smoking cessation led to a gradual normalization of SAF levels as the years since smoking abstinence increase. Moreover, we have demonstrated that urinary cotinine N-oxide, a biomarker for environmental tobacco smoke, is significantly associated with higher SAF lev- els and might therefore be used as an alternative way for questionnaires to examine the effect secondhand smoking on SAF. These findings should be taken into consideration in future studies on SAF or using SAF as a screening tool for populations at risk for diabetes and cardio- vascular diseases. Supporting information S1 Table. Sensitivity and specificity for cotinine markers in current smokers (QMDiab Study). Missing saliva collection (n = 44), plasma collection (n = 16), urine collection (n = 14). Missing smoking data (n = 10). (DOCX) S1 Table. Sensitivity and specificity for cotinine markers in current smokers (QMDiab Study). Missing saliva collection (n = 44), plasma collection (n = 16), urine collection (n = 14) Missing smoking data (n = 10). Smoking behavior and skin autofluorescence Our study has some strengths and limitations. Firstly, part of this study is based on a large number of participants, resulting in a good statistical power and the ability to perform analysis for different smoking status and classes. A limitation of the LifeLines Study is the potential misclassification of individuals with regards to their smoking status as we cannot completely rule out misreporting of smoking habits or history. Secondly, we were not able to stratify the analyses for diabetes status since the number of subjects with type 2 diabetes was too small. A limitation of the QMDiab study is the small number of study participants which may have caused a reduced statistical power in finding significant associations. Since the QMDiab study did not include questions regarding secondhand smoking, we were not able to assess any correlation with cotinine biomarkers. Acknowledgments This work was supported by Netherlands Consortium for Healthy Ageing (NCHA), Bio- SHaRE-EU, Biobank Standardisation and Harmonisation for research excellence in the Euro- pean Union. Bioresource research impact factor BRIF4568. PLOS ONE \| https://doi.org/10.1371/journal.pone.0179330 June 20, 2017 Conceptualization: RvW MMK DOMK BHRW. Investigation: RvW MMK DOMK. After around 15 years of smoking abstinence, SAF Z-scores had returned to levels of never smokers. Ohkuma et. al. have shown a decrease in HbA1c levels in subjects with type 2 diabe- tes as the years since quitting smoking increased [26]. Since HbA1c is actually an intermediate product of glycation, our findings might be considered in line with their results. Our data provide some indication that at least part of the effect of tobacco smoking on AGEs accumulation is reversible in individuals who quit smoking, indicating a beneficial effect of smoking cessation on both metabolic and glycaemic control. It has been reported that smoking cessation reduces the risk of type 2 diabetes [50] and car- diovascular disease [51]. Paradoxically, smoking cessation is commonly associated with subse- quent weight gain [52] which in turn might attenuate the beneficial effects of smoking cessation. Several potential mechanisms may contribute to a lower risk of type 2 diabetes and cardiovascular disease associated with smoking cessation, including improved insulin sensitiv- ity and lipoprotein levels as well as reduced inflammation [53–55]. Considering the role of increased AGE accumulation as a consequence of tobacco smoking in its association with type 2 diabetes and cardiovascular disease, it is likely that reduced formation and accumulation of AGEs may result in reduced type 2 diabetic and cardiovascular risk. However, our hypothesis needs to be confirmed by future translational studies. 11 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0179330 June 20, 2017 References 1. Baynes JW, Thorpe SR. Glycoxidation and lipoxidation in atherogenesis. Free Radic Biol Med. 2000; 28(12):1708–1716 PMID: 10946212. 2. Monnier VM. Nonenzymatic glycosylation, the Maillard reaction and the aging process. J Gerontol. 1990; 45(4):B105–11:PMID: 2195101. 3. Dunn JA, McCance DR, Thorpe SR, Lyons TJ, Baynes JW. Age-dependent accumulation of N epsilon- (carboxymethyl)lysine and N epsilon-(carboxymethyl)hydroxylysine in human skin collagen. Biochemis- try. 1991; 30(5):1205–1210:PMID: 1899338. 4. Dyer DG, Dunn JA, Thorpe SR, Bailie KE, Lyons TJ, McCance DR, et al. Accumulation of Maillard reac- tion products in skin collagen in diabetes and aging. J Clin Invest. 1993; 91(6):2463–2469 https://doi. org/10.1172/JCI116481 PMID: 8514858. 5. Brownlee M. Lilly Lecture 1993. Glycation and diabetic complications. Diabetes. 1994; 43(6):836–841: PMID: 8194672. 6. Meerwaldt R, Zeebregts CJ, Navis G, Hillebrands JL, Lefrandt JD, Smit AJ. Accumulation of advanced glycation end products and chronic complications in ESRD treated by dialysis. Am J Kidney Dis. 2009; 53(1):138–150 https://doi.org/10.1053/j.ajkd.2008.08.031 PMID: 19036487. 7. Meerwaldt R, Graaff R, Oomen PH, Links TP, Jager JJ, Alderson NL, et al. Simple non-invasive assess- ment of advanced glycation endproduct accumulation. Diabetologia. 2004; 47(7):1324–1330 https:// doi.org/10.1007/s00125-004-1451-2 PMID: 15243705. 8. Meerwaldt R, Lutgers HL, Links TP, Graaff R, Baynes JW, Gans RO, et al. Skin autofluorescence is a strong predictor of cardiac mortality in diabetes. Diabetes Care. 2007; 30(1):107–112 https://doi.org/ 10.2337/dc06-1391 PMID: 17192342. 9. Lutgers HL, Graaff R, Links TP, Ubink-Veltmaat LJ, Bilo HJ, Gans RO, et al. Skin autofluorescence as a noninvasive marker of vascular damage in patients with type 2 diabetes. Diabetes Care. 2006; 29(12): 2654–2659 https://doi.org/10.2337/dc05-2173 PMID: 17130200. 10. Meerwaldt R, Hartog JW, Graaff R, Huisman RJ, Links TP, den Hollander NC, et al. Skin autofluores- cence, a measure of cumulative metabolic stress and advanced glycation end products, predicts mortal- ity in hemodialysis patients. J Am Soc Nephrol. 2005; 16(12):3687–3693 https://doi.org/10.1681/ASN. 2005020144 PMID: 16280473. 11. de Vos LC, Boersema J, Mulder DJ, Smit AJ, Zeebregts CJ, Lefrandt JD. Skin autofluorescence as a measure of advanced glycation end products deposition predicts 5-year amputation in patients with peripheral artery disease. Arterioscler Thromb Vasc Biol. 2015; 35(6):1532–1537 https://doi.org/10. 1161/ATVBAHA.115.305407 PMID: 25882066. 12. Hoonhorst SJ, Lo Tam Loi AT, Hartman JE, Telenga ED, van den Berge M, Koenderman L, et al. Advanced glycation end products in the skin are enhanced in COPD. Metabolism. 2014; 63(9): 1149–1156 https://doi.org/10.1016/j.metabol.2014.06.006 PMID: 25034386. 13. Koetsier M, Lutgers HL, de Jonge C, Links TP, Smit AJ, Graaff R. Supervision: BHRW DOMK. 12 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0179330 June 20, 2017 Smoking behavior and skin autofluorescence PLOS ONE \| https://doi.org/10.1371/journal.pone.0179330 June 20, 2017 References Reference values of skin autofluores- cence. Diabetes Technol Ther. 2010; 12(5):399–403 https://doi.org/10.1089/dia.2009.0113 PMID: 20388050. 14. van Waateringe RP, Slagter SN, van der Klauw MM, van Vliet-Ostaptchouk JV, Graaff R, Paterson AD, et al. Lifestyle and clinical determinants of skin autofluorescence in a population-based cohort study. Eur J Clin Invest. 2016; 46(5):481–490 https://doi.org/10.1111/eci.12627 PMID: 27002914. 15. Cerami C, Founds H, Nicholl I, Mitsuhashi T, Giordano D, Vanpatten S, et al. Tobacco smoke is a source of toxic reactive glycation products. Proc Natl Acad Sci U S A. 1997; 94(25):13915–13920: PMID: 9391127. 16. Connor Gorber S, Schofield-Hurwitz S, Hardt J, Levasseur G, Tremblay M. The accuracy of self- reported smoking: a systematic review of the relationship between self-reported and cotinine-assessed smoking status. Nicotine Tob Res. 2009; 11(1):12–24 https://doi.org/10.1093/ntr/ntn010 PMID: 19246437. PLOS ONE \| https://doi.org/10.1371/journal.pone.0179330 June 20, 2017 13 / 15 Smoking behavior and skin autofluorescence 17. Benowitz NL. Biomarkers of environmental tobacco smoke exposure. Environ Health Perspect. 1999; 107 Suppl 2:349–355:PMID: 10350520. 18. Benowitz NL. Cotinine as a biomarker of environmental tobacco smoke exposure. Epidemiol Rev. 1996; 18(2):188–204:PMID: 9021312. 19. Seccareccia F, Zuccaro P, Pacifici R, Meli P, Pannozzo F, Freeman KM, et al. Serum cotinine as a marker of environmental tobacco smoke exposure in epidemiological studies: the experience of the MATISS project. Eur J Epidemiol. 2003; 18(6):487–492:PMID: 12908713. 20. Stolk RP, Rosmalen JG, Postma DS, de Boer RA, Navis G, Slaets JP, et al. Universal risk factors for multifactorial diseases: LifeLines: a three-generation population-based study. Eur J Epidemiol. 2008; 23(1):67–74 https://doi.org/10.1007/s10654-007-9204-4 PMID: 18075776. 21. Eny KM, Lutgers HL, Maynard J, Klein BE, Lee KE, Atzmon G, et al. GWAS identifies an NAT2 acetyla- tor status tag single nucleotide polymorphism to be a major locus for skin fluorescence. Diabetologia. 2014; 57(8):1623–1634 https://doi.org/10.1007/s00125-014-3286-9 PMID: 24934506. 22. Mook-Kanamori DO, Selim MM, Takiddin AH, Al-Homsi H, Al-Mahmoud KA, Al-Obaidli A, et al. 1,5- Anhydroglucitol in saliva is a noninvasive marker of short-term glycemic control. J Clin Endocrinol Metab. 2014; 99(3):E479–83 https://doi.org/10.1210/jc.2013-3596 PMID: 24423354. 23. Mook-Kanamori MJ, Selim MM, Takiddin AH, Al-Homsi H, Al-Mahmoud KA, Al-Obaidli A, et al. Ethnic and gender differences in advanced glycation end products measured by skin auto-fluorescence. Der- matoendocrinol. 2013; 5(2):325–330 https://doi.org/10.4161/derm.26046 PMID: 24194974. 24. Cockcroft DW, Gault MH. Prediction of creatinine clearance from serum creatinine. Nephron. 1976; 16(1):31–41:PMID: 1244564. 25. Evans AM, DeHaven CD, Barrett T, Mitchell M, Milgram E. References Integrated, nontargeted ultrahigh perfor- mance liquid chromatography/electrospray ionization tandem mass spectrometry platform for the identi- fication and relative quantification of the small-molecule complement of biological systems. Anal Chem. 2009; 81(16):6656–6667 https://doi.org/10.1021/ac901536h PMID: 19624122. 26. Ohkuma T, Iwase M, Fujii H, Kaizu S, Ide H, Jodai T, et al. Dose- and time-dependent association of smoking and its cessation with glycemic control and insulin resistance in male patients with type 2 dia- betes mellitus: the Fukuoka Diabetes Registry. PLoS One. 2015; 10(3):e0122023 https://doi.org/10. 1371/journal.pone.0122023 PMID: 25822499. 27. Nicholl ID, Stitt AW, Moore JE, Ritchie AJ, Archer DB, Bucala R. Increased levels of advanced glycation endproducts in the lenses and blood vessels of cigarette smokers. Mol Med. 1998; 4(9):594–601: PMID: 9848076. 28. Yanbaeva DG, Dentener MA, Creutzberg EC, Wesseling G, Wouters EF. Systemic effects of smoking. Chest. 2007; 131(5):1557–1566 https://doi.org/10.1378/chest.06-2179 PMID: 17494805. 29. Unoki H, Bujo H, Yamagishi S, Takeuchi M, Imaizumi T, Saito Y. Advanced glycation end products attenuate cellular insulin sensitivity by increasing the generation of intracellular reactive oxygen species in adipocytes. Diabetes Res Clin Pract. 2007; 76(2):236–244 https://doi.org/10.1016/j.diabres.2006.09. 016 PMID: 17097186. 30. Cai W, Ramdas M, Zhu L, Chen X, Striker GE, Vlassara H. Oral advanced glycation endproducts (AGEs) promote insulin resistance and diabetes by depleting the antioxidant defenses AGE receptor-1 and sirtuin 1. Proc Natl Acad Sci U S A. 2012; 109(39):15888–15893 https://doi.org/10.1073/pnas. 1205847109 PMID: 22908267. 31. Goldin A, Beckman JA, Schmidt AM, Creager MA. Advanced glycation end products: sparking the development of diabetic vascular injury. Circulation. 2006; 114(6):597–605 https://doi.org/10.1161/ CIRCULATIONAHA.106.621854 PMID: 16894049. 32. Schmidt AM, Hasu M, Popov D, Zhang JH, Chen J, Yan SD, et al. Receptor for advanced glycation end products (AGEs) has a central role in vessel wall interactions and gene activation in response to circu- lating AGE proteins. Proc Natl Acad Sci U S A. 1994; 91(19):8807–8811:PMID: 8090728. 33. Basta G, Lazzerini G, Massaro M, Simoncini T, Tanganelli P, Fu C, et al. Advanced glycation end prod- ucts activate endothelium through signal-transduction receptor RAGE: a mechanism for amplification of inflammatory responses. Circulation. 2002; 105(7):816–822:PMID: 11854121. 34. den Dekker MA, Zwiers M, van den Heuvel ER, de Vos LC, Smit AJ, Zeebregts CJ, et al. Skin autofluor- escence, a non-invasive marker for AGE accumulation, is associated with the degree of atherosclero- sis. PLoS One. 2013; 8(12):e83084 https://doi.org/10.1371/journal.pone.0083084 PMID: 24376641. 35. PLOS ONE \| https://doi.org/10.1371/journal.pone.0179330 June 20, 2017 References Mulder DJ, van Haelst PL, Gross S, de Leeuw K, Bijzet J, Graaff R, et al. Skin autofluorescence is ele- vated in patients with stable coronary artery disease and is associated with serum levels of neopterin and the soluble receptor for advanced glycation end products. Atherosclerosis. 2008; 197(1):217–223 https://doi.org/10.1016/j.atherosclerosis.2007.03.027 PMID: 17499742. 14 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0179330 June 20, 2017 Smoking behavior and skin autofluorescence 36. de Vos LC, Noordzij MJ, Mulder DJ, Smit AJ, Lutgers HL, Dullaart RP, et al. Skin autofluorescence as a measure of advanced glycation end products deposition is elevated in peripheral artery disease. Arter- ioscler Thromb Vasc Biol. 2013; 33(1):131–138 https://doi.org/10.1161/ATVBAHA.112.300016 PMID: 23139292. 36. de Vos LC, Noordzij MJ, Mulder DJ, Smit AJ, Lutgers HL, Dullaart RP, et al. Skin autofluorescence as a measure of advanced glycation end products deposition is elevated in peripheral artery disease. Arter- ioscler Thromb Vasc Biol. 2013; 33(1):131–138 https://doi.org/10.1161/ATVBAHA.112.300016 PMID: 23139292. 37. Avila-Tang E, Al-Delaimy WK, Ashley DL, Benowitz N, Bernert JT, Kim S, et al. Assessing secondhand smoke using biological markers. Tob Control. 2013; 22(3):164–171 https://doi.org/10.1136/ tobaccocontrol-2011-050298 PMID: 22940677. 38. Pirkle JL, Flegal KM, Bernert JT, Brody DJ, Etzel RA, Maurer KR. Exposure of the US population to environmental tobacco smoke: the Third National Health and Nutrition Examination Survey, 1988 to 1991. JAMA. 1996; 275(16):1233–1240:PMID: 8601954. 39. Benowitz NL, Bernert JT, Caraballo RS, Holiday DB, Wang J. Optimal serum cotinine levels for distin- guishing cigarette smokers and nonsmokers within different racial/ethnic groups in the United States between 1999 and 2004. Am J Epidemiol. 2009; 169(2):236–248 https://doi.org/10.1093/aje/kwn301 PMID: 19019851. 40. Wagenknecht LE, Cutter GR, Haley NJ, Sidney S, Manolio TA, Hughes GH, et al. Racial differences in serum cotinine levels among smokers in the Coronary Artery Risk Development in (Young) Adults study. Am J Public Health. 1990; 80(9):1053–1056:PMID: 2382740. 41. Taylor AE, Johnson DC, Kazemi H. Environmental tobacco smoke and cardiovascular disease. A posi- tion paper from the Council on Cardiopulmonary and Critical Care, American Heart Association. Circula- tion. 1992; 86(2):699–702:PMID: 1638735. 42. Flouris AD, Vardavas CI, Metsios GS, Tsatsakis AM, Koutedakis Y. Biological evidence for the acute health effects of secondhand smoke exposure. Am J Physiol Lung Cell Mol Physiol. 2010; 298(1): L3–L12 https://doi.org/10.1152/ajplung.00215.2009 PMID: 19767410. 43. Schick S, Glantz S. Philip Morris toxicological experiments with fresh sidestream smoke: more toxic than mainstream smoke. Tob Control. 2005; 14(6):396–404 https://doi.org/10.1136/tc.2005.011288 PMID: 16319363. 44. PLOS ONE \| https://doi.org/10.1371/journal.pone.0179330 June 20, 2017 References Wang Y, Ji J, Liu YJ, Deng X, He QQ. Passive smoking and risk of type 2 diabetes: a meta-analysis of prospective cohort studies. PLoS One. 2013; 8(7):e69915 https://doi.org/10.1371/journal.pone. 0069915 PMID: 23922856. 45. Barnoya J, Glantz SA. Cardiovascular effects of secondhand smoke: nearly as large as smoking. Circu- lation. 2005; 111(20):2684–2698 https://doi.org/10.1161/CIRCULATIONAHA.104.492215 PMID: 15911719. 46. Iversen B, Jacobsen BK, Lochen ML. Active and passive smoking and the risk of myocardial infarction in 24,968 men and women during 11 year of follow-up: the Tromso Study. Eur J Epidemiol. 2013; 28(8):659–667 https://doi.org/10.1007/s10654-013-9785-z PMID: 23443581. 47. Howard DJ, Ota RB, Briggs LA, Hampton M, Pritsos CA. Oxidative stress induced by environmental tobacco smoke in the workplace is mitigated by antioxidant supplementation. Cancer Epidemiol Bio- markers Prev. 1998; 7(11):981–988:PMID: 9829705. 48. Facchini FS, Hollenbeck CB, Jeppesen J, Chen YD, Reaven GM. Insulin resistance and cigarette smoking. Lancet. 1992; 339(8802):1128–1130:PMID: 1349365. 49. Chowdhury P, Rayford PL, Chang LW. Pathophysiological effects of nicotine on the pancreas. Proc Soc Exp Biol Med. 1998; 218(3):168–173:PMID: 9648934. 50. Wannamethee SG, Shaper AG, Perry IJ, British Regional Heart Study. Smoking as a modifiable risk factor for type 2 diabetes in middle-aged men. Diabetes Care. 2001; 24(9):1590–1595:PMID: 11522704. 51. Jha P, Ramasundarahettige C, Landsman V, Rostron B, Thun M, Anderson RN, et al. 21st-century haz- ards of smoking and benefits of cessation in the United States. N Engl J Med. 2013; 368(4):341–350 https://doi.org/10.1056/NEJMsa1211128 PMID: 23343063. 52. Filozof C, Fernandez Pinilla MC, Fernandez-Cruz A. Smoking cessation and weight gain. Obes Rev. 2004; 5(2):95–103 https://doi.org/10.1111/j.1467-789X.2004.00131.x PMID: 15086863. 53. Gepner AD, Piper ME, Johnson HM, Fiore MC, Baker TB, Stein JH. Effects of smoking and smoking cessation on lipids and lipoproteins: outcomes from a randomized clinical trial. Am Heart J. 2011; 161(1):145–151 https://doi.org/10.1016/j.ahj.2010.09.023 PMID: 21167347. 54. Eliasson B, Attvall S, Taskinen MR, Smith U. Smoking cessation improves insulin sensitivity in healthy middle-aged men. Eur J Clin Invest. 1997; 27(5):450–456:PMID: 9179554. 55. Johnson HM, Gossett LK, Piper ME, Aeschlimann SE, Korcarz CE, Baker TB, et al. Effects of smoking and smoking cessation on endothelial function: 1-year outcomes from a randomized clinical trial. J Am Coll Cardiol. 2010; 55(18):1988–1995 https://doi.org/10.1016/j.jacc.2010.03.002 PMID: 20236788. 15 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0179330 June 20, 2017
https://openalex.org/W1980933686	https://molecularneurodegeneration.biomedcentral.com/counter/pdf/10.1186/1750-1326-7-S1-L25	English	null	Selenite benefits embryonic stem cells therapy in the animal models of Parkinson’s disease through inhibiting inflammation	Molecular neurodegeneration	2,012	cc-by	513	* Correspondence: jqding18@yahoo.com; chen_sd@medmail.com.cn † Contributed equally Department of Neurology & Institute of Neurology, Ruijin Hospital affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China LECTURE PRESENTATION Open Access Open Access Tian et al. Molecular Neurodegeneration 2012, 7(Suppl 1):L25 http://www.molecularneurodegeneration.com/content/7/S1/L25 Published: 7 February 2012 Selenite benefits embryonic stem cells therapy in the animal models of Parkinson’s disease through inhibiting inflammation From 2011 International Conference on Molecular Neurodegeneration Shanghai, China. 22-24 September 2011 Embryonic stem cells (ESCs) transplantation is a potential therapeutic approach for Parkinson’s disease. However, the key problems the therapy is facing are the efficiency of differentiation into dopaminergic (DA) neurons and the low survival of the transplanted DA neurons. In the pre- sent study, mouse ESC were effectively differentiated into DA neurons by serum free method and were transplanted into 6-OHDA lesioned striatum of PD rats. We found reduced viability of DA neurons after graft, being accom- panied by activated microglia and high levels of TNF-a and iNOS. This suggested that inflammation might be an underlying mechanism for decreased cells viability. In the following in vitro assay, selenite, the source of essential micronutrient selenium, was tested to inhibit inflamma- tory activation of BV2 microglia cells. Furthermore, the anti-inflammatory effects of selenite in animals after cells transplantation were investigated. In PD rats treated by selenite, microglia activation after transplantation was inhibited in the graft niche, and the levels of TNF-a and iNOS were effectively abated nearly by 30% and 50%. The viability of implanted DA neurons was also remarkably improved after selenite treatment, with favored behavior recovery of PD rats. Therefore, selenite might benefit embryonic stem cells therapy in Parkinson’s disease through inhibiting inflammation. doi:10.1186/1750-1326-7-S1-L25 Cite this article as: Tian et al.: Selenite benefits embryonic stem cells therapy in the animal models of Parkinson’s disease through inhibiting inflammation. Molecular Neurodegeneration 2012 7(Suppl 1):L25. 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 his is an Open Access article distributed under the terms of the Creative Commons icenses/by/2.0), which permits unrestricted use, distribution, and reproduction in erly cited. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: © 2012 Tian et al; licensee BioMed Central Ltd. Selenite benefits embryonic stem cells therapy in the animal models of Parkinson’s disease through inhibiting inflammation 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.
https://openalex.org/W2115673262	https://www.repo.uni-hannover.de/bitstream/123456789/1451/1/adgeo-27-91-2010.pdf	English	null	Influence of spatial interpolation methods for climate variables on the simulation of discharge and nitrate fate with SWAT	Advances in geosciences	2,010	cc-by	6,086	1 Introduction Climate data as input for ecohydrological modeling are usu- ally measured at individual points. For the modeling of larger catchments, these data are needed on subbasin or raster basis, consistently covering the whole study area. Thus, spatial in- terpolation is necessary to upscale the data from point to area. A large number of different interpolation methods has been proposed over time (for a recent review see e.g. Tveito et al., 2006). From the early, simple nearest neighbour technique or Thiessen polygon method (Thiessen, 1911) via the inverse distance weighting (e.g. Shepard, 1968), interpolation me- thods evolved to more sophisticated geostatistical methods like kriging in all its variants (Goovaerts, 1997), which are becoming more and more popular. Many other techniques e.g. based on splines (Hutchinson, 1998a,b) or genetic algo- rithms (Demyanov et al., 1998; Huang et al., 1998) have also been applied recently. I. Automated calibration of the model with a mixed cli- mate data set and consecutive application of the four interpolated data sets. II. Consecutive automated calibration of the model with each of the four climate data sets. III. Random generation of 1000 model parameter sets and consecutive application of the four interpolated climate data sets on each of the 1000 realisations, evaluating the number of realisations above a certain quality criterion threshold. With such abundance of methods to select from the ques- tion arises which method a modeler should apply for his par- ticular case. Many studies have been carried out comparing the predictive performance of interpolation methods via cross validation (e.g. Dubois et al., 1998; Vicente-Serrano et al., 2003; Stahl et al., 2006; Hofstra et al., 2008), often on a monthly or annual basis. However, to our knowledge only few studies so far examined the inﬂuence of different inter- polation methods on the modeling results of rainfall-runoff models on a daily or sub-daily basis concerning hydrolog- ical validation (e.g. Kneis and Heistermann, 2009), and no studies concerning the validation of interpolation methods relating to nitrogen loads. Results show that strategies I and II are not suitable for eva- luation of the quality of the interpolated data. Strategy III however proves a signiﬁcant inﬂuence of the interpolation method on nitrate modeling. A rank order from the simplest to the most sophisticated method is visible, with kriging with external drift (KED) outperforming all others. Inﬂuence of spatial interpolation methods for climate variables on the simulation of discharge and nitrate fate with SWAT S. van der Heijden and U. Haberlandt Institute for Water Resources Management, Hydrology and Agricultural Hydraulic Engineering, Leibniz University of Hanover, Hanover, Germany Received: 19 January 2010 – Revised: 3 June 2010 – Accepted: 9 June 2010 – Published: 10 September 2010 Received: 19 January 2010 – Revised: 3 June 2010 – Accepted: 9 June 2010 – Published: 10 September 2010 is the main driving force for the nitrate cycle. The missing in- ﬂuence of the interpolation methods on discharge modeling is explained by a much higher measuring network density for precipitation than for all other climate variables. Abstract. For ecohydrological modeling climate variables are needed on subbasin basis. Since they usually originate from point measurements spatial interpolation is required during preprocessing. Different interpolation methods yield data of varying quality, which can strongly inﬂuence mo- deling results. Four interpolation methods to be compared were selected: nearest neighbour, inverse distance, ordinary kriging, and kriging with external drift (Goovaerts, 1997). This study presents three strategies to evaluate the inﬂuence of the interpolation method on the modeling results of dis- charge and nitrate load in the river in a mesoscale river catch- ment (∼1000 km2) using the Soil and Water Assessment Tool (SWAT, Neitsch et al., 2005) model: Advances in Geosciences Advances in Geosciences Adv. Geosci., 27, 91–98, 2010 www.adv-geosci.net/27/91/2010/ doi:10.5194/adgeo-27-91-2010 © Author(s) 2010. CC Attribution 3.0 License. Adv. Geosci., 27, 91–98, 2010 www.adv-geosci.net/27/91/2010/ doi:10.5194/adgeo-27-91-2010 © Author(s) 2010. CC Attribution 3.0 License. Advances in Geosciences Advances in Geosciences 1 Introduction Responsible for this behaviour is the variable temperature, which beneﬁts most from more sophisticated methods and at the same time Correspondence to: S. van der Heijden (vdheijden@iww.uni-hannover.de) Correspondence to: S. van der Heijden (vdheijden@iww.uni-hannover.de) Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. S. van der Heijden and U. Haberlandt: Inﬂuence of climate interpolation methods on SWAT simulations 92 S. van der Heijden and U. Haberlandt: Inﬂuence of climate interpolation methods on SWAT simulations 1 Aller Catchment Fig. 1. Study area. 1 Aller Catchment Fig. 1. Study area. 1 Fig. 1. Study area. This study focuses on four interpolation techniques, namely nearest neighbour, inverse distance weighting, or- dinary kriging, and kriging with external drift, and aims to evaluate their inﬂuence on daily discharge and monthly nitrate simulations with the ecohydrological model SWAT, when all necessary climate input data is interpolated with each of the four methods. Three strategies of model calibra- tion and application have been developed in order to evaluate the inﬂuence of the interpolation techniques. geologic conditions Cambisols, Luvisols and gley soils deve- loped as predominant soils, as well as Rendzinas and Rankers on the more elevated terrain. The main landuse is rain-fed agriculture, predominantly in the ﬂatter low-lying regions of the Leine valley, while the more sloped terrain is dominated by semi-natural deciduous and mixed forest. The main settlement in the area is the city of G¨ottingen. For this study, the investigation area is divided into 25 subcatchments (Fig. 1). Climate data is available from the climate and precipitation network of the German Weather Service (DWD), with a total of 94 climate stations and 378 precipitation gauges for the whole Aller catchment. Only about 10 climate and 40 precipitation stations are near or within the actual study area, but spatial interpolation was done for the larger catchment. Out of the total number of sta- tions about 50 climate stations and 250 precipitation gauges were recording at any one time step, which were available for interpolation. Six climate variables are needed by the selected numerical model and thus were selected for inter- polation. These are daily values for the sum of precipita- tion, minimum and maximum temperature, relative humidity, wind speed, and sunshine duration (which is converted into solar radiation after interpolation). 2.1 Study area and data The investigation area is the upper Leine river catchment with a size of approximately 1000 km2 (see Fig. 1). It is part of the larger Aller river catchment, which covers most of the south-eastern part of the federal state of Lower Sa- xony in Northern Germany. The average annual precipitation is about 700 mm and the mean annual temperature is around 8 ◦C. The elevation is between 533 m on the southern edge of the catchment and 115 m at the gauge Leineturm, which is the outlet of the catchment. The geology in the study area is dominated by the Meso- zoic sedimentary rock strata of the Muschelkalk and the Buntsandstein west and east of the Leine trough. The Keu- per, which is found in the subsurface of the actual trough, is mostly covered by loess of several meters thickness. Also, the broad Leine ﬂoodplains are ﬁlled with the ﬂuvial sed- iments gravel and coarse sand. Under these climatic and The digital elevation model is the freely available SRTM grid (USGS, 2004) with 90 m cell size, land use information is taken from the CORINE Land Cover data set (Umweltbun- desamt, 2004). Daily discharge measurements are available from ﬁve gauges with four of them on the Leine river itself and one on the tributary Garte. Out of the nine available Adv. Geosci., 27, 91–98, 2010 www.adv-geosci.net/27/91/2010/ S. van der Heijden and U. Haberlandt: Inﬂuence of climate interpolation methods on SWAT simulations S. van der Heijden and U. Haberlandt: Inﬂuence of climate interpolation methods on SWAT simulations 93 water quality sampling sites with biweekly or monthly mea- surements only three correspond to a discharge gage and could be used in the study (see Fig. 1). Discharge and wa- ter quality data were obtained from the Lower Saxony Wa- ter Management, Coastal Defence and Nature Conservation Agency (NLWKN). Due to the large spans of time between the irregular samplings it was impossible to examine nitrate on a daily basis. Since no dependence between nitrate con- centration and discharge value was found, measured nitrate concentration values were simply interpolated linearly and converted into monthly nitrate load values. can be calculated for every time step, but an average exper- imental semivariogram over all time steps has been found to be sufﬁcient (Haberlandt, 2007). The average semivar- iogram is obtained by weighting the daily semivariograms with the variance for each day. 2.2.5 Interpolation software The six climate variables were interpolated using the pro- gram SAINT (Haberlandt, unpublished), which allows the user to chose from several interpolation methods, including the four selected NN, IDW, OK, and KED. In addition to interpolation, SAINT also allows to compare the prediction quality of the methods by crossvalidation. Interpolation was done over the whole Aller river catchment on a grid with 900×900 m2 cell size (10×10 cells of the SRTM DEM). The program interpolates one time series for every cell, and sub- sequently aggregates all the cells within a given subbasin to a mean areal time series for the subbasin. Thus, for every method, the spatial interpolation returns one time series for each of the 25 subbasins. This procedure yielded four sets of equally interpolated climate data, one for each interpolation method. zIDW(u) = 1 n(u) P i=1 λi (u) n(u) X i=1 λi (u)z(ui) with λi (u) = 1 \|u−ui\|2 (2) 2.2.3 Ordinary kriging (OK) zIDW(u) = 1 n(u) P i=1 λi (u) n(u) X i=1 λi (u)z(ui) with (2) 2.2 Interpolation methods Four established spatial interpolation methods were selected for this study. The two simple methods of nearest neighbour (abbr. NN) and inverse distance weighting (IDW), as well as the more sophisticated methods ordinary kriging (OK) and kriging with external drift (KED). Let {z(ui),i = 1,...,n} be the set of a measured climate variable at n locations ui. The four interpolation methods are used to estimate the climate variables at unsampled locations u. zOK(u) = n(u) X i=1 λOK i (u)z(ui) (3) (3) 2.2.4 Kriging with external drift (KED) While the three former methods are univariate, the fourth method KED is bivariate and uses secondary information to interpolate the climate data. Additionally to the spatial de- pendence of a variable, the linear relation to another variable can be processed. The basic assumption is that the expected value of the target variable Z(u) is a linear function of an additional variable Y(u): 2.1 Study area and data Then a theoretical semivar- iogram model is ﬁtted to the average experimental semivar- iogram. Different theoretical semivariograms were used for different climate data, with the spherical semivariogram used the most. When the theoretical semivariogram is ﬁtted, it is used to determine weights λOK i (u) in a way to ensure unbiased- ness of the estimator, E{ZOK(u)−Z(u)} = 0, and to min- imize the estimation variance, Var{ZOK(u)−Z(u)}. Like with IDW, these weights are needed to estimate the unknown value at an unsampled location u as a linear combination of neighbouring observations: 2.2.1 Nearest neighbour (NN) The simplest approach for this task is the Thiessen polygon method. Each unsampled location u is assigned the value of the nearest observation ui′. zNN(u) = z(ui′) with \|u−ui′\| < \|u−ui\|∀i ̸= i′ (1) (1) E[Z(u)\|Y (u)] = a +b·Y (u) (4) (4) This method produces unrealistic maps with sharp bound- aries between polygons of equal value, but preserves the vari- ance of the observed data. This method produces unrealistic maps with sharp bound- aries between polygons of equal value, but preserves the vari- ance of the observed data. The KED estimate is the same as for OK (see Eq. 3), but the weights are determined as the solution of a system of linear equations (the kriging system) under the employ of the sec- ondary information y(u). For further details the reader is re- ferred to geostatistical textbooks (e.g. Isaaks and Srivastava, 1989; Goovaerts, 1997). 2.2.2 Inverse distance weighting (IDW) To avoid sudden jumps in values between neighbouring loca- tions, the variable z can be estimated taking several surround- ing observations into account. The estimation is a weighted linear combination, with the weights λi being inversely pro- portional to the square distance between sampled location ui and the point of estimation u: www.adv-geosci.net/27/91/2010/ 2.2.3 Ordinary kriging (OK) Kriging techniques are generalized least-squares regression algorithms. Kriging considers the spatial variability based on the semivariogram. The experimental semivariogram ˆγ (h) www.adv-geosci.net/27/91/2010/ Adv. Geosci., 27, 91–98, 2010 S. van der Heijden and U. Haberlandt: Inﬂuence of climate interpolation methods on SWAT simulations 94 j p Table 1. SWAT Parameters used for automated calibration. Number Variable Description Min Max 1 CN2∗∗ Curve Number 25 90 2, 3, 4 ALPHA BF∗ L,G,R Base ﬂow recession constant 0 1 5, 6, 7 GW DELAY∗ L,G,R Delay in percolation to groundwater 0 500 8, 9, 10 GW REVAP∗ L,G,R Groundwater revap coefﬁcient 0.02 0.2 11 SFTMP Snow fall temperature –3 3 12 SMTMP Snow melt temperature –3 5 13 ESCO Soil evaporation compensation factor 0 1 14 EPCO Plant uptake compensation factor 0 1 15 OV N∗∗ Manning’s n for overland ﬂow 0.01 30 16 CANMX∗∗ Maximum canopy storage 0 10 17 HRU SLP∗∗ Average slope steepness of HRU 0 2 18 N UPDIS Nitrogen uptake distribution parameter 0 30 19 NPERCO Nitrate percolation coefﬁcient 0.01 1 20 HLIFE NGW Nitrate half life in groundwater 0 200 ∗The study area was divided into three hydrological landscapes for groundwater, R: from spring to gauge Reckershausen, G: from Reckers- hausen to G¨ottingen, L: from G¨ottingen to Leineturm. ∗∗Variable is HRU-speciﬁc, but all HRUs were calibrated at the same time keeping the ratio to each other constant, thus it represents only a single degree of freedom during calibration. Table 1. SWAT Parameters used for automated calibration. ∗The study area was divided into three hydrological landscapes for groundwater, R: from spring to gauge Re hausen to G¨ottingen, L: from G¨ottingen to Leineturm. ∗The study area was divided into three hydrological landscapes for groundwater, R: from spring to gauge Reckershausen, G: from Reckers- hausen to G¨ottingen, L: from G¨ottingen to Leineturm. ∗∗Variable is HRU-speciﬁc, but all HRUs were calibrated at the same time keeping the ratio to each other constant, thus it represents only a single degree of freedom during calibration. g , g ∗∗Variable is HRU-speciﬁc, but all HRUs were calibrated at the same time keeping the ratio to each other constant, thus it represents only a single degree of freedom during calibration. 2.3 The SWAT model In this study the inﬂuence of the different interpolation me- thods on modeling results of the Soil and Water Assessment Tool (Neitsch et al., 2005) is examined. The SWAT model is a semi-distributed, process-oriented model capable of simu- lating runoff, nutrient and other agricultural chemicals dy- namics as well as sediment yield in large complex water- sheds with varying soils, land use, and management condi- tions. The simulated water balance comprises interception, evapotranspiration, snow melt, surface runoff, inﬁltration, soil percolation, lateral ﬂow, groundwater ﬂow, and river routing. Evapotranspiration is calculated here after Penman- Monteith, snow melt with the degree day method, inﬁltration based on the SCS curve number method, runoff transforma- tion using a surface runoff lag method and ﬂood routing is calculated with the variable storage method. Simulations are carried out on the basis of hydrotopes (Hydrologic Response Units, HRUs), which are characterized by unique combina- tions of land use, soil, and topography in each subbasin. Cal- culated ﬂows are aggregated at the subbasin outlet and routed through the stream network. 2.2.3 Ordinary kriging (OK) For OK and KED semivariograms were calculated for each climate variable with a FORTRAN program based on the Geostatistical Software Library (GSLIB, Deutsch and Jour- nel, 1992) using all available stations during the time period from 1960 to 2006. Interpolation was then carried out em- ploying those semivariograms. Since many climate variables correlate with terrain elevation, this was taken as secondary information for KED. To facilitate the use of GIS-based data for model setup, the ArcSWAT interface (Winchell et al., 2008) has been cre- ated to link the SWAT model and ArcGIS 9. For this study ArcGIS 9.2 and ArcSWAT 2.0.0 were employed, which in- cludes the SWAT model in its 2005 version. Altogether 20 parameters were used for automated calibra- tion using the PEST program (Doherty, 2004) for strategies I and II, as described below. Seventeen were responsible for calibration of the hydrology, while the remaining three go- verned the nitrate calibration (see Table 1). Modeling time period was from 1979 till 2005. The ﬁrst two years were taken as warm-up period for the model. For discharge, cal- ibration was from 1981 to 1986, validation from 1987 to 1991. For nitrate, since only monthly values were available, calibration was from 1981 to 1995 and validation from 1996 to 2005. 3.1 Strategy I Strategy I did not show any signiﬁcant differences between interpolation methods, neither for discharge nor for nitrate simulation. Figure 3 shows results for gauges Leineturm and Reckershausen, the other gauges are similar. Nash- Sutcliffe efﬁciency (NSE) is higher for the discharge and ni- trate gauges with larger catchment areas, as it is usually the 3 Results and discussion As strategy I before, the second approach neither showed any signiﬁcant differences between interpolation methods (Fig. 4, gauges not shown are similar). Again NSE for both discharge and nitrate varies between gauges, but not be- tween interpolation methods. Although the inﬂuence of a biased calibration has been removed, this outcome is plau- sible, since separate calibration might compensate for dif- ferences resulting from the climate data sets. Furthermore, the inﬂuence of the PEST optimization start parameters is very strong, as it turned out in a follow-up investigation. Strategy II was carried out several times using different PEST start parameters, and each time the result was different, and no realization clearly favoured any of the interpolation me- thods in modeling results. In Table 2 some exemplary results of the spatial interpolation are shown. The numbers represent average long-term val- ues of precipitation sum and maximum daily temperature for the Upper Leine catchment. The presented numbers already show clearly the variation in the different interpolation me- thods, while the differences can be much more pronounced for smaller regions or on individual days. Simulation results were generally good with the calibrated SWAT model. Figure 2 exemplarily shows two graphs, one for daily discharge and one for monthly nitrate load at gauge Leineturm. 2.4 Three strategies to evaluate the inﬂuence ∗Long-term average sum of precipitation (PCP) and long-term average maximum daily temperature (TMAX) for May–October (summer), November–April (winter) and whole year, averaged from 1960 to 2006 over the Upper Leine catchment. ∗∗Four interpolation methods were compared: kriging with exter- nal drift (KED), ordinary kriging (OK), inverse distance weighting (IDW) and nearest neighbour (NN). ∗∗Four interpolation methods were compared: kriging with exter- nal drift (KED), ordinary kriging (OK), inverse distance weighting (IDW) and nearest neighbour (NN). Strategy III : to overcome the possible problem of compen- sation of poor climate data by separate calibration the third strategy was developed. No calibration is necessary for this approach. Of the 20 model parameters used for calibration in strategies I and II, 1000 random parameter sets were gener- ated, uniformly distributed between the minimum and max- imum values allowed by SWAT. These 1000 parameter sets were applied to each of the four interpolated climate data sets. As evaluation the number of all realisations above a selected threshold in Nash-Sutcliffe-Efﬁciency (determined over a period of 10 years from 1981 to 1990) for both dis- charge and nitrate load was counted for each interpolation method. case, but there is no conclusive difference between interpo- lation methods at each gauge. This is surprising, since it was expected that this approach would perform better for inter- polated climate data used for calibration. A reason might be the random inﬂuence of automated calibration. Not only the choice of the climate data set for calibration, but also the PEST optimization start parameters have a strong effect on the ﬁnal calibrated model, and thus the performance of the modeling results. This means that strategy I is not suitable to detect any differences in modeling results arising from the interpolation method for the climate data. 2.4 Three strategies to evaluate the inﬂuence Three strategies were developed to examine the inﬂuence of the four different interpolation methods on runoff and nitrate simulation results: Strategy I : in this simple approach, the SWAT model was calibrated only one time, using PEST and one selected set of interpolated climate data from mixed interpolation me- thods. For each climate variable for the mixed set the best performing interpolation method during cross-validation was selected. Then, all four interpolation climate data sets were subsequently applied to the calibrated model and the mo- deling results were compared running the SWAT model for both the calibration and the validation periods. The climate Adv. Geosci., 27, 91–98, 2010 www.adv-geosci.net/27/91/2010/ S. van der Heijden and U. Haberlandt: Inﬂuence of climate interpolation methods on SWAT simulations 95 data for calibration was selected from the interpolated data sets via crossvalidation. Data resulting from the method which performed best in crossvalidation considering correla- tion and RMSE were chosen for each climate variable. This was KED for temperature, humidity, and wind speed, OK for precipitation, and IDW for sunshine duration. One possible problem with this strategy is that it might be biased towards one or more of those interpolation methods used for calibra- tion. Table 2. Exemplary results of the spatial interpolation of the climate variables. Table 2. Exemplary results of the spatial interpolation of the climate variables. Climate variable∗ KED∗∗ OK∗∗ IDW∗∗ NN∗∗ PCP (summer) [mm] 385.11 381.04 379.64 379.19 PCP (winter) [mm] 341.34 337.20 334.50 333.95 PCP (year) [mm] 726.45 718.25 714.13 713.14 TMAX (summer) [◦C] 18.67 19.07 19.25 19.32 TMAX (winter) [◦C] 5.91 6.22 6.36 6.42 TMAX (year) [◦C] 12.34 12.69 12.85 12.92 Strategy II : to circumvent a biased calibration the sec- ond approach was designed as fourfold calibration of the model, separately with each of the four interpolated climate data sets. Again automated calibration with the PEST tool was employed. Results of those four calibrated models were compared, again for simulations using both the calibration and validation periods. Individual calibration however might cover the differences in modeling results arising from the dif- ferent interpolation methods. ∗Long-term average sum of precipitation (PCP) and long-term average maximum daily temperature (TMAX) for May–October (summer), November–April (winter) and whole year, averaged from 1960 to 2006 over the Upper Leine catchment. 3.3 Strategy III The results of strategy III are presented in Fig. 5 (threshold value 0.5, other threshold values show similar results). As the other approaches before, there is no visible inﬂuence of the www.adv-geosci.net/27/91/2010/ Adv. Geosci., 27, 91–98, 2010 S. van der Heijden and U. Haberlandt: Inﬂuence of climate interpolation methods on SWAT simulations S. van der Heijden and U. Haberlandt: Inﬂuence of climate interpolation methods on SWAT simulations 96 1 0 20 40 60 80 100 120 01.11.1981 01.11.1983 01.11.1985 discharge [m³/s] observed simulated 0 100 200 300 400 500 600 700 Nov. 81 Nov. 84 Nov. 87 Nov. 90 Nov. 93 nitrate load [t/month] observed simulated 1 0 20 40 60 80 100 120 01.11.1981 01.11.1983 01.11.1985 discharge [m³/s] observed simulated 0 100 200 300 400 500 600 700 Nov 81 Nov 84 Nov 87 Nov 90 Nov 93 nitrate load [t/month] observed simulated Fig. 2. Simulated and observed time series of daily discharge (left) and monthly nitrate load (right) at gauge Leineturm. 0 20 40 60 80 100 120 01.11.1981 01.11.1983 01.11.1985 discharge [m³/s] observed simulated 0 20 40 60 80 100 120 01.11.1981 01.11.1983 01.11.1985 discharge [m³/s] observed simulated 0 100 200 300 400 500 600 700 Nov. 81 Nov. 84 Nov. 87 Nov. 90 Nov. 93 nitrate load [t/month] observed simulated 0 100 200 300 400 500 600 700 Nov 81 Nov 84 Nov 87 Nov 90 Nov 93 nitrate load [t/month] observed simulated mulated and observed time series of daily discharge (left) and monthly nitrate load (right) at gauge Leineturm. 1 0.0 0.2 0.4 0.6 0.8 1.0 Leine cal. Leine val. Reck cal. Reck val. NN IDW OK KED 0.0 0.2 0.4 0.6 0.8 1.0 Leine cal. Leine val. Reck cal. Reck val. NN IDW OK KED 1 0.0 0.2 0.4 0.6 0.8 1.0 Leine cal. Leine val. Reck cal. Reck val. NN IDW OK KED 0.0 0.2 0.4 0.6 0.8 1.0 Leine cal. Leine val. Reck cal. Reck val. NN IDW OK KED Fig. 3. Results of strategy I, NSE for discharge (left) and nitrate load (right). Gauges Leineturm (Leine) and Reckershausen (Reck) shown for calibration (cal.) and validation (val.) periods. 0.0 0.2 0.4 0.6 0.8 1.0 Leine cal. Leine val. Reck cal. Reck val. NN IDW OK KED W 0.0 0.2 0.4 0.6 0.8 1.0 Leine cal. Leine val. Reck cal. Reck val. NN IDW OK KED Fig. 3. 4 Conclusions and discussions Although strategies I and II did not show any signiﬁcant dif- ference between interpolation methods, strategy III proves that there is a strong inﬂuence at least on nitrate simula- tion. KED clearly outperforms all other evaluated interpo- lation methods, and a well-deﬁned rank order from the sim- plest to the most sophisticated method is visible. The ques- tion arises, why this only shows in the modeling results for nitrate, and not for discharge. Most likely the measuring network density plays a major role in this. Previous stud- ies proved an inﬂuence of the network density on modeling results (e.g. B´ardossy and Das, 2008). The main driving force for discharge is precipitation, while nitrate dynamics strongly depend on plant growth, with temperature as its main driving force. The precipitation measuring network for the Aller river catchment has a four times higher den- sity than the measuring network for the other climate vari- ables, which even raises to six times when taking the average number of “non-missing” stations into account. It is self- evident that the higher the network density, the smaller the differences between interpolation methods. Further analy- sis showed that indeed temperature makes up for the largest part of the differences in performance between NN and KED in strategy III. Since temperature also is the climate vari- able beneﬁtting most from more sophisticated interpolation methods, especially KED with elevation as secondary infor- mation, as shown by cross-validation, the large difference in performance between interpolation methods regarding ni- trate simulation in strategy III can be explained. To test the inﬂuence of measuring network density, the analysis could be extended considering different densities of the precipitation network. Automated calibration could become suitable for eva- luation of the interpolation methods with some alterations and improvements of the evaluation strategies. For one, the permitted parameter space could be narrowed down to phys- ically meaningful limits for the examined study area instead of employing the maximum limits set by the model. Further- more, a Monte Carlo-like automated calibration with varying optimization starting parameters seems necessary to elim- inate the random inﬂuence of the starting parameters. A Monte Carlo-like automated calibration might also reveal other aspects of the inﬂuence of the interpolation methods, e.g. if a more robust parameter calibration would be pos- sible using climate data from more sophisticated interpola- tion methods. 3.3 Strategy III van der Heijden and U. Haberlandt: Inﬂuence of climate interpolation methods on SWAT simulations S. van der Heijden and U. Haberlandt: Inﬂuence of climate interpolation methods on SWAT simulations 97 Even when one climate data set is used for calibration, as in strategy II, it does in no way guarantee that this data set performs best with the calibrated model. Subsequent tri- als revealed that different optimization starting parameters yield totally different calibrated model parameters and dif- ferent rank orders of performance of the interpolation me- thods. These ﬁndings allow two major conclusions: ﬁrstly, any strategies involving automated calibration in the way presented in this paper (one random set of optimization start- ing parameters, minimum and maximum parameter values as allowed by the model) are not suitable to evaluate the per- formance of interpolation methods. Even a combination of strategies I and II did not give any other results (fourfold cal- ibration with each climate data set, application of all four data sets to all four calibrated models in turn). Secondly, if for a model application a single automated calibration as shown is used, it is very unlikely that the modeler will get the best out of his climate input data, even when testing several interpolation methods. interpolation method on discharge simulation. Although this strategy is not biased towards any method and no calibration is carried out, all discharge gauges show a similar number of “good” realizations with a Nash-Sutcliffe efﬁciency above the chosen threshold for each method. In agreement with the previous methods, higher numbers of good realizations are found for gauges with larger catchment areas. This holds true for all tested threshold values. Nitrate simulation on the other hand shows an extreme and clear inﬂuence of the interpolation method. All three gauges consistently reveal a rising number of good realizations from the simplest to the most sophisticated interpolation method, with KED clearly performing best, often with about twice as much good realizations than the second-best method. This again holds true for all tested threshold values. www.adv-geosci.net/27/91/2010/ 3.3 Strategy III Results of strategy I, NSE for discharge (left) and nitrate load (right). Gauges Leineturm (Leine) and Reckershausen (Reck) shown for calibration (cal.) and validation (val.) periods. 0.0 0.2 0.4 0.6 0.8 1.0 Leine cal. Leine val. Reck cal. Reck val. NN IDW OK KED W D 0.0 0.2 0.4 0.6 0.8 1.0 Leine cal. Leine val. Reck cal. Reck val. NN IDW OK KED 1 0.0 0.2 0.4 0.6 0.8 1.0 Leine cal. Leine val. Reck cal. Reck val. NN IDW OK KED 0.0 0.2 0.4 0.6 0.8 1.0 Leine cal. Leine val. Reck cal. Reck val. NN IDW OK KED 1 0.0 0.2 0.4 0.6 0.8 1.0 Leine cal. Leine val. Reck cal. Reck val. NN IDW OK KED 0.0 0.2 0.4 0.6 0.8 1.0 Leine cal. Leine val. Reck cal. Reck val. NN IDW OK KED Fig. 4. Results of strategy II, NSE for discharge (left) and nitrate load (right). Gauges Leineturm (Leine) and Reckershausen (Reck) shown for calibration (cal.) and validation (val.) periods. Fig. 4. Results of strategy II, NSE for discharge (left) and nitrate load (right). Gauges Leineturm (Leine) and Reckershausen (Reck) shown for calibration (cal.) and validation (val.) periods. 1 0 100 200 300 400 500 600 700 Leine Gött Reck Arens Garte NN IDW OK KED 0 50 100 150 200 Leine Reck Garte NN IDW OK KED 1 0 100 200 300 400 500 600 700 Leine Gött Reck Arens Garte NN IDW OK KED 0 50 100 150 200 Leine Reck Garte NN IDW OK KED Fig. 5. Results of strategy III, number of “good” realisations for each gauge (Leineturm (Leine), G¨ottingen (G¨ott), Reckershausen (Reck), Arenshausen (Arens), and Gartem¨uhle (Garte)) and interpolation method with NSE ≥0.5, discharge on the left, nitrate load on the right. 1 0 100 200 300 400 500 600 700 Leine Gött Reck Arens Garte NN IDW OK KED 1 0 100 200 300 400 500 600 700 Leine Gött Reck Arens Garte NN IDW OK KED 0 50 100 150 200 Leine Reck Garte NN IDW OK KED 0 50 100 150 200 Leine Reck Garte NN IDW OK KED Fig. 5. Results of strategy III, number of “good” realisations for each gauge (Leineturm (Leine), G¨ottingen (G¨ott), Reckershausen (Reck), Arenshausen (Arens), and Gartem¨uhle (Garte)) and interpolation method with NSE ≥0.5, discharge on the left, nitrate load on the right. Adv. Geosci., 27, 91–98, 2010 www.adv-geosci.net/27/91/2010/ S. Edited by: A. Wahren, F. Tavares Wahren, and K.-H. Feger Reviewed by: two anonymous referees References Isaaks, E. H. and Srivastava, R. M.: Applied Geostatistics, Oxford University Press, New York, 1989. B´ardossy, A. and Das, T.: Inﬂuence of rainfall observation network on model calibration and application, Hydrol. Earth Syst. Sci., 12, 77–89, doi:10.5194/hess-12-77-2008, 2008. Kneis, D. and Heistermann, M.: Bewertung der G¨ute einer Radar- basierten Niederschlagssch¨atzung am Beispiel eines kleinen Einzugsgebiets, Hydrologie und Wasserbewirtschaftung, 53, 160–171, 2009 (in German). Demyanov, V., Kanevski, M., Chernov, S., Savelieva, E., and Timo- nin, V.: Neural network residual kriging application for climatic data, Journal of Geographic Information and Decision Analysis, 2, 215–232, 1998. Neitsch, S. L., Arnold, J. G., Kiniry, J. R., and Williams, J. R.: Soil and Water Assessment Tool Theoretical Documentation, Black- land Research Center, Temple, Texas, 2005. Deutsch, C. V. and Journel, A. G.: GSLIB: Geostatistical software library and user’s guide, Oxford University Press, New York, 1992. Shepard, D.: A two-dimensional interpolation function for irregu- larly spaced data, Proc. of ACM National Conference, 1968. Stahl, K., Moore, R. D., Floyer, J. A., Asplin, M. G., and McK- endry, I. G.: Comparison of approaches for spatial interpolation of daily air temperature in a large region with complex topogra- phy and highly variable station density, Agricultural and Forest Meteorology, 139, 224–236, 2006. Doherty, J.: PEST Model – Independent Parameter Estimation User Manual, 5th edn., Watermark Numerical Computing, 2004. Dubois, G., Malczewski, J., and Cort, M. D.: Spatial Interpolation Comparison 97, 2(1–2), 1998. p Goovaerts, P.: Geostatistics for natural resources evaluation, Oxford University Press, New York, Oxford, 1997. Thiessen, A. H.: Precipitation averages for large areas, Mon. Weather Rev., 39, 1082–1084, 1911. Haberlandt, U.: Geostatistical interpolation of hourly precipitation from rain gauges and radar for a large-scale extreme rainfall event, J. Hydrol., 332, 144–157, 2007. Tveito, O. E., Wegehenkel, M., van der Wel, F., and Dobesch, H.: Spatialisation of climatological and meteorological information with the support of GIS (Working Group 2), COST Action 719, 37–172, 2006. Haberlandt, U.: SpAtial INterpolation of climatological Time series (SAINT), unpublished. Umweltbundesamt: CORINE Land Cover, DLR-DFD, 2004. Hofstra, N., Haylock, M., New, M., Jones, P., and Frei, C.: Comparison of six methods for the interpolation of daily, European climate data, J. Geophys. Res., 113, D21110, doi:10.1029/2008JD010100, 2008. USGS: Shuttle Radar Topography Mission (SRTM), U.S. Geologi- cal Survey, available at: http://www.landcover.org/data/srtm (last access: 4 March 2008), 2004. Vicente-Serrano, S. M., Saz-S´anchez, M. A., and Cuadrat, J. 4 Conclusions and discussions Other aspects for future research are to ana- lyze how well the ﬁndings from this study are transferable to other investigation areas and/or other models. Also, if dif- ferences arise, it is worthwhile to invesigate which climate variables contribute most to the differences, when interpo- lated with different methods. This may lead to conclusions about whether one interpolation method is more suitable for a speciﬁc climate variable than another, with different me- thods performing best for different variables. A useful fur- ther study would be to establish a guideline for modelers how to interpolate the climate input data for their models in order to receive the best performance and the most robust model parameters, while minimizing the amount of time needed for calibration. Acknowledgements. The authors thank two anonymous reviewers who helped to improve the quality of the paper. One major problem with the ﬁrst two strategies is the ran- dom inﬂuence of the automated calibration. The optimiza- tion starting parameters for PEST have a strong inﬂuence on the ﬁnal calibrated model parameters, which of course inﬂuences which interpolated climate data performs best. Edited by: A. Wahren, F. Tavares Wahren, and K.-H. Feger Reviewed by: two anonymous referees Adv. Geosci., 27, 91–98, 2010 www.adv-geosci.net/27/91/2010/ S. van der Heijden and U. Haberlandt: Inﬂuence of climate interpolation methods on SWAT simulations 98 www.adv-geosci.net/27/91/2010/ References M.: Comparative analysis of interpolation methods in the middle Ebro Valley (Spain): application to annual precipitation and tem- perature, Climate Research, 24, 161–180, 2003. Huang, Y., Wong, P., and Gedeon, T.: Spatial interpolation using fuzzy reasoning and genetic algorithms., Journal of Geographic Information and Decision Analysis, 2, 204–214, 1998. Hutchinson, M. F.: Interpolation of rainfall data with thin plate smoothing splines: I Two dimensional smoothing of data with short range correlation, Journal of Geographic Information and Decision Analysis, 2, 139–151, 1998a. Winchell, M., Srinivasan, R., Di Luzio, M., and Arnold, J. G.: ArcSWAT 2.0 Interface For SWAT User’s Guide, Blackland Re- search Center, Temple, Texas, 2008. y Hutchinson, M. F.: Interpolation of rainfall data with thin plate smoothing splines: II Analysis of topographic dependence, Jour- nal of Geographic Information and Decision Analysis, 2, 152– 167, 1998b. www.adv-geosci.net/27/91/2010/ Adv. Geosci., 27, 91–98, 2010
https://openalex.org/W2340334834	https://europepmc.org/articles/pmc4849238?pdf=render	English	null	Acromioclavicular joint reconstruction using a tendon graft: a biomechanical study comparing a novel “sutured throughout” tendon graft to a standard tendon graft	SICOT-J	2,016	cc-by	4,577	Qais Naziri, Nadine Williams, Westley Hayes, Bhaveen H. Kapadia, Dipal Chatterjee, and William P. Urban Department of Orthopaedics, SUNY Downstate Medical Center, 450 Clarkson Avenue, MSC 30, Brooklyn, NY 11203, USA Department of Orthopaedics, SUNY Downstate Medical Center, 450 Clarkson Avenue, MSC 30, Brooklyn, NY 11203, USA Received 17 April 2015 Accepted 17 February 2016 Published online 20 April 2016 Received 17 April 2015, Accepted 17 February 2016, Published online 20 April 2016 Abstract – Background: With a recurrence rate of over 30%, techniques that offer stronger acromioclavicular (AC) joint reconstruction through increased graft strength may provide longevity. The purpose of our study was to deter- mine the biomechanical strength of a novel tendon graft sutured throughout compared to a native tendon graft in Grade 3 anatomical AC joint reconstruction. Methods: For this in vitro experiment, nine paired (n = 18) embalmed cadaveric AC joints of three males and six females (age 86 years, range 51–94 years) were harvested. Anatomic repair with fresh bovine Achilles tendon grafts without bone block was simulated. Specimens were divided into two groups; with group 1 using grafts with ultra-high molecular-weight polyethylene (UHMWPE) suture ran throughout the entire length. In group 2, reconstruction with only native allografts was performed. The distal scapula and humerus were casted in epoxy compound and mounted on the mechanical testing machine. Tensile tests were performed using a mechanical testing machine at the rate of 50 mm/min. Maximum load and displacement to failure were collected. Results: The average load to failure was signiﬁcantly higher for group 1 compared to group 2, with mean values of 437.5 N ± 160.7 N and 94.4 N ± 43.6 N, ( p = 0.001). The average displacement to failure was not signiﬁcantly dif- ferent, with 29.7 mm ± 10.6 mm in group 1 and 25 mm ± 9.1 mm in group 2 ( p = 0.25). Conclusion: We conclude that a UHMWPE suture reinforced graft can provide a 3.6 times stronger AC joint recon- struction compared to a native graft. Key words: Biomechanics, Acromioclavicular joint, UHMWPE suture, Coracoclavicular ligament, Graft augmentation. Key words: Biomechanics, Acromioclavicular joint, UHMWPE suture, Coracoclavicular ligament, Gra augmentation. 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. Corresponding author: dipal.chatterjee@gmail.com SICOT J 2016, 2, 17 The Authors, published by EDP Sciences, 2016 DOI: 10.1051/sicotj/2016013 SICOT J 2016, 2, 17 The Authors, published by EDP Sciences, 2016 DOI: 10.1051/sicotj/2016013 Available online at: www.sicot-j.org Available online at: www.sicot-j.org OPEN ACCESS OPEN ACCESS RESEARCH Introduction different types which are (1) primary AC and coracoclavicular (CC) ﬁxation, (2) the Weaver-Dunn procedure, (3) anatomic reconstruction, and (4) arthroscopic reconstructions [6]. Acromioclavicular (AC) joint injuries are common in the active population [1–3]. The injury often involves direct trauma to the superior aspect of the acromion, which causes an inferior and anterior translation of the acromion in relation to the distal clavicle [4]. Conservative management is used for the treatment of Rockwood Type I–III (in nonathletes) separa- tions [3, 5]. Operative management is often indicated in ath- letes and for chronic symptomatic Type III separations that have not responded to conservative treatment. Type IV–VI AC separations generally require operative management [1]. Primary AC and CC ﬁxation accomplished with Kirschner wires, sutures, and Bosworth screws are one of the ﬁrst ﬁxation methods. Though good results have been reported, these meth- ods have fallen into disfavor due to hardware migration, of which some have been devastating [7–9]. The proper Weaver-Dunn procedure demands the excision of the distal clavicle and transfer of the CA ligament onto the CC ligament. Biomechanically this construct seemed weaker and led to greater displacement [10]. More than 60 surgical procedures to treat AC joint separa- tion have been reported. These can be categorized into four Subsequently, this method has been more commonly aug- mented with sutures, wire cerclage, or tendon grafts for rein- forcement. In order to replicate biomechanical behavior of the native ligament, anatomic reconstructions, wherein the Q. Naziri et al.: SICOT J 2016, 2, 17 2 Figure 1. Graft with UHMWPE suture ran throughout the entire length for reinforcement. conoid and trapezoid ligaments are recreated, have become more popular [2]. However, anatomic reconstruction has been reported to fail via coracoid process fractures, clavicular frac- tures of the bone tunnels, or AC joint separations [10, 11]. Newer arthroscopic techniques involve suture and screw ﬁxa- tion. In a comparative study by Mazzocca et al. arthroscopic ﬁxation showed less anterior displacement and laxity than the Weaver-Dunn procedure, compared to anatomic recon- structions the outcomes were similar [12]. Open procedures are becoming increasingly harder to justify due to the greater risk of iatrogenic injury to adjacent neurovascular structures [13]. So far, the orthopedic literature does not contain recom- mendations for a single operative technique as the optimal reconstruction method for AC joint separations. Specimen preparation Fresh-frozen bovine Achilles tendon grafts, which mea- sured in average 5 mm · 127 mm, were obtained. All tendon specimens were stored at 20 C for no longer than four weeks and thawed at room temperature for 12 h prior to test- ing. Nine grafts were whipstitched with UHMWPE suture (#5 FibreWireTM, Arthrex, Naples, FL) across the entire length (Figure 1) on a graft preparation station. All specimens were wrapped in saline-soaked gauze. While tendon graft repair techniques augmented with cer- clage cables have been described, to our knowledge, no study has evaluated the effect of an intra-tendinous suture that func- tions similarly to a coracoid cerclage construction, on the strength of the AC joint repair [27]. The clavicular bone tunnel sites were carefully and accu- rately measured prior to drilling as previously described by Caroﬁno and Mazzocca [2]. A drill tip guide pin was used for placement of the tunnels. The ﬁrst tunnel was created in the posterior aspect of the clav- icle approximately 45 mm from the distal end. After measur- ing 20 mm distally from the center of the ﬁrst tunnel, the second tunnel was created in the anterior aspect of the clavicle. Both tunnels were then reamed through the full thickness of the clavicle using a 5.5 mm reamer. We hypothesized that running a suture throughout the ten- don graft itself would increase load to failure and the longevity of reconstruction techniques using tendon grafts. Therefore, the purpose of our study was to determine the difference in ten- sile strength and displacement to failure, between a novel ten- don graft with ultra-high molecular-weight polyethylene (UHMWPE) suture incorporated throughout and a standard nonaugmented tendon graft in an anatomic AC joint reconstruction. The matched pairs of AC joints were separated. Randomly, either the left or the right shoulder was assigned a graft sutured throughout with UHMWPE suture and became group 1 (n = 9). The contralateral AC joint received the standard native bovine Achilles tendon graft and represented group 2 (n = 9). Prior to joint reconstruction all grafts were tensioned to 10 N to remove any creep. Introduction Postsurgical recurrence rates of AC joint separation after reconstruction can range between 20 and 30% or higher and occur frequently within one year of the initial surgery [14–18]. Recently the focus has been on reconstruction techniques utilizing tendon grafts, as the need for implant removal is obviated and implant fracture, loosening, and migration are eliminated. Therefore, various tendon grafts, including palmaris longus, semitendino- sus, anterior tibialis, or gracilis tendon, have been utilized [4, 19, 20]. However, one of the limiting factors for the longevity of reconstruction techniques using tendon grafts is the biome- chanical strength of the tendon graft itself as they do not rees- tablish the strength and stiffness of the native AC joint [21–23]. Several authors have advocated more resilient ﬁxation to improve surgical outcomes [11, 24–26]. Figure 1. Graft with UHMWPE suture ran throughout the entire length for reinforcement. Material and methods For this biomechanical laboratory study evaluating tensile load to failure and displacement to failure of a novel graft aug- mentation technique, nine paired (n = 18) cadaveric AC joints (three males and six females) were harvested. After thorough inspection, none of the 18 specimens showed signs of prior injury to the shoulder or prior operative intervention and thus were all included for biomechanical testing. The mean age of the embalmed cadaveric donors at the time of death was 86 years (range, 51–94 years). Surrounding muscles and other soft tissues were removed. The AC and coracoclavicular (CC) ligaments were severed in order to simulate a Type III dislocation. In both groups, the tendon graft was looped around the base of the coracoid process. Then the lateral limb of the ten- don graft was placed through the posteromedial bone tunnel, which recreated the conoid ligament medially. The medial limb of the graft was passed through the anterolateral bone tunnel, which recreated the trapezoid ligament laterally. This method of passing the graft through creates a crossing pattern as described by Shin et al. [27]. In all specimens the grafts were ﬁxed in the bone tunnels using 5.5 mm · 8 mm Bio-Tenodesis Screws (PEEK, Arthrex, Naples, FL). The ends of the graft were sutured to each other in a side-to-side fashion in both groups (Figure 2). Figure 3. The testing construct, which is attached to the load cell and the base of the mechanical testing machine. COT J 2016, 2, 17 3 Q. Naziri et al.: SICOT J 2016, 2, 17 Figure 2. Schematic diagram of anatomic AC joint reconstruction. In group 1 suture was placed into the entire graft (not depicted) while group 2 lacked suture within the graft. Q. Naziri et al.: S 3 Figure 2. Schematic diagram of anatomic AC joint reconstruction. In group 1 suture was placed into the entire graft (not depicted) while group 2 lacked suture within the graft. Figure 3. The testing construct, which is attached to the load cell and the base of the mechanical testing machine. Figure 3. The testing construct, which is attached to the load cell and the base of the mechanical testing machine. The distal ends of the scapula and humerus were casted in epoxy compound (BondoTM, Atlanta, GA). Results All failures except two were graft failures at the mid- substance. Each group had one clavicular fracture just distal to the grip. These specimens were not included in the statistical analyses. Load to failure was signiﬁcantly higher in group 1 compared to group 2 (Figure 4), with mean values of 437.5 N ± 160.7 N and 94.4 N ± 43.6 N, respectively ( p = 0.001). The difference in displacement to failure (Figure 5), though higher in group 1 compared to group 2, with mean values of 29.7 mm ± 10.6 mm and 25 mm ± 9.1 mm, did not reach clinical signiﬁcance ( p = 0.25). Material and methods The epoxy end was mounted in a shop vise that was bolted to the mechanical test- ing machine (InstronTM 8874 Biaxial Testing Machine, Norwood, MA) with calibrated load cells. Custom-made ﬁx- ture and grips, made from a bronze alloy ASTM B150, were used to grab the clavicle (Figure 3). The Z-axis was deﬁned as the tensile axis and was arranged longitudinally to the ten- don graft. Tensile test was performed at a deformation rate of 50 mm/min. Maximum load to failure and displacement to failure were collected. Similar to Elenes et al. failure was deﬁned at the breaking point of the failure test curve [28]. Samples that experienced fracture of the clavicle during load- ing were excluded from statistical analysis. Figure 4. Maximum load to failure of eight specimens in each group. Difference between groups is statistically signiﬁcant (p = 0.001). Figure 4. Maximum load to failure of eight specimens in each group. Difference between groups is statistically signiﬁcant (p = 0.001). Data was stored in Microsoft Excel Spreadsheet (Microsoft Corp, Redmond, Washington). For statistical analysis a paired Student’s t test was performed using SPSS 22.0 (2013, IBM Corp, Armonk, NY). There was a 363% increase in average load to failure in UHMWPE augmented tendon grafts compared to the non- augmented group. An increase in average displacement to fail- ure was noted to be 19% in the augmented group. Discussion Recently the focus has been on repair using tendon grafts. In this study, we determined whether a difference in strength of AC joint repair exists between the use of a standard tendon graft and a tendon graft augmented throughout with UHMWPE suture. As expected, our data supports the proposed novel tech- nique to run UHMWPE suture throughout the entire length of the tendon graft for reinforcement. One explanation for why constructs using such augmented tendon grafts are stron- ger than nonaugmented is the added material that leads to greater time zero strength. Results of this study show that the proposed tendon graft augmentation technique signiﬁcantly increases load to failure (356%). In this biomechanical study, none of our samples fractured at the bone bridge between the tunnels. Each group experi- enced one clavicular fracture just distal to the part where the custom-made grips grabbed the clavicle. This may be due to the fact that our cadaver specimens had a higher average age (86 years) than the patients who generally require this proce- dure. To compensate for advanced age and possible osteoporo- sis the fractured specimens were excluded from statistical analysis. Grutter and Petersen [30] compared various AC joint reconstructive techniques (modiﬁed Weaver-Dunn, anatomic reconstruction using palmaris longus tendon graft, and ana- tomic reconstruction using ﬂexor carpi radialis tendon graft). One of their conclusions was that though the anatomical recon- struction is superior to the modiﬁed W-D reconstruction, the tendon graft used limited its strength. Load to failure using a ﬂexor carpi radialis tendon graft was 774 N as opposed to 326 N using a palmaris longus tendon. The strength in the native AC joint (815 N) was greater than in any of their inves- tigated reconstruction methods [30]. Current graft augmentations, such as metal or suture cer- clage techniques, have been linked to erosion of the coracoid and clavicle [33–35]. The incorporation of the suture into a tendon graft may potentially prevent the suture from cutting through clavicular tunnels or coracoid and thereby also prevent subsequent reoperations. Secondary surgeries for hardware removal, needed when employing coracoclavicular screw and plate techniques, are also obviated with our method [25, 35]. In addition, complications associated with breakage and migra- tion of metal implants are avoided. In this current study a substantial improvement in graft per- formance when augmented with a suture within the graft was observed. Discussion The AC ligament, the CC ligaments (trapezoid and con- oid), and to some degree the coracoacromial (CA) ligament, are the primary static stabilizers of the AC joints [2]. There- fore, high-energy injury to these structures will result in clini- cal instability and disability. Various operative techniques exist Q. Naziri et al.: SICOT J 2016, 2, 17 Q. Naziri et al.: SICOT J 2016, 2, 17 4 Figure 5. Displacement to failure of eight specimens in each group. Difference between groups is not statistically signiﬁcant (p = 0.25). suture and subsequently provides protection of the graft from screw damage and increases pullout strength [31]. Our sug- gested method of placing sutures continuously through the entire length of the graft offers greater load to failure and may also lead to greater pull out strength when used with screws in anatomic AC joint reconstruction. Concerns have been raised regarding clavicular fractures. Costic et al. [11] reported in their cadaver study two failed specimens due to clavicular fracture. One fracture occurred at the site where the clavicle was anchored in the epoxy com- pound. The second was due to inadequate bone bridge between the two bone tunnels in the clavicle. Turman et al. [32] reported on clavicular fractures after CC ligament reconstruction with a tendon graft. Suggested explanation of fractures includes placement of bioabsorbable screws that have the potential for osteolysis, insufﬁcient patient compliance with the postopera- tive protocol, and imperfect communication between the sur- geon and the patient regarding patient compliance with the postoperative protocol. Another factor seems to be the rela- tively large bone tunnels and subsequent cortical breach. Caroﬁno and Mazzocca [2] noted that spacing the bony tunnels at least 20–25 mm apart could prevent clavicular fractures. If tendon grafts are reinforced with UHMWPE sutures, the graft diameter could be decreased without losing graft strength. Decreased graft and bone tunnel diameter will result in less clavicular substance loss and a larger bone bridge between the tunnels. This is something to be explored in future biome- chanical studies. Figure 5. Displacement to failure of eight specimens in each group. Difference between groups is not statistically signiﬁcant (p = 0.25). for management of AC joint separation. The goal is to maintain the AC joint reduction for an adequate period of time to allow healing [29]. However, recurrence rates of AC joint separation after reconstruction can range between 20–30% and higher [3, 14–18]. Discussion The maximum load to failure recorded in the aug- mented group was 438 N. The tremendous increase compared to the nonaugmented group (94 N) suggests that our method may be a viable option to improve the biomechanical strength of tendon grafts and provide longevity to AC joint repairs. Our method may offer greater biologic ﬁxation compared to all synthetic grafts and may also restore normal arthrokine- matics because it is strong but of nonrigid nature, which is shown by the lack of statistically signiﬁcant displacement to failure when compared to the nonaugmented tendon graft. Almost half of all shoulder injuries in athletes involved in contact sports are AC joint injuries [36–38]. Also, surgical treatment of Type III is preferred in young active patients, man- ual workers, and high-level athletes [39]. This shows that higher energy injuries are more likely the cause of AC joint separations. Therefore, due to the limited number of specimens available, the decision was made to determine load to failure Charlick and Caborn [31] developed a graft preparation technique for cruciate ligament reconstruction usable in a wide range of grafts. The basis of their method is the circular place- ment of sutures (Whip stitch, Krackow stitch, or Baseball stitch) at the portion of the graft that will pass through a bone tunnel. This permits interdigitation of screw threads and the Q. Naziri et al.: SICOT J 2016, 2, 17 5 6. Geaney LE, Miller MD, Ticker JB, Romeo AA, Guerra JJ, Bollier M, Arciero RA, DeBerardino TM, Mazzocca A (2010) Management of the failed AC joint reconstruction: causation and treatment. Sports Med Arthrosc 18(3), 167–172. without number of cycles to failure. Even though the relatively stiff #5 FibreWireTM has been added to the tendon graft, dis- placement to failure did not change signiﬁcantly. This might be partially explained by the relatively circular shape of the stitch loops that changed to an oval shape with increasing ten- sion. Cyclic loading may provide greater insight and should be the focus of future investigations. 7. Inman VTMH, Neviaser J, Rowe C (1962) Treatment of complete acromioclavicular dislocation. J Bone Joint Surg Am 44, 1008–1011. 8. Phemister DB (1942) The treatment of dislocation of acromi- oclavicular joint by open reduction and threaded-wire ﬁxation. J Bone Joint Surg 24, 166–168. Another limitation was the use of calf tendon for recon- struction, which may be slightly different from human-derived tendon grafts. Conclusion 12. Mazzocca AD, Santangelo SA, Johnson ST, Rios CG, Dumonski ML, Arciero RA (2006) A biomechanical evaluation of an anatomical coracoclavicular ligament reconstruction. Am J Sports Med 34(2), 236–246. The results of our tendon graft augmentation method are very promising in terms of reconstructive strength in both max- imum load and displacement to failure. This method may be used with different AC joint recon- struction techniques that use tendon grafts for repair. However, further biomechanical and clinical studies using human allo- grafts are warranted to explore the feasibility of our novel method. 13. Fraser-Moodie JA, Shortt NL, Robinson CM (2008) Injuries to the acromioclavicular joint. J Bone Joint Surg Br 90(6), 697–707. 14. Kirchhoff C, Braunstein V, Buhmann S, Mutschler W, Bibert- haler P (2008) A salvage procedure for failed Weaver-Dunn reconstruction. Oper Orthop Traumatol 20(2), 176–181. 15. Lim YW (2008) Triple endobutton technique in acromiocla- vicular joint reduction and reconstruction. Ann Acad Med Singapore 37(4), 294–299. Discussion Ideally, tendon grafts similar to those used in actual patient surgeries, for example semitendinosus allografts should have been used. However, since the basic microarchi- tecture of collagen ﬁbrils is shared among mammals, there is no difference between the strength of individual bovine tendon ﬁbers compared to human ﬁbers. Therefore the grafts should be of similar strength, if the dimensions of the grafts are sim- ilar [40]. Also the focus of this study, being on a graft augmen- tation method and not on reconstruction techniques, allows the usage of bovine Achilles tendon. 9. Urist MR (1946) Complete dislocations of the acromiclavicular joint; the nature of the traumatic lesion and effective methods of treatment with an analysis of forty-one cases. J Bone Joint Surg Am 28(4), 813–837. 10. Lee SJ, Nicholas SJ, Akizuki KH, McHugh MP, Kremenic IJ, Ben-Avi S (2003) Reconstruction of the coracoclavicular ligaments with tendon grafts: a comparative biomechanical study. Am J Sports Med 31(5), 648–655. 11. Costic RS, Labriola JE, Rodosky MW, Debski RE (2004) Biomechanical rationale for development of anatomical recon- structions of coracoclavicular ligaments after complete acro- mioclavicular joint dislocations. Am J Sports Med 32(8), 1929–1936. Conﬂict of interest 16. Milewski MD, Tompkins M, Giugale JM, Carson EW, Miller MD, Diduch DR (2012) Complications related to anatomic reconstruction of the coracoclavicular ligaments. Am J Sports Med 40(7), 1628–1634. All authors certify that they have no ﬁnancial conﬂict of interest (e.g., consultancies, stock ownership, equity interest, patent/licensing arrangements, etc.) in connection with this article. No beneﬁts in any form have been received or will be received from a commercial party related directly or indi- rectly to the subject of this article. 17. Salzmann GM, Walz L, Buchmann S, Glabgly P, Venjakob A, Imhoff AB (2010) Arthroscopically assisted 2-bundle anatom- ical reduction of acute acromioclavicular joint separations. Am J Sports Med 38(6), 1179–1187. 18. Thiel E, Mutnal A, Gilot GJ (2011) Surgical outcome following arthroscopic ﬁxation of acromioclavicular joint disruption with the tightrope device. Orthopedics 34(7), e267–e274. References Jari R, Costic RS, Rodosky MW, Debski RE (2004) Biome- chanical function of surgical procedures for acromioclavicular joint dislocations. Arthroscopy 20(3), 237–245. 34. Ladermann A, Grosclaude M, Lubbeke A, Christoﬁlopoulos P, Stern R, Rod T, Hoffmeyer P (2011) Acromioclavicular and coracoclavicular cerclage reconstruction for acute acromiocla- vicular joint dislocations. J Shoulder Elbow Surg 20(3), 401–408. 24. Kiefer H, Claes L, Burri C, Holzwarth J (1986) The stabilizing effect of various implants on the torn acromioclavicular joint. A biomechanical study. Arch Orthop Trauma Surg 106(1), 42–46. 35. Morrison DS, Lemos MJ (1995) Acromioclavicular separation. Reconstruction using synthetic loop augmentation. Am J Sports Med 23(1), 105–110. 25. Lemos MJ (1998) The evaluation and treatment of the injured acromioclavicular joint in athletes. Am J Sports Med 26(1), 137–144. 36. Agel J, Dompier TP, Dick R, Marshall SW (2007) Descriptive epidemiology of collegiate men’s ice hockey injuries: national collegiate athletic association injury surveillance system, 1988–1989 through 2003–2004. J Athl Train 42(2), 241–248. 26. Motamedi AR, Blevins FT, Willis MC, McNally TP, Shahinpoor M (2000) Biomechanics of the coracoclavicular ligament complex and augmentations used in its repair and reconstruction. Am J Sports Med 28(3), 380–384. 37. Dick R, Romani WA, Agel J, Case JG, Marshall SW (2007) Descriptive epidemiology of collegiate men’s lacrosse injuries: national collegiate athletic association injury surveillance system, 1988–1989 through 2003–2004. J Athl Train 42(2), 255–261. 27. Shin SJ, Campbell S, Scott J, McGarry MH, Lee TQ (2014) Simultaneous anatomic reconstruction of the acromioclavicular and coracoclavicular ligaments using a single tendon graft. Knee Surg Sports Traumatol Arthrosc 22(9), 2216–2222. 38. Kaplan LD, Flanigan DC, Norwig J, Jost P, Bradley J (2005) Prevalence and variance of shoulder injuries in elite collegiate football players. Am J Sports Med 33(8), 1142–1146. 28. Elenes EY, Hunter SA. Gamma irradiated soft tissue allografts are biomechanically equivalent to aseptic, non-sterilized tissues. Available at: http://www.communitytissue.org/ﬂipbooks/SM-700- F-10/ﬁles/inc/74b81ca5cc.pdf [accessed 2 March 2015]. 39. Guy DK, Wirth MA, Grifﬁn JL, Rockwood CA Jr (1998) Reconstruction of chronic and complete dislocations of the acromioclavicular joint. Clin Orthop Relat Res 347, 138–149. 29. Dimakopoulos P, Panagopoulos A, Syggelos SA, Panagiotopoulos E, Lambiris E (2006) Double-loop suture repair for acute acromioclavicular joint disruption. Am J Sports Med 34(7), 1112–1119. 40. Gentleman E, Lay AN, Dickerson DA, Nauman EA, Livesay GA, Dee KC (2003) Mechanical characterization of collagen ﬁbers and scaffolds for tissue engineering. Biomaterials 24(21), 3805–3813. 30. References 1. Beim GM (2000) Acromioclavicular joint injuries. J Athl Train 35(3), 261–267. 19. Mazzocca A, Arciero R, Romeo A Anatomic coracoclavicular reconstruction surgical technique: for treatment of chronic acromioclavicular (AC) instabilities. Available at: http://secure. cdn.arthrex.com/pdfs/sjjgb_kEEeCRTQBQVoRHOw/sjjgb_ kEEeCRTQBQVoRHOw.pdf?Expires=1444351498&Signature= AlPUgzoCgIQA2u0yxFoR4bcHtaAppFj0fwhFvWaN41gr5D2 tiaH6cC8P8J9PhQP0gxKtnl8CGvN4eePfhBXmbr1uedCbLDzi UjVLTaeKhZR3bM~tYepPbV55LDVgByDJE2cv72ZTC7UHkK SenNaiC7AuM0WRuZ8u2vGaAvGdGJeUWxaWzo0yrphqiIW 23hDgT82hDux8drw-XjNIzX7aQnXT9a~XWZ1LDZsPKNfGX k3QUWRHTUUeSx-18-K0WGNed1ZsfqZfqyi0MfSqg0cjvc2 CkrX8-nhQHl8v-901-vzNomU3V-zs6dzgtIT0ZwDf-CZ3NrpAo QMUHh9EMpXnUw__&Key-Pair-Id=APKAJMGJRW6JX5 OBM5LA [accessed 03 March 2015]. 2. Caroﬁno BC, Mazzocca AD (2010) The anatomic coracocla- vicular ligament reconstruction: surgical technique and indica- tions. J Shoulder Elbow Surg 19(2 Suppl), 37–46. 3. Trainer G, Arciero RA, Mazzocca AD (2008) Practical management of Grade III acromioclavicular separations. Clin J Sport Med 18(2), 162–166. 4. Luis GE, Yong CK, Singh DA, Sengupta S, Choon DS (2007) Acromioclavicular joint dislocation: a comparative biomechan- ical study of the palmaris-longus tendon graft reconstruction with other augmentative methods in cadaveric models. J Orthop Surg Res 2, 22. 4. Luis GE, Yong CK, Singh DA, Sengupta S, Choon DS (2007) Acromioclavicular joint dislocation: a comparative biomechan- ical study of the palmaris-longus tendon graft reconstruction with other augmentative methods in cadaveric models. J Orthop Surg Res 2, 22. 5. Rockwood CA Jr, Williams GR, Young CD (1996) Injuries of the acromioclavicular joint, in Fractures in adults. Rockwood CA Jr, Malsen FA, Editors. Philadelphia, Lippincott-Raven. 20. Ranne JO, Sarimo JJ, Rawlins MI, Heinonen OJ, Orava SY (2012) All-arthroscopic double-bundle coracoclavicular 20. Ranne JO, Sarimo JJ, Rawlins MI, Heinonen OJ, Orava SY (2012) All-arthroscopic double-bundle coracoclavicular Q. Naziri et al.: SICOT J 2016, 2, 17 6 ligament reconstruction using autogenous semitendinosus graft: a new technique. Arthrosc Tech 1(1), e11–e14. 31. Charlick DA, Caborn DN (2000) Technical note: alternative soft-tissue graft preparation technique for cruciate ligament reconstruction. Arthroscopy 16(8), E20. 21. Deshmukh AV, Wilson DR, Zilberfarb JL, Perlmutter GS (2004) Stability of acromioclavicular joint reconstruction: biomechanical testing of various surgical techniques in a cadaveric model. Am J Sports Med 32(6), 1492–1498. 32. Turman KA, Miller CD, Miller MD (2010) Clavicular fractures following coracoclavicular ligament reconstruction with tendon graft: a report of three cases. J Bone Joint Surg Am 92(6), 1526–1532. 22. Harris RI, Wallace AL, Harper GD, Goldberg JA, Sonnabend DH, Walsh WR (2000) Structural properties of the intact and the reconstructed coracoclavicular ligament complex. Am J Sports Med 28(1), 103–108. 33. Baker JE, Nicandri GT, Young DC, Owen JR, Wayne JS (2003) A cadaveric study examining acromioclavicular joint congruity after different methods of coracoclavicular loop repair. J Shoulder Elbow Surg 12(6), 595–598. 23. Cite this article as: Naziri Q, Williams N, Hayes W, Kapadia BH, Chatterjee D & Urban WP (2016) Acromioclavicular joint reconstruction using a tendon graft: a biomechanical study comparing a novel ‘‘sutured throughout’’ tendon graft to a standard tendon graft. SICOT J, 2, 17 References Grutter PW, Petersen SA (2005) Anatomical acromioclavicular ligament reconstruction: a biomechanical comparison of reconstructive techniques of the acromioclavicular joint. Am J Sports Med 33(11), 1723–1728.
https://openalex.org/W3195554629	https://www.researchsquare.com/article/rs-1033150/latest.pdf	English	null	The Airway Microbiota of Non-Small-Cell Lung Cancer Patients and Its Relationship to Tumor Stage and EGFR Mutation	Research Square (Research Square)	2,021	cc-by	10,817	The Airway Microbiota of Non-Small-Cell Lung Cancer Patients and Its Relationship to Tumor Stage and EGFR Mutation DanHui Huang Southern Medical University Nanfang Hospital Jing He Southern Medical University Nanfang Hospital Xiaofang Su Southern Medical University Nanfang Hospital YaNa Wen Southern Medical University Nanfang Hospital ShuJia Zhang Southern Medical University Nanfang Hospital LaiYu Liu Southern Medical University Nanfang Hospital Haijin Zhao Southern Medical University Nanfang Hospital CuiPin Ye Southern Medical University Nanfang Hospital JianHua Wu Southern Medical University Nanfang Hospital Shaoxi Cai Southern Medical University Nanfang Hospital Hangming Dong (  dhm@smu.edu.cn ) Southern Medical University https://orcid.org/0 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. Research Page 1/26 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 2/26 Abstract Background: Accumulating studies have suggested the airway microbiota of lung cancer was significantly different from healthy controls. However, little was known about the relationship between airway microbiota and important clinical parameters of lung cancer. In this study, we aimed to explore the association between sputum microbiota and lung cancer stage, lymph node metastasis, intrathoracic metastasis, and Epidermal growth factor receptor (EGFR) gene mutation. Methods: The microbiota of sputum samples from 85 newly diagnosed NSCLC patients were sequenced via 16S rRNA sequencing with V3-V4 region. Sequencing reads were filtered using QIIME2 and clustered against UPARSE. Results: The α diversity and β diversity was significantly different between patients in stage I to II (early stage, ES) and patients in stage III to IV (advanced stage, AS). Lefse identified that genera Granulicatella and Actinobacillus were significantly enriched in ES, and genus Actinomyces were significantly enriched in AS. PICRUSt2 identified NAD salvage pathway was significantly enriched in AS, which was positively associated with Granulicatella. Patients with intrathoracic metastasis were associated with increased genus Peptostreptococcus and incomplete reductive TCA cycle, which was associated with increased Peptostreptococcus. Genera Parvimonas, Pseudomona and L-valine biosynthesis were positively associated with lymph node metastasis. L-valine biosynthesis was related with increased Pseudomona. Finally, genus Parvimonas were significantly upregulated in adenocarcinoma patients with EGFR mutation. Conclusion: Taxonomy structure differed between different lung cancer stage. The tumor stage, intrathoracic metastasis, lymph node metastasis, and EGFR mutation were associated with alteration of specific airway genera and metabolic function of sputum microbiota. Background Lung cancer is the second leading malignancy for morbidity and the first for cancer deaths worldwide[1]. Although with the development of target therapy and immunotherapy, the 5-year survival rate of lung cancer remain low, especially in metastatic disease[2]. Historically speaking, the lung has been considered sterile in health. However, with the advent of novel culture-independent techniques, subsequent studies identified that healthy lung was inhabited by distinct commensal microbiota, which was altered under multiple lung diseases [3]. Therefore, it is of great interest to explore the relationship between lung microbiome and lung cancer. Accumulating studies have suggested that the airway microbiota of lung cancer patients was significantly different from healthy or benign control [4-11], suggesting that airway microbiota may contribute to the development of lung cancer or be affected during the progression of lung cancer. More specifically, the α diversity[6, 8, 9, 11], β diversity[5, 6, 9-11] , and some specific genera [4-11] were changed among non-small cell lung cancer (NSCLC). As of yet, the study of lung microbiota and lung Page 3/26 Page 3/26 cancer remained in its infancy and deep knowledge of the interplay between lung cancer with different clinical parameters and lung microbiota needed to be further explored. TNM stage remains the most important prognostic factor in predicting recurrence rates and survival time. The 5-year-survival rate of lung cancer is significantly affected by tumor anatomic stages, from 87%-97% of stage I to 10%-23% of stage IV[12]. Through the analysis of 165 cases of normal tissue adjacent to lung cancer, an early study found that the α diversity and genus Thermus was more abundant in late-stage (stage IIIB and IV) than that in early-stage[13](13)(13)[13], suggesting that lung microbiota participated in the development of different stages of lung cancer. However, the study only included 7 subjects in IIIB stage and 7 subjects in IV stage. Therefore, more studies regarding the association between tumor anatomic stage and lung microbiota should be conducted to find out more potential bacterial markers linked with the stepwise change of lung cancer from early-stage to late-stage. Lung cancer staging traditionally relies on the TNM staging system. Since the stage of lung cancer was associated with lung microbiota, detailed understanding regarding the association between N, M classifications and lung microbiota should be explored. Previous studies suggested that specific genera might be engaged with the metastasis of lung cancer patients[13, 14]. Background In vivo mechanistic investigations found that certain species might contribute to the development of extrathoracic or intrathoracic metastasis via enhancement of adhesion of lung cancer cell or regulation of lung immune system[15-17]. Therefore, it is plausible to hypothesize that the lung microbiota may be identified as relevant to N and M classification. Epidermal growth factor receptor (EGFR) is a paramount therapeutic target for the treatment of lung cancer. Tyrosine kinase inhibitors (TKIs) which target the kinase domain of EGFR are especially effective in NSCLC patients whose tumors harbor activating mutations in the tyrosine kinase domain of the EGFR gene. Bacterium that carried genotoxic markers could promote the accumulation of genetic lesions and initiated cancer development[18]. Current evidences suggested that some pathogens might play a role in driving EGFR gene mutation. A retrospective study found lung adenocarcinoma patients who had tuberculosis lesions had a higher probability of having EGFR gene mutations[19]. Another early study demonstrated an association between human papillomavirus and EGFR gene mutation in lung cancer patients[20]. Conversely, EGFR mutation might also regulate lung microbiome since it played a role in maintaining airway epithelial barrier via activation of Claudin 1, a member of tight junction protein[21]. However, the association between EGFR gene mutation and lung microbiota was unknown. Thus, it is plausible that lung microbiota may have a connection with EGFR gene mutation among NSCLC patients. In this study, we used next-generation sequencing to identify airway microbiota in spontaneous sputum of NSCLC patients, aiming to characterize airway microbiota in NSCLC patients with different tumor stages (included tumor stage and TNM classification), and EGFR gene mutation. Materials And Methods 2.1 Patients and samples 2.2 DNA extraction, 16S rRNA amplification, 16S rRNA sequencing Sputum samples kept on dry ice were transferred to Sagene Biotechnology Company, GuangZhou. DNA was extracted from samples using Hipure Bacterial DNA kit (Mageon, China) using standard techniques. The V3-V4 region of 16S rRNA gene was amplified using specific primers(16S_341F:5’- CCTAYGGGRBGCASCAG-3’;16S_806R:5-GGACTACNNGGGTATCTAAT). PrimeSTAR HS DNA Polymerase was used for PCR reaction. The concentration and length of the PCR products were detected by 1% agarose gel electrophoresis. Samples with a bright main strip were used for further experiments. Sequencing libraries were conducted using the NEBNext® UltraTM DNA Library Prep Kit for Illumina® sequencing (New England Biolabs, United States). The quality of the library was evaluated under a Qubit@ 2.0 Fluorometer (Thermo Scientific) and Agilent Bioanalyzer 2100 system. Sequencing was conducted to generate 250-bp paired-end reads using an Illumina HiSeq 2500 sequencer according to the manufacturer’s instructions. 2.1 Patients and samples Page 4/26 Page 4/26 The study was approved by the Ethics Committee of Nanfang Hospital, Southern Medical University. First diagnosed NSCLC patients were prospectively admitted in this study at NanFang Hospital, Southern Medical University between April 2017 and September 2019. The inclusion criteria were as follows: pathologically diagnosed of NSCLC; aged 30-80; did not receive any anti-tumor therapy such as surgery, radiotherapy, chemotherapy, targeted therapy or immunotherapy; no evidence of community-acquired pneumonia, acute exacerbation of chronic obstructive pulmonary disease, bronchiectasis with infection, acute bronchitis or asthma; had no fever or purulent or gray sputum; without a history of other malignant diseases or multiple primary lung cancer. We conducted a questionnaire and reviewed the electronic medical records to obtain demographic and clinical data including age, sex, smoking status, antibiotics usage, TNM stage, systemic or pulmonary comorbidities and tumor EFGR mutation. Tumor anatomic stage and TNM classification was based on NCNN clinical practice Guidelines of NSCLC (Version 2020. V1). The EGFR mutation was detected based on the ARMS technology in the pathology department of Nanfang Hospital. Participants were asked to rinse their mouths before sampling. The first mouthful of phlegm in the morning was collected within 24 hours of hospitalization and transferred into -20℃ refrigerators within 2 hours and then transferred into -80℃ within 1 week. 2.3 Microbiota analysis P value ≤ 0.05 and SparCC correlation scores ≥ 0.5 or ≤ -0.5 were included for networks inference. (http://huttenhower.sph.harvard.edu/galaxy). PICRUSt2 was used to predict the functional profiling of microbial communities based on the 16S rRNA sequence[25]. Metabolic function predictions were based on MetaCyc [26] database. Differentially present pathways between groups were analyzed with welch t test using STAMP[27]. The network analysis on the genus level was carried out with SparCC[28]. P value ≤ 0.05 and SparCC correlation scores ≥ 0.5 or ≤ -0.5 were included for networks inference. 2.4 Statistical analysis The software SPSS (V 23.0) was used for statistical analysis. The continuous variables were compared between two groups by Mann-Whitney U test or independent t test. The categorical variables were compared by chi-square test, continuity-adjusted chi-square test, Fisher’s exact test. P value<0.05 was considered statistically significant. 3.1 Subjects clinical characteristics and sputum microbiota in NSCLC Spontaneous sputum samples were collected from 116 NSCLC patients preliminarily. After carefully assessment, 85 patients who met eligible criteria were finally taken into further analysis. The procedure of patients’ recruitment and exclusion was shown in Figure 1. The average number of trimmed sequences reads number of the 85 subjects was 33271 (7869, 44193). OTU rarefaction curve was constructed to evaluate sequence depth (Supplementary figure S1). The result indicated that sequence depth of sputum samples was sufficient enough to reach a reliable estimate of microbiome structure. The median age of all patients was 59.21±8.75 years. The clinical characteristics of the 85 patients were listed in supplementary table S1. Among the 85 patients, 66 (78%) were adenocarcinoma, 18 (21%) were squamous cell carcinoma, 1 was unidentified type of NSCLC. 13(15%) patients were in tumor stage Ⅰ, 9 (11%) were in stage Ⅱ, 11 (13%) were in stage Ⅲ and 40 (47%) were in stage Ⅳ. Phyla and genera that were ≥ 1% were considered as dominant. At the phylum level, the dominant phyla in the sputum samples of NSCLC were Firmicutes (40%), Bacteroidetes (20%), Actinobacteria (17%), Proteobacteria (13%), Fusobacteria (6%), and TM7 (3%) (Supplementary figure S2 A). At the genus level, the dominant genera in the sputum samples of NSCLC were Streptococcus (21%), Prevotella (12%), Rothia (9%) and Neisseria (7%), Actinomyces (5%), Leptotrichia (4%), Porphyromona (4%), Veillonella (4%), Granulicatella (3%), Haemophilus (3%), Atopobium (2%), Peptostreptococcus (2%), Capnocytophaga (2%) and Fusobacterium (1%) (Supplementary figure S2 B). 3.2 The Association between sputum microbiota and NSCLC clinical stage 2.3 Microbiota analysis Raw data was obtained and then further filtered to eliminate reads with adapter pollution and low quality to obtain clean reads by using QIIME2[22]. Clean sequences were clustered by 97% identity into operational taxonomic units (OTUs) using UPARSE[23]. Representative sequence of each OTU was annotated into taxonomy against Greengenes database[24]. We applied OTUs data in online microbiome data analyze platform (MicrobiomeAnalyst) (https://www.microbiomeanalyst.ca/) to compare microbiota community structure at both inter- community and α-diversity level and β-diversity level. For α diversity, we chose Chao1 value, Simpson Page 5/26 index and Shannon index to evaluate. For β diversity, we estimated using Bray-Curtis distance and visualized by principal coordinate analysis (PCoA). Differential taxonomy was identified by LEfSe (Linear discriminant analysis (LDA) effect size) analysis in an online platform (GALAXY) index and Shannon index to evaluate. For β diversity, we estimated using Bray-Curtis distance and visualized by principal coordinate analysis (PCoA). Differential taxonomy was identified by LEfSe (Linear discriminant analysis (LDA) effect size) analysis in an online platform (GALAXY) index and Shannon index to evaluate. For β diversity, we estimated using Bray-Curtis distance and visualized by principal coordinate analysis (PCoA). Differential taxonomy was identified by LEfSe (Linear discriminant analysis (LDA) effect size) analysis in an online platform (GALAXY) (http://huttenhower.sph.harvard.edu/galaxy). PICRUSt2 was used to predict the functional profiling of microbial communities based on the 16S rRNA sequence[25]. Metabolic function predictions were based on MetaCyc [26] database. Differentially present pathways between groups were analyzed with welch t test using STAMP[27]. The network analysis on the genus level was carried out with SparCC[28]. P value ≤ 0.05 and SparCC correlation scores ≥ 0.5 or ≤ -0.5 were included for networks inference. index and Shannon index to evaluate. For β diversity, we estimated using Bray-Curtis distance and visualized by principal coordinate analysis (PCoA). Differential taxonomy was identified by LEfSe (Linear discriminant analysis (LDA) effect size) analysis in an online platform (GALAXY) (http://huttenhower.sph.harvard.edu/galaxy). PICRUSt2 was used to predict the functional profiling of microbial communities based on the 16S rRNA sequence[25]. Metabolic function predictions were based discriminant analysis (LDA) effect size) analysis in an online platform (GALAXY) (http://huttenhower.sph.harvard.edu/galaxy). PICRUSt2 was used to predict the functional profiling of microbial communities based on the 16S rRNA sequence[25]. Metabolic function predictions were based on MetaCyc [26] database. Differentially present pathways between groups were analyzed with welch t test using STAMP[27]. The network analysis on the genus level was carried out with SparCC[28]. .2 The Association between sputum microbiota and NSCLC clinical stage A previous study found that the α diversity of microbiota of non-malignant tissues adjacent to stage IIIB lung tumor tissues was similar with that of stage IV[13], suggesting that the airway microbiota may be similar between stage III and stage IV lung cancer patients. To evaluate the similarity of microbiota between stage III and stage IV patients, we compare the sputum microbiota between these 2 groups via diversity analysis and differential analysis. Among the 11 NSCLC patients in stage III, 4 patients were in stage IIIA, 5 patients were in stage IIIB, 2 patients were in stage IIIC. Baseline information included age (independent samples T test, P=0.21), BMI index (Mann-Whitney U test, P=0.536), smoking status (continuity-adjusted chi-square test, P=0.249), antibiotics treatment before sampling (continuity- adjusted chi-square test, P=0.121), pathological type (continuity-adjusted chi-square test, P=0.191) was comparable between the groups. Chao1, Simpson index, and Shannon index were selected to estimate the α diversity of the lung microbiome community. α diversity between stage III and stage IV patients was similar (Mann-Whitney U test, P=0.519 for Shannon; P=0.783 for chao1; P=0.261 for Simpson index) (Supplementary figure S3 A-C). β diversity based on Bray Curtis distance was used to estimate the β diversity of lung taxonomy community structure in different groups. The result showed that there was no significant difference in taxonomy structure between stage III and stage IV patients (PERMANOVA test, P=0.905) (Supplementary figure S3 D). LEfSe analysis was conducted to identify whether differential taxonomy existed between stage III and stage IV patients. Only genus Paludibacter was found to be significantly different between the 2 groups (Supplementary figure S3 E). The relative abundance of Paludibacter was only 0.01% in stage III and 0.05% in stage IV patients. Taken together, the results above suggested that sputum microbiome of stage III and stage IV patients was similar. Since the sputum microbiome of stage III and stage IV was similar, we divided the lung cancer patients into 2 groups: stage I and stage II (Early stage, ES) and Stage III and stage IV (Advanced stage, AS) and evaluate the microbiota difference between these 2 groups. Baseline information included demographic and clinical characteristic were comparable between AS and ES groups (supplementary table S2). The relative abundance of phylum level and genus level of ES and AS group were shown in Figure 2 A-B. .2 The Association between sputum microbiota and NSCLC clinical stage Page 6/26 Stage III and stage IV lung cancer patients are on a continuum with respect to tumor burden. It is well accepted that a great number of lung cancer patients with anatomical stage III also harbor micro- metastases. A previous study found that the α diversity of microbiota of non-malignant tissues adjacent to stage IIIB lung tumor tissues was similar with that of stage IV[13], suggesting that the airway microbiota may be similar between stage III and stage IV lung cancer patients. To evaluate the similarity of microbiota between stage III and stage IV patients, we compare the sputum microbiota between these 2 groups via diversity analysis and differential analysis. Among the 11 NSCLC patients in stage III, 4 patients were in stage IIIA, 5 patients were in stage IIIB, 2 patients were in stage IIIC. Baseline information included age (independent samples T test, P=0.21), BMI index (Mann-Whitney U test, P=0.536), smoking status (continuity-adjusted chi-square test, P=0.249), antibiotics treatment before sampling (continuity- adjusted chi-square test, P=0.121), pathological type (continuity-adjusted chi-square test, P=0.191) was comparable between the groups. Chao1, Simpson index, and Shannon index were selected to estimate the α diversity of the lung microbiome community. α diversity between stage III and stage IV patients was similar (Mann-Whitney U test, P=0.519 for Shannon; P=0.783 for chao1; P=0.261 for Simpson index) (Supplementary figure S3 A-C). β diversity based on Bray Curtis distance was used to estimate the β diversity of lung taxonomy community structure in different groups. The result showed that there was no significant difference in taxonomy structure between stage III and stage IV patients (PERMANOVA test, P=0.905) (Supplementary figure S3 D). LEfSe analysis was conducted to identify whether differential taxonomy existed between stage III and stage IV patients. Only genus Paludibacter was found to be significantly different between the 2 groups (Supplementary figure S3 E). The relative abundance of Paludibacter was only 0.01% in stage III and 0.05% in stage IV patients. Taken together, the results above suggested that sputum microbiome of stage III and stage IV patients was similar. Stage III and stage IV lung cancer patients are on a continuum with respect to tumor burden. It is well accepted that a great number of lung cancer patients with anatomical stage III also harbor micro- metastases. .2 The Association between sputum microbiota and NSCLC clinical stage For the α diversity, there was a significantly difference in the Chao1 index between ES and AS group. Chao1 index was 221.529 (42.976) in ES group and 198.752398 (42.20770) in AS group (Mann-Whitney U test, P=0.038) (Figure 3 A). Simpson index was 0.907 (0.064) in ES patients and 0.919 (0.050) in AS patients (P=0.705) (Figure 3 B). Shannon index was 3.398 (0.541) in ES group and 3.419 (0.448) in AS group (P=0.815) (Figure 3 C). For the β diversity, Bray Curtis distance based on genus level was performed. The result showed that there was significantly different taxonomy structure between patients in ES group and AS group (genus level, PERMANOVA test, P=0.045) (Figure 3D). Differentia analysis using Lefse identified that phylum Firmicutes, genera Peptoniphilus, Granulicatella, Hylemonella, Actinobacillus, SMB53 and Gemella were significantly enriched in ES group, and phylum Actinobacteria, genus Actinomyces were significantly enriched in AS group (Figure 4A). The relative abundance of phyla Firmicutes and Actinobacteria, genera Granulicatella, Actinomyces and Actinobacillus were ≥ 0.1% and were shown in Figure 4 B, C. Page 7/26 Page 7/26 Page 7/26 Functional analysis based on metaCyc database identified 29 differentially abundant pathways (Figure 4 D). The largest 3 pathways which had higher proportion in ES patients were anhydromuropeptides recycling, gondoate biosynthesis (anaerobic) and L-lysine biosynthesis II. For the differential abundant pathways had higher relative abundance in AS group, incomplete reductive tricarboxylic acid (TCA) cycle, NAD salvage pathway I and phosphopantothenate biosynthesis I were the top 3 differentially abundant pathways. Genus Actinomyces was positively correlated with the NAD salvage pathway (Spearman rank correlation, P value < 0.0001, r=0.547). Functional analysis based on metaCyc database identified 29 differentially abundant pathways (Figure 4 D). The largest 3 pathways which had higher proportion in ES patients were anhydromuropeptides recycling, gondoate biosynthesis (anaerobic) and L-lysine biosynthesis II. For the differential abundant pathways had higher relative abundance in AS group, incomplete reductive tricarboxylic acid (TCA) cycle, NAD salvage pathway I and phosphopantothenate biosynthesis I were the top 3 differentially abundant pathways. Genus Actinomyces was positively correlated with the NAD salvage pathway (Spearman rank correlation, P value < 0.0001, r=0.547). Co-abundance analysis based on SparCC was conducted. The sputum microbiota structure of ES lung cancer patients was more complex and better organized than the taxonomy structure inferred for patients in AS group (Figure 5 A, B). .2 The Association between sputum microbiota and NSCLC clinical stage The taxonomy structure of ES group was composed of 33 genera while the structure inferred for AS group was composed of 19 genera. The number of inter-genus correlations in ES group was 78, while only 44 in AS group. The interactions between genus Streptococcus and other genera (Porphyromonas, Prevotella, Capnocytophaga, Veillonella, Atopobium, Actinomyces, Rothia, Granulicatella) were exclusively co-occurrence in the AS group. Co-occurrence between Actinomyces and genera Rothia and Atopobium was ubiquitous among 2 groups, while co-occurrence between Actinomyces and genera Granulicatella, Veillonella, Prevotella and Streptococcus were exclusive in AS group. Co-abundance analysis based on SparCC was conducted. The sputum microbiota structure of ES lung cancer patients was more complex and better organized than the taxonomy structure inferred for patients in AS group (Figure 5 A, B). The taxonomy structure of ES group was composed of 33 genera while the structure inferred for AS group was composed of 19 genera. The number of inter-genus correlations in ES group was 78, while only 44 in AS group. The interactions between genus Streptococcus and other genera (Porphyromonas, Prevotella, Capnocytophaga, Veillonella, Atopobium, Actinomyces, Rothia, 3.3 The role of sputum microbiota on NSCLC intrathoracic metastasis and lymph node metastasis Since tumor stage is associated with the organism metastasis and lymph node metastasis, a further analysis was conducted to explore the linkage between sputum microbiota and these clinical parameters. Previous mouse studies suggested that the homeostasis of commensal lung microbiota may affect intrathoracic metastasis[17] and extrathoracic metastasis[15] and these 2 phenomena may depend on different mechanisms. Thus, we hypothesized that airway microbiota associated with intrathoracic (ipsilateral or contralateral lung metastasis or pleural metastasis) and extrathoracic metastasis was different. Among the 85 NSCLC patients, 15 were with intrathoracic metastasis and without extrathoracic metastasis (Intra group), only 3 patients were with extrathoracic metastasis and without intrathoracic metastasis, 28 were without neither intrathoracic nor extrathoracic metastasis (Non_M group). We further explore the characterization of sputum microbiota among Intra and Non_M patients. Baseline information was comparable between Intra and Non_M group (Supplementary table S3). α diversity index between these 2 groups was similar (P=0.6192 for chao1; P=0.1668 for Simpson index; P=0.2193 for Shannon) (Supplementary Figure S4 A-C). β diversity based on Bray Curtis distance was used and the result showed that taxonomy structure between Intra group and Non_M group was similar (PERMANOVA test, P=0.197) (Supplementary Figure S4 D). LEfse analysis showed that compared with Non-M patients, genera Peptostreptococcus, Peptococcus, Parabacteroides, and Escherichia were significantly enriched in Intra patients, while phylum Firmicutes and genus Streptococcus were significantly decreased (Supplementary Figure S5 A). The relative Page 8/26 Page 8/26 abundance of phylum Firmicutes and genera Peptostreptococcus and Streptococcus were ≥ 0.1% and were listed in Supplementary Figure S5 B, C. Functional analysis based on metaCyc database identified 31 differentially abundant pathways (Supplementary Figure S5 D). The largest 3 pathways which had higher proportion in Intra patients were incomplete reductive TCA cycle, tetrapyrrole biosynthesis II (from glycine), tetrapyrrole biosynthesis I (from glutamate). For the differential abundant pathways had higher relative abundance in Non_M group, purine ribonucleosides degradation, lactose and galactose degradation I, L-lysine biosynthesis II were the top 3 differentially abundant pathways. Genus Peptostreptococcus was positively correlated with the incomplete reductive TCA cycle (Spearman rank correlation, P value=0.017, r=0.5779). Next, we explored the association between sputum microbiota and lymph node metastasis. Among 28 patients in M0 stage, 12 were in N1-3 stage (LNM_Y) and 16 were in N0 stage (LNM_N). Baseline information was comparable between LNM_Y and LNM_N group (Supplementary table S4). 3.3 The role of sputum microbiota on NSCLC intrathoracic metastasis and lymph node metastasis α diversity analysis indicated that Chao 1 (P=0.0593), Simpson (P=1.000), and Shannon (P=0.9818) index were similar between the 2 groups (Supplementary Figure S6 A-C). β diversity analysis based on Bray Curtis distance showed that there was no significant difference in the bacterial community between the 2 groups (PERMANOVA test, P=0.091) (Supplementary Figure S6 D). Compared with LNM_N, genera Parvimonas and Pseudomona were significantly increased in LNM_Y, while phylum Proteobacteria and genera Neisseria, Actinobacillus, Eikenella were significantly declined in LNM_Y (Supplementary Figure S7 A). All the above-mentioned differential taxonomy except for genus Eikenella was ≥0.1%. The relative abundance of each differential genus and phylum were listed in Supplementary Figure S7 B, C Functional profile prediction based on Metacyc database identified 23 differential metabolic pathways (Supplementary Figure S7 D). L-valine biosynthesis, L-isoleucine biosynthesis I (from threonine), L- isoleucine biosynthesis II were the top 3 differential pathways that were more abundant in LNM_Y group. Anhydromuropeptides recycling, 8-amino-7-oxononanoate biosynthesis I, biotin biosynthesis I and ppGpp biosynthesis were the top 3 differential pathways that were more enriched in LNM_N group. Genus Pseudomonas was associated with L-valine biosynthesis (Spearman rank correlation, P value= 0.012, r=0.468). 3.4 The Association between sputum microbiota and NSCLC EGFR gene mutation Among the 65 lung adenocarcinoma patients, 44 patients with EGFR mutation testing were available in subgroup analysis. Finally, 21 were with EGFR mutation-positive (EGFR+), 23 were with EGFR mutation- negative (EGFR-). Patients with EGFR mutation were more likely to be never smoker (Fisher exact test, P=0.036) and female (Fisher exact test, P=0.031). Other baseline information included age, BMI, tumor stage, antibiotics usage was comparable between 2 groups (Supplementary table S5). Page 9/26 Page 9/26 α diversity between EGFR+ and EGFR- was similar (P=0.1054 for chao1; P=0.1532 for Simpson index; P=0.0820 for Shannon) (Supplementary Figure S8 A-C). β diversity based on Bray Curtis distance was conducted to estimate the bacterial community composition in different groups. The result showed that there was no association between EGFR mutation and airway taxonomy structure (PERMANOVA test, P=0.212) (Supplementary Figure S8 D). LEfse analysis identified that EGFR mutation was associated with significantly enriched level of phyla Bacteroidetes and Tenericutes, genera Sharpea, Prevotella, Porphyromonas, Parvimonas, Desulfovibrio, Mycoplasma, Actinobacillus, Dialister, and Eikenella (Figure 6 A). Subgroup analysis limited to non- smoker subjects was conducted. The result showed similarly that phylum Bacteroidetes and genera Parvimonas and Actinobacillus were associated with EGFR mutation (Figure 6 B). The relative abundance of both genera Parvimonas and Actinobacillus and phylum Bacteroidetes were ≥0.1% and were shown in Figure6 C, D PICRUSt2 based on Metacyc prediction identified that superpathway of L-aspartate and L-asparagine biosynthesis, preQ0 biosynthesis and queuosine biosynthesis were the most 3 significantly abundant pathways in EGFR mutation non-smoking group and L-isoleucine biosynthesis II, L-isoleucine biosynthesis II III and superpathway of branched amino acid biosynthesis were the top 3 pathways that were significantly enriched in EGFR negative non-smoking group (Figure6 E). Discussion Growing pieces of evidence suggested that the development of cancer is affected by human commensal microbiota through inflammation, immunity, metabolism pathways[29]. Recently, various studies identified the alteration of airway microbiota among NSCLC patients[5, 7, 9, 11, 30-34]. The interplay between microbiota and lung cancer is complex. However, only few studies focused on the association between airway microbiota and tumor clinical parameters, includes tumor anatomic stage, metastasis, and gene mutation. In this study, we reported the characterization of sputum microbiota among NSCLC patients with early stage (stage I and stage II) and advanced stage (stage III and stage IV). More deeply, we explored the association between sputum microbiota and tumor N stage and intrathoracic metastasis. Besides, we investigated the linkage between EGFR mutation of lung adenocarcinoma and sputum microbiota. Using 16S rRNA sequencing to profile the sputum microbiota in NSCLC patients, we found that the most abundant phylum and genus in NSCLC sputum samples were Firmicutes (40%) and Streptococcus (21%), which was consistent with the previous 2 studies analyzed sputum microbiota in lung cancer patients [11, 33]. TNM stage is the most predominant factor in predicting NSCLC survival time(35). The stepwise development of NSCLC from early-stage to late-stage was the results of various genetic and epigenetic alterations[36, 37], which may be associated with alteration of airway microbiota. Lung cancer staging system is categorical, however stage III and stage IV lie on a continuum with respect to tumor burden[38]. A great proportion of stage III patients had occult metastasis. The difference of stage III and stage IV lung Page 10/26 Page 10/26 Page 10/26 cancer patients lie on the tumor burden of distant sites, instead of the tumor burden of local reginal sites[38]. Among the 11 III stage NSCLC patients enrolled in this study, 7 (63%) patients were in stage IIIB or IIIC. We found that the α diversity and β diversity between stage III and stage IV patients were not significantly different, suggesting that the sputum microbiota might not sensitively reflect the tumor burden of distant site. Similarly, Yu et al collected adjacent tumor tissues from lung cancer patients and found that the α diversity among NSCLC patients in IIIB and IV stage was similar[13]. Discussion However, considering that III stage NSCLC is a heterogeneous disease, the difference of sputum or lung tissue microbiota between stage III and stage IV lung cancer should be interpreted in a larger scale study in the future. Compared with ES stage patients, we found a significant reduction of α diversity in AS patients. The significant decrease of α diversity in lung cancer patients compared with healthy or non-malignant control was evident in several studies, among which 2 studies used sputum samples[39, 40], 1 study used protected brush samples[41] and 1 study used surgical lung tissues[7]. Taken above, the results suggested that the reduction of α diversity might be a potential marked indicated the development and progression of lung cancer. β diversity between ES and AS lung cancer patients were significant different in our study, indicating that the taxonomy community structure differed during the progression of lung cancer. The results of genus network analysis also supported the difference of taxonomy community structure. The SparCC results indicated that the sputum microbiota structure of ES lung cancer patients was more complex and better organized than the taxonomy structure inferred for AS patients. We reported differential abundant taxonomy among NSCLC patients in AS stage and ES stage. More precisely, phylum Firmicutes, genera Granulicatella, Actinobacillus were significantly enriched in ES group, and phylum Actinobacteria, genus Actinomyces were significantly enriched in AS group. Granulicatella has been previously identified as a member of the normal bacterial flora of the respiratory tract[42] and was implicated in clinical infection such as sinusitis[43]. A study enrolled female lung cancer patients in China and found significantly enriched genus Granulicatella in sputum samples of lung cancer patients compared with healthy control[11]. Another pilot study using metagenomic sequencing technology identified Granulicatella adicens, a species belongs to genus Granulicatella, in sputum of lung cancer patients compared with benign diseases[4]. Taken together, our result and the previous studied mentioned above suggested that genus Granulicatella might played a role in the early development of NSCLC. Actinobacillus was a common member of human oral commensal microbiota. Previous studies found that Actinobacillus might influence the production of inflammatory cytokines[16] and was associated with COPD [44]. COPD is a widely recognized risk factor of lung cancer. Chronic inflammation is a key feature of COPD and could be a potential driver for lung cancer development[45].Thus, genus Actinobacillus might serve as a linkage between COPD and lung cancer. Discussion It is plausible that the inhabitation of Actinobacillus lead to a chronic inflammation of the lung and enhance the initiation and early development of lung cancer. An early study identified genus Actinomyces was a common anaerobe colonizing in the airway of lung cancer patients[46]. It is interesting to note that in our study the co- occurrence of Actinomyces and genus Veillonella exclusively existed in AS group. Thus, in AS lung cancer patients, the increase of genus Actinomyces could increase the abundance of genus Veillonella. A Page 11/26 Page 11/26 previous study found that the lower airway of lung cancer patients was enriched for genus Veillonella, which was further found to be associated with upregulation of ERK and PI3K signaling pathways[47]. It was recognized that PI3K and ERK pathways activation was involved in lung cancer metastasis[48]. Besides, we found genus Actinomyces was positively related with NAD salvage pathway, which was significantly enriched in AS patients. Cancer cells have enhanced glycolysis for sustaining rapid proliferation. Increased NAD levels enhance glycolysis and fuel cancer cells and is associated with cancer cell survival and enhanced invasion capacity [49, 50]. In fact, rate-limiting enzyme, such as nicotinamide phosphoribosyltransferase, was frequently amplified in several cancer cells[51]. Thus, in addition to its possible indirect influence on cancer related signaling pathway, genus Actinomyces might enhance lung cancer progression partly via enhanced NAD production. previous study found that the lower airway of lung cancer patien which was further found to be associated with upregulation of E was recognized that PI3K and ERK pathways activation was invo Besides, we found genus Actinomyces was positively related wit significantly enriched in AS patients. Cancer cells have enhanced proliferation. Increased NAD levels enhance glycolysis and fuel c cell survival and enhanced invasion capacity [49, 50]. In fact, rate phosphoribosyltransferase, was frequently amplified in several c possible indirect influence on cancer related signaling pathway, g cancer progression partly via enhanced NAD production. Lung microbiota was reported to have influence on proliferation or metastasis of intrathoracic cancer via regulation of immune system[17, 52]. In this study, we reported intrathoracic metastasis was associated with enriched sputum genus peptostreptococcus and decreased Streptococcus. Peptostreptococcus was associated with colon cancer progression[53, 54]. However, its relationship with lung cancer remained largely unknown. We noticed that genus Peptostreptococcus are obligate anaerobes. It has been suggested that tumor microenvironment condition such as hypoxia may enhance tumor invasion and metastasis[55]. Discussion Valine and isoleucine belong to branched chain amino acids, which play critical role in the regulation of energy homeostasis, nutrition metabolism, immunity and disease in humans[66]. They can act as signaling molecules regulating metabolism of glucose, lipid, and protein synthesis and serve as potential biomarkers in cancer[66](66)(66)[66]. Since genus Pseudomona was positively associated with L-valine biosynthesis, it was plausible that Pseudomona might apply valine for lung cancer cell and enhance its proliferation and invasiveness. EGFR mutation was a strong prognostic factor among lung adenocarcinoma patients. The present data here showed that certain sputum bacterium had a close link with EGFR mutation among lung adenocarcinoma. Both in the overall analysis and subgroup analysis limited to non-smoker subjects, the results showed that the relative abundance of phylum Bacteroidetes and genera Parvimonas and Actinobacillus were positively associated with EGFR mutation. The increased EGFR signaling pathway was identified as relevant to airway mucin production, epithelial cell repairment [67], thus may have an influence on the abundance of phylum Bacteroidetes, genus Parvimonas and genus Actinobacillus. On the other hand, other evidences suggested that some specific bacterium such as genus Parvimonas may cause the EGFR mutation. Currently, several evidences suggested that Parvimonas micra, a member of genus Parvimonas, was enriched in patients with colon cancer[68, 69]. Interestingly, in vitro study demonstrated that infection of Parvimonas micra could enhance the ability of human inflammatory cells to generate reactive oxygen species and caused DNA damage of human cells[70], which could cause oncogene mutation and carcinogenesis. Our study provided novel insight into the association between sputum microbiota, its predicted metabolic function and lung cancer stage, intrathoracic metastasis, lymph node metastasis and EGFR mutation. However, there are some limitations in our study. Firstly, the number of patients enrolled in this study is not large enough, so there may be heterogeneity. Secondly, the use of sputum can not surrogate lung cancer tissue. It should be caution to interpret intratumor microbiota using our results. Thirdly, the discovery of specific bacterial genera to distinguish lung cancer with various important clinical parameters hypothesis lacked validation cohorts, which may result in the false positive value and unreliability. Fourthly, the study is a cross-sectional study and only illustrates the phenomenon from microbiology. The mechanism of the microbiota and the causal relationship needed further exploration. Discussion Therefore, it was plausible to speculate that the anoxic lung tumor condition, which can facilitate intrathoracic metastasis, may favor the growth of some obligate anaerobe, such as genus Peptostreptococcus. It is of interest to notice that incomplete reductive TCA cycle of sputum microbiota was significantly enriched in Intra group and was positively related with genus Peptostreptococcus. Reductive TCA cycle existed in anaerobe, including some deeply rooted bacteria, is one alternative strategy for fixing CO2[56]. During this reaction, oxaloacetate is finally produced[57] and may participate in TCA cycle in cancer cell. Current evidences demonstrated that certain cancer cells, including lung cancer with specific genome subtype[58, 59], rely heavily on the TCA cycle for energy production [60]. A recent study reported that enhanced TCA cycle might promote lung metastasis of certain cancer[61]. In the absence of distant metastasis, the existence of lung cancer spread to a regional lymph node affects clinical treatment options and prognosis. In this study, we found that the α diversity and β diversity were similar between LNM_Y and LNM_N, which indicated that the sputum taxonomy structure did not vary during the progression of lymph node metastasis. LEfse analysis revealed genera Parvimonas, Pseudomona was positively correlated with lymph node metastasis, while genera Neisseria and Actinobacillus was associated with depression of lymph node metastasis. Genus Pseudomonas showed a correlation with adenocarcinoma[62]. A clinical study identified that genus Pseudomonas was positively associated with matrix metalloproteinase in transplant lung patients[63], which was associated with metastasis and invasiveness of cancer cell[64]. Genus Neisseria was found to be negatively associated with lymph node metastasis. A previous study discovered that compared with healthy control, the relative abundance of salivary Neisseria was significantly decreased among lung cancer patients, which suggested that it might serve as a protective role in lung cancer progression[65]. Metabolic function prediction identified L-valine biosynthesis and L-isoleucine were increased in sputum microbiota Page 12/26 Page 12/26 of LNM_Y patients. Valine and isoleucine belong to branched chain amino acids, which play critical role in the regulation of energy homeostasis, nutrition metabolism, immunity and disease in humans[66]. They can act as signaling molecules regulating metabolism of glucose, lipid, and protein synthesis and serve as potential biomarkers in cancer[66](66)(66)[66]. Since genus Pseudomona was positively associated with L-valine biosynthesis, it was plausible that Pseudomona might apply valine for lung cancer cell and enhance its proliferation and invasiveness. of LNM_Y patients. Abbreviations Abbreviations NSCLC: non-small-cell lung cancer EGFR: Epidermal growth factor receptor ES: early stage AS: advanced stage TKIs: Tyrosine kinase inhibitors OTUs: operational taxonomic units TCA: reductive tricarboxylic acid Intra: lung cancer patients with intrathoracic metastasis but without extrathoracic metastasis Non-M: lung cancer patients without neither intrathoracic nor extrathoracic metastasis LNM_Y: M0 lung cancer patients with lymph node metastasis LNM_N: M0 lung cancer patients without lymph node metastasis EGFR+ lung cancer with EGFR mutation EGFR-: lung cancer without EGFR mutation l Data availability statement Public database with 16S rRNA sequencing data could be obtained online at the Sequence Read Archive (SRA). The BioProject number is PRJNA741774. Ethics approve and consent to participate The study was approved by the Ethics Committee of Nanfang Hospital, Southern Medical University and all analysis were performed after obtaining individual written informed consent. Conclusions Collectively, the present data showed association between important clinical parameters of lung cancer and airway microbiota. The taxonomy structure differed between patients in early stage and advanced stage. The tumor stage, intrathoracic metastasis, lymph node metastasis, and EGFR mutation were associated with alteration of specific airway genera and predicted metabolic function of sputum microbiota. Our study shed light that airway microbiota might participate in various pathophysiological processes that were importantly related to lung cancer development. Further studies with large scale and multi-omics are needed to achieve a better understanding of the role of microbiota in the development Page 13/26 Page 13/26 and progression of lung cancer could pave a new way for exploring new therapeutic options and biomarkers. and progression of lung cancer could pave a new way for exploring new therapeutic options and biomarkers. biomarkers. Abbreviations NSCLC: non-small-cell lung cancer EGFR: Epidermal growth factor receptor ES: early stage AS: advanced stage TKIs: Tyrosine kinase inhibitors OTUs: operational taxonomic units TCA: reductive tricarboxylic acid Intra: lung cancer patients with intrathoracic metastasis but without extrathoracic metastasis Non-M: lung cancer patients without neither intrathoracic nor extrathoracic metastasis LNM_Y: M0 lung cancer patients with lymph node metastasis LNM_N: M0 lung cancer patients without lymph node metastasis EGFR+ lung cancer with EGFR mutation EGFR-: lung cancer without EGFR mutation Consent for publication Obtained. Obtained. Page 14/26 Acknowledgements Not applicable. Funding This work was supported by National Natural Science foundation of China (No. 81970032; 81670026; 8187011256). Contribution (I) Conceptualization: Hangming Dong, Shaoxi Cai, DanHui Huang. (II) Formal analysis: DanHui Huang, Jing He, XiaoFang Su; (III) Data curation: DanHui Huang, YaNa Wen, ShuJia Zhang. (IV) Project administration: Jing He, XiaoFang Su, YaNa Wen, ShuJia Zhang, LaiYu Liu, Haijin Zhao, CuiPin Ye, JianHua Wu. (V)Funding acquisition: Shaoxi Cai, Hangming Dong; (VI) Writing-original draft: DanHui Huang, Jing He. (VII) Writing-review and editing: Shaoxi Cai, Hangming Dong. Declaration of Competing Interest There are no interests to declare. 2020, 9(3):693-704. 8. Liu HX, Tao LL, Zhang J, Zhu YG, Zheng Y, Liu D, Zhou M, Ke H, Shi MM, Qu JM: Difference of lower airway microbiome in bilateral protected specimen brush between lung cancer patients with unilateral lobar masses and control subjects. INT J CANCER 2018, 142(4):769-778. 9. Greathouse KL, White JR, Vargas AJ, Bliskovsky VV, Beck JA, von Muhlinen N, Polley EC, Bowman ED, Khan MA, Robles AI et al: Interaction between the microbiome and TP53 in human lung cancer. GENOME BIOL 2018, 19(1):123. 10. Apopa PL, Alley L, Penney RB, Arnaoutakis K, Steliga MA, Jeffus S, Bircan E, Gopalan B, Jin J, 10. Apopa PL, Alley L, Penney RB, Arnaoutakis K, Steliga MA, Jeffus S, Bircan E, Gopalan B, Jin J, Patumcharoenpol P et al: PARP1 Is Up-Regulated in Non-small Cell Lung Cancer Tissues in the Presence of the Cyanobacterial Toxin Microcystin. FRONT MICROBIOL 2018, 9:1757. Patumcharoenpol P et al: PARP1 Is Up-Regulated in Non-small Cell Lung Cancer Tissues in the Presence of the Cyanobacterial Toxin Microcystin. FRONT MICROBIOL 2018, 9:1757. 11. Hosgood HR, Sapkota AR, Rothman N, Rohan T, Hu W, Xu J, Vermeulen R, He X, White JR, Wu G et al: The potential role of lung microbiota in lung cancer attributed to household coal burning exposures. ENVIRON MOL MUTAGEN 2014, 55(8):643-651. 12. Rusch VW, Chansky K, Kindler HL, Nowak AK, Pass HI, Rice DC, Shemanski L, Galateau-Salle F, McCaughan BC, Nakano T et al: The IASLC Mesothelioma Staging Project: Proposals for the M Descriptors and for Revision of the TNM Stage Groupings in the Forthcoming (Eighth) Edition of the TNM Classification for Mesothelioma. J THORAC ONCOL 2016, 11(12):2112-2119. 12. Rusch VW, Chansky K, Kindler HL, Nowak AK, Pass HI, Rice DC, Shemanski L, Galateau-Salle F, 13. Yu G, Gail MH, Consonni D, Carugno M, Humphrys M, Pesatori AC, Caporaso NE, Goedert JJ, Ravel J, Landi MT: Characterizing human lung tissue microbiota and its relationship to epidemiological and clinical features. GENOME BIOL 2016, 17(1):163. 14. Huang D, Su X, Yuan M, Zhang S, He J, Deng Q, Qiu W, Dong H, Cai S: The characterization of lung microbiome in lung cancer patients with different clinicopathology. AM J CANCER RES 2019, 9(9):2047- 2063. 15. Gowing SD, Chow SC, Cools-Lartigue JJ, Chen CB, Najmeh S, Goodwin-Wilson M, Jiang HY, Bourdeau 5. References 1. Siegel RL, Miller KD, Jemal A: Cancer statistics, 2020. CA Cancer J Clin 2020, 70(1):7-30. 2. Herbst RS, Morgensztern D, Boshoff C: The biology and management of non-small cell lung cancer. NATURE 2018, 553(7689):446-454. 3. Dickson RP, Erb-Downward JR, Huffnagle GB: The role of the bacterial microbiome in lung disease. Expert Rev Respir Med 2013, 7(3):245-257. 4. Cameron S, Lewis KE, Huws SA, Hegarty MJ, Lewis PD, Pachebat JA, Mur L: A pilot study using metagenomic sequencing of the sputum microbiome suggests potential bacterial biomarkers for lung cancer. PLOS ONE 2017, 12(5):e177062. 5. Tsay JJ, Wu BG, Badri MH, Clemente JC, Shen N, Meyn P, Li Y, Yie TA, Lhakhang T, Olsen E et al: Airway Microbiota Is Associated with Upregulation of the PI3K Pathway in Lung Cancer. Am J Respir Crit Care Med 2018, 198(9):1188-1198. 6. Lee SH, Sung JY, Yong D, Chun J, Kim SY, Song JH, Chung KS, Kim EY, Jung JY, Kang YA et al: Characterization of microbiome in bronchoalveolar lavage fluid of patients with lung cancer comparing with benign mass like lesions. LUNG CANCER 2016, 102:89-95. Page 15/26 7. Cheng C, Wang Z, Wang J, Ding C, Sun C, Liu P, Xu X, Liu Y, Chen B, Gu B: Characterization of the lung microbiome and exploration of potential bacterial biomarkers for lung cancer. Transl Lung Cancer Res 2020, 9(3):693-704. 2020, 9(3):693-704. Gowing SD, Chow SC, Cools-Lartigue JJ, Chen CB, Najmeh S, Goodwin-W F, Beauchamp A, Angers I et al: Gram-Negative Pneumonia Augments Non-Small Cell Lung Cancer Metastasis through Host Toll-like Receptor 4 Activation. J THORAC ONCOL 2019, 14(12):2097-2108. 16. Hessle CC, Andersson B, Wold AE: Gram-positive and Gram-negative bacteria elicit different patterns of pro-inflammatory cytokines in human monocytes. CYTOKINE 2005, 30(6):311-318. 17. Le Noci V, Guglielmetti S, Arioli S, Camisaschi C, Bianchi F, Sommariva M, Storti C, Triulzi T, Castelli C, Balsari A et al: Modulation of Pulmonary Microbiota by Antibiotic or Probiotic Aerosol Therapy: A Strategy to Promote Immunosurveillance against Lung Metastases. CELL REP 2018, 24(13):3528-3538. 18. Chen J, Pitmon E, Wang K: Microbiome, inflammation and colorectal cancer. SEMIN IMMUNOL 2017, 32:43-53. 18. Chen J, Pitmon E, Wang K: Microbiome, inflammation and colorectal cancer. SEMIN IMMUNOL 2017, 32:43-53. Page 16/26 Page 16/26 19. Luo YH, Wu CH, Wu WS, Huang CY, Su WJ, Tsai CM, Lee YC, Perng RP, Chen YM: Association between tumor epidermal growth factor receptor mutation and pulmonary tuberculosis in patients with adenocarcinoma of the lungs. J THORAC ONCOL 2012, 7(2):299-305. 20. Li M, Deng F, Qian LT, Meng SP, Zhang Y, Shan WL, Zhang XL, Wang BL: Association between human papillomavirus and EGFR mutations in advanced lung adenocarcinoma. ONCOL LETT 2016, 12(3):1953- 1958. 21. Jia Z, Bao K, Wei P, Yu X, Zhang Y, Wang X, Wang X, Yao L, Li L, Wu P et al: EGFR activation-induced decreases in claudin1 promote MUC5AC expression and exacerbate asthma in mice. MUCOSAL IMMUNOL 2021, 14(1):125-134. 22. Hall M, Beiko RG: 16S rRNA Gene Analysis with QIIME2. Methods Mol Biol 2018, 1849:113-129. 23. Edgar RC: UPARSE: highly accurate OTU sequences from microbial amplicon reads. NAT METHODS 2013, 10(10):996-998. 24. DeSantis TZ, Hugenholtz P, Larsen N, Rojas M, Brodie EL, Keller K, Huber T, Dalevi D, Hu P, Andersen GL: Greengenes a chimera-checked 16S rRNA gene database and workbench compatible with ARB Appl 24. DeSantis TZ, Hugenholtz P, Larsen N, Rojas M, Brodie EL, Keller K, Huber T, Dalevi D, Hu P, Andersen GL: Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB. Appl Environ Microbiol 2006 72(7):5069 5072 24. DeSantis TZ, Hugenholtz P, Larsen N, Rojas M, Brodie EL, Keller K, Huber T, Dalevi D, Hu P, Andersen GL: Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB. Appl Environ Microbiol 2006, 72(7):5069-5072. Microorganisms 2020, 8(6). Microorganisms 2020, 8(6). 32. Jin J, Gan Y, Liu H, Wang Z, Yuan J, Deng T, Zhou Y, Zhu Y, Zhu H, Yang S et al: Diminishing microbiome richness and distinction in the lower respiratory tract of lung cancer patients: A multiple comparative study design with independent validation. LUNG CANCER 2019, 136:129-135. 33. Liu HX, Tao LL, Zhang J, Zhu YG, Zheng Y, Liu D, Zhou M, Ke H, Shi MM, Qu JM: Difference of lower airway microbiome in bilateral protected specimen brush between lung cancer patients with unilateral lobar masses and control subjects. INT J CANCER 2018, 142(4):769-778. 34. Cameron S, Lewis KE, Huws SA, Hegarty MJ, Lewis PD, Pachebat JA, Mur L: A pilot study using metagenomic sequencing of the sputum microbiome suggests potential bacterial biomarkers for lung cancer. PLOS ONE 2017, 12(5):e177062. 35. Woodard GA, Jones KD, Jablons DM: Lung Cancer Staging and Prognosis. Cancer Treat Res 2016, 170:47-75. 36. Tessema M, Yingling CM, Liu Y, Tellez CS, Van Neste L, Baylin SS, Belinsky SA: Genome-wide unmasking of epigenetically silenced genes in lung adenocarcinoma from smokers and never smokers. CARCINOGENESIS 2014, 35(6):1248-1257. 37. Inamura K: Clinicopathological Characteristics and Mutations Driving Development of Early Lung Adenocarcinoma: Tumor Initiation and Progression. INT J MOL SCI 2018, 19(4). 38. Marks LB, Saynak M, Christodouleas JP: Stage III vs. stage IV lung cancer: "Crossing a Great Divide". LUNG CANCER 2010, 67(1):1-3. 39. Hosgood HR, Sapkota AR, Rothman N, Rohan T, Hu W, Xu J, Vermeulen R, He X, White JR, Wu G et al: The potential role of lung microbiota in lung cancer attributed to household coal burning exposures. ENVIRON MOL MUTAGEN 2014, 55(8):643-651. 40. Hosgood HR, Mongodin EF, Wan Y, Hua X, Rothman N, Hu W, Vermeulen R, Seow WJ, Rohan T, Xu J et al: The respiratory tract microbiome and its relationship to lung cancer and environmental exposures found in rural china. ENVIRON MOL MUTAGEN 2019, 60(7):617-623. 40. Hosgood HR, Mongodin EF, Wan Y, Hua X, Rothman N, Hu W, Vermeulen R, Seow WJ, Rohan T, Xu J et 40. Hosgood HR, Mongodin EF, Wan Y, Hua X, Rothman N, Hu W, Vermeulen R, Seow WJ, Rohan T, Xu J et al: The respiratory tract microbiome and its relationship to lung cancer and environmental exposures found in rural china. ENVIRON MOL MUTAGEN 2019, 60(7):617-623. 41. 2020, 9(3):693-704. GL: Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB. Appl Environ Microbiol 2006, 72(7):5069-5072. 25. Douglas GM, Maffei VJ, Zaneveld JR, Yurgel SN, Brown JR, Taylor CM, Huttenhower C, Langille M: PICRUSt2 for prediction of metagenome functions. NAT BIOTECHNOL 2020, 38(6):685-688. 26. Caspi R, Altman T, Billington R, Dreher K, Foerster H, Fulcher CA, Holland TA, Keseler IM, Kothari A, Kubo A et al: The MetaCyc database of metabolic pathways and enzymes and the BioCyc collection of Pathway/Genome Databases. NUCLEIC ACIDS RES 2014, 42(Database issue):D459-D471. 27. Parks DH, Tyson GW, Hugenholtz P, Beiko RG: STAMP: statistical analysis of taxonomic and functional profiles. BIOINFORMATICS 2014, 30(21):3123-3124. Page 17/26 p ( ) 28. Friedman J, Alm EJ: Inferring correlation networks from genomic survey data. PLOS COMPUT BIOL 2012, 8(9):e1002687. 29. Chen J, Domingue JC, Sears CL: Microbiota dysbiosis in select human cancers: Evidence of association and causality. SEMIN IMMUNOL 2017, 32:25-34. 30. Druzhinin VG, Matskova LV, Demenkov PS, Baranova ED, Volobaev VP, Minina VI, Apalko SV, Churina MA, Romanyuk SA, Shcherbak SG et al: Taxonomic diversity of sputum microbiome in lung cancer patients and its relationship with chromosomal aberrations in blood lymphocytes. Sci Rep 2020, 0(1):9681. 31. Reinhold L, Mollering A, Wallis S, Palade E, Schafer K, Dromann D, Rupp J, Graspeuntner S, Dalhoff K: Dissimilarity of Airway and Lung Tissue Microbiota in Smokers undergoing Surgery for Lung Cancer. 28. Friedman J, Alm EJ: Inferring correlation networks from genomic survey data. PLOS COMPUT BIOL 2012, 8(9):e1002687. 29. Chen J, Domingue JC, Sears CL: Microbiota dysbiosis in select human cancers: Evidence of association and causality. SEMIN IMMUNOL 2017, 32:25-34. 30. Druzhinin VG, Matskova LV, Demenkov PS, Baranova ED, Volobaev VP, Minina VI, Apalko SV, Churina 0. Druzhinin VG, Matskova LV, Demenkov PS, Baranova ED, Volobaev VP, Mi MA, Romanyuk SA, Shcherbak SG et al: Taxonomic diversity of sputum microbiome in lung cancer patients and its relationship with chromosomal aberrations in blood lymphocytes. Sci Rep 2020, 10(1):9681. 31. Reinhold L, Mollering A, Wallis S, Palade E, Schafer K, Dromann D, Rupp J, Graspeuntner S, Dalhoff K: Dissimilarity of Airway and Lung Tissue Microbiota in Smokers undergoing Surgery for Lung Cancer. Page 17/26 Page 17/26 Microorganisms 2020, 8(6). Liu HX, Tao LL, Zhang J, Zhu YG, Zheng Y, Liu D, Zhou M, Ke H, Shi MM, Qu JM: Difference of lower airway microbiome in bilateral protected specimen brush between lung cancer patients with unilateral lobar masses and control subjects. INT J CANCER 2018, 142(4):769-778. 42. Harris JK, De Groote MA, Sagel SD, Zemanick ET, Kapsner R, Penvari C, Kaess H, Deterding RR, Accurso FJ, Pace NR: Molecular identification of bacteria in bronchoalveolar lavage fluid from children with cystic fibrosis. Proc Natl Acad Sci U S A 2007, 104(51):20529-20533. Page 18/26 43. De Luca M, Amodio D, Chiurchiu S, Castelluzzo MA, Rinelli G, Bernaschi P, Calo CF, D'Argenio P: Granulicatella bacteraemia in children: two cases and review of the literature. BMC PEDIATR 2013, 13:61. 44. Haldar K, George L, Wang Z, Mistry V, Ramsheh MY, Free RC, John C, Reeve NF, Miller BE, Tal-Singer R et al: The sputum microbiome is distinct between COPD and health, independent of smoking history. Respir Res 2020, 21(1):183. 45. Parris BA, O'Farrell HE, Fong KM, Yang IA: Chronic obstructive pulmonary disease (COPD) and lung cancer: common pathways for pathogenesis. J THORAC DIS 2019, 11(Suppl 17):S2155-S2172. 46. Rybojad P, Los R, Sawicki M, Tabarkiewicz J, Malm A: Anaerobic bacteria colonizing the lower airways in lung cancer patients. Folia Histochem Cytobiol 2011, 49(2):263-266. 47. Tsay JJ, Wu BG, Badri MH, Clemente JC, Shen N, Meyn P, Li Y, Yie TA, Lhakhang T, Olsen E et al: Airway Microbiota Is Associated with Upregulation of the PI3K Pathway in Lung Cancer. Am J Respir Crit Care Med 2018, 198(9):1188-1198. 48. Popper HH: Progression and metastasis of lung cancer. Cancer Metastasis Rev 2016, 35(1):75-91. 49. Yaku K, Okabe K, Hikosaka K, Nakagawa T: NAD Metabolism in Cancer Therapeutics. FRONT ONCOL 2018, 8:622. 50. Zhu Y, Liu J, Park J, Rai P, Zhai RG: Subcellular compartmentalization of NAD(+) and its role in cancer: A sereNADe of metabolic melodies. Pharmacol Ther 2019, 200:27-41. 51. Yaku K, Okabe K, Hikosaka K, Nakagawa T: NAD Metabolism in Cancer Therapeutics. FRONT ONCOL 2018, 8:622. 52. Jin C, Lagoudas GK, Zhao C, Bullman S, Bhutkar A, Hu B, Ameh S, Sandel D, Liang XS, Mazzilli S et al: Commensal Microbiota Promote Lung Cancer Development via gammadelta T Cells. CELL 2019, 176(5):998-1013. 53. Microorganisms 2020, 8(6). Sheng Q, Du H, Cheng X, Cheng X, Tang Y, Pan L, Wang Q, Lin J: Characteristics of fecal gut microbiota in patients with colorectal cancer at different stages and different sites. ONCOL LETT 2019, 18(5):4834-4844. 54. Tsoi H, Chu E, Zhang X, Sheng J, Nakatsu G, Ng SC, Chan A, Chan F, Sung J, Yu J: Peptostreptococcus anaerobius Induces Intracellular Cholesterol Biosynthesis in Colon Cells to Induce Proliferation and Causes Dysplasia in Mice. GASTROENTEROLOGY 2017, 152(6):1419-1433. 54. Tsoi H, Chu E, Zhang X, Sheng J, Nakatsu G, Ng SC, Chan A, Chan F, Sung J, Yu J: Peptostreptococcus anaerobius Induces Intracellular Cholesterol Biosynthesis in Colon Cells to Induce Proliferation and Causes Dysplasia in Mice. GASTROENTEROLOGY 2017, 152(6):1419-1433. 55. Quail DF, Joyce JA: Microenvironmental regulation of tumor progression and metastasis. NAT MED 2013, 19(11):1423-1437. 55. Quail DF, Joyce JA: Microenvironmental regulation of tumor progression and metastasis. NAT MED 2013, 19(11):1423-1437. Page 19/26 56. Romano AH, Conway T: Evolution of carbohydrate metabolic pathways. RES MICROBIOL 1996, 147(6- 7):448-455. 57. Evans MC, Buchanan BB, Arnon DI: A new ferredoxin-dependent carbon reduction cycle in a photosynthetic bacterium. Proc Natl Acad Sci U S A 1966, 55(4):928-934. 58. Strohecker AM, White E: Autophagy promotes BrafV600E-driven lung tumorigenesis by preserving mitochondrial metabolism. AUTOPHAGY 2014, 10(2):384-385. 59. Hoque MO, Kim MS, Ostrow KL, Liu J, Wisman GB, Park HL, Poeta ML, Jeronimo C, Henrique R, Lendvai A et al: Genome-wide promoter analysis uncovers portions of the cancer methylome. CANCER RES 2008, 68(8):2661-2670. 60. Anderson NM, Mucka P, Kern JG, Feng H: The emerging role and targetability of the TCA cycle in cancer metabolism. PROTEIN CELL 2018, 9(2):216-237. 61. Cai Z, Li CF, Han F, Liu C, Zhang A, Hsu CC, Peng D, Zhang X, Jin G, Rezaeian AH et al: Phosphorylation of PDHA by AMPK Drives TCA Cycle to Promote Cancer Metastasis. MOL CELL 2020, 80(2):263-278. 62. Kovaleva OV, Romashin D, Zborovskaya IB, Davydov MM, Shogenov MS, Gratchev A: Human Lung Microbiome on the Way to Cancer. J IMMUNOL RES 2019, 2019:1394191. 63. Mouraux S, Bernasconi E, Pattaroni C, Koutsokera A, Aubert JD, Claustre J, Pison C, Royer PJ, Magnan A, Kessler R et al: Airway microbiota signals anabolic and catabolic remodeling in the transplanted lung. J Allergy Clin Immunol 2018, 141(2):718-729. 64. Conlon GA, Murray GI: Recent advances in understanding the roles of matrix metalloproteinases in tumour invasion and metastasis. J PATHOL 2019, 247(5):629-640. 65. Microorganisms 2020, 8(6). Yan X, Yang M, Liu J, Gao R, Hu J, Li J, Zhang L, Shi Y, Guo H, Cheng J et al: Discovery and validation of potential bacterial biomarkers for lung cancer. AM J CANCER RES 2015, 5(10):3111-3122. 66. Nie C, He T, Zhang W, Zhang G, Ma X: Branched Chain Amino Acids: Beyond Nutrition Metabolism. INT J MOL SCI 2018, 19(4). 67. Burgel PR, Nadel JA: Epidermal growth factor receptor-mediated innate immune responses and their roles in airway diseases. EUR RESPIR J 2008, 32(4):1068-1081. 68. Lowenmark T, Lofgren-Burstrom A, Zingmark C, Eklof V, Dahlberg M, Wai SN, Larsson P, Ljuslinder I, Edin S, Palmqvist R: Parvimonas micra as a putative non-invasive faecal biomarker for colorectal cancer. Sci Rep 2020, 10(1):15250. Page 20/26 69. Xu J, Yang M, Wang D, Zhang S, Yan S, Zhu Y, Chen W: Alteration of the abundance of Parvimonas micra in the gut along the adenoma-carcinoma sequence. ONCOL LETT 2020, 20(4):106. 70. Matsui A, Jin JO, Johnston CD, Yamazaki H, Houri-Haddad Y, Rittling SR: Pathogenic bacterial species associated with endodontic infection evade innate immune control by disabling neutrophils. INFECT IMMUN 2014, 82(10):4068-4079. 69. Xu J, Yang M, Wang D, Zhang S, Yan S, Zhu Y, Chen W: Alteration of the abundance of Parvimonas micra in the gut along the adenoma-carcinoma sequence. ONCOL LETT 2020, 20(4):106. 69. Xu J, Yang M, Wang D, Zhang S, Yan S, Zhu Y, Chen W: Alteration of the abundance of Parvimonas micra in the gut along the adenoma-carcinoma sequence. ONCOL LETT 2020, 20(4):106. 70. Matsui A, Jin JO, Johnston CD, Yamazaki H, Houri-Haddad Y, Rittling SR: Pathogenic bacterial species associated with endodontic infection evade innate immune control by disabling neutrophils. INFECT IMMUN 2014, 82(10):4068-4079. 70. Matsui A, Jin JO, Johnston CD, Yamazaki H, Houri-Haddad Y, Rittling SR: Pathogenic bacterial species associated with endodontic infection evade innate immune control by disabling neutrophils. INFECT IMMUN 2014, 82(10):4068-4079. Figures Fi 1 Figure 1 Study flow diagram of patients’ recruitment and exclusion. Study flow diagram of patients’ recruitment and exclusion. Page 21/26 Page 21/26 Page 21/26 Figure 2 Taxonomic composition of sputum microbiota of the patients in ES and AS group. (A) Sputum phyla of the patients in ES and AS group; (B) Sputum Genera of the patients in ES and AS group. Figure 2 Taxonomic composition of sputum microbiota of the patients in ES and AS group. (A) Sputum phyla of the patients in ES and AS group; (B) Sputum Genera of the patients in ES and AS group. Taxonomic composition of sputum microbiota of the patients in ES and AS group. (A) Sputum phyla of the patients in ES and AS group; (B) Sputum Genera of the patients in ES and AS group. Taxonomic composition of sputum microbiota of the patients in ES and AS group. (A) Sputum phyla of the patients in ES and AS group; (B) Sputum Genera of the patients in ES and AS group. Page 22/26 Figure 3 Difference of sputum microbiota between NSCLC patients in ES and AS group. (A) Chao 1 index; (B) Simpson index; (C) Shannon index among NSCLC patients in ES and AS group; (D) PCOA plot based on Bray-Curtis distance of sputum genus among NSCLC patients in ES and AS group. P＜0.05, P was calculated using Mann-Whitney U test. Figure 3 Difference of sputum microbiota between NSCLC patients in ES and AS group. (A) Chao 1 index; (B) Simpson index; (C) Shannon index among NSCLC patients in ES and AS group; (D) PCOA plot based on Bray-Curtis distance of sputum genus among NSCLC patients in ES and AS group. P＜0.05, P was calculated using Mann-Whitney U test. Page 23/26 Figure 4 Differentially abundant taxonomy and predicted metabolic function of sputum microbiota between NSCLC patients in ES and AS group. (A) Differentially abundant taxonomy between patients in ES and AS group identified by LEFse; (B) Differentially abundant of phyla Actinobacteria, Firmicutes between ES and AS group; (C) Differentially abundant of genera Actinobacillus, Actinomyces and Granulicatella between SCC_M1 and AD_M1; (D) Differential predicted metabolic function based on MetaCyc database between patients in ES and AS group. P＜0.05, P was calculated using Mann-Whitney U test. Figure 4 Differentially abundant taxonomy and predicted metabolic function of sputum microbiota between NSCLC patients in ES and AS group. (A) Differentially abundant taxonomy between patients in ES and AS group identified by LEFse; (B) Differentially abundant of phyla Actinobacteria, Firmicutes between ES and AS group; (C) Differentially abundant of genera Actinobacillus, Actinomyces and Granulicatella between SCC_M1 and AD_M1; (D) Differential predicted metabolic function based on MetaCyc database between patients in ES and AS group. P＜0.05, P was calculated using Mann-Whitney U test. Page 24/26 Page 24/26 Figure 5 Genera Co-occurrence network based on SparCC of patients in (A) ES group; (B) AS group. Only P value ≤ 0.05 and SparCC correlation scores ≥ 0.5 or ≤ -0.5 were included for networks inference. The genus nodes were colored based on phylum level. The size of each node was determined by the relative abundance of each genus. Figure 5 Figure 5 Figure 6 Differentially abundant taxonomy and predicted metabolic function of sputum microbiota between lung adenocarcinoma patients with and without EGFR mutation. (A) Differentially abundant taxonomy between EGFR- lung adenocarcinoma and EGFR+ lung adenocarcinoma identified by LEFse; (B) Differentially abundant taxonomy between EGFR- non-smoker lung adenocarcinoma and EGFR+ non- smoker lung adenocarcinoma identified by LEFse; (C) Differentially abundant of phylum Bacteroidetes between EGFR- non-smoker lung adenocarcinoma and EGFR+ non-smoker lung adenocarcinoma; (D) Differentially abundant of genera Actinobacillus and Parvimonas between EGFR- non-smoker lung adenocarcinoma and EGFR+ non-smoker lung adenocarcinoma; (E) Differential predicted metabolic function based on MetaCyc database between EGFR- non-smoker lung adenocarcinoma and EGFR+ non- smoker lung adenocarcinoma. P＜0.05, P was calculated using Mann-Whitney test. Differentially abundant taxonomy and predicted metabolic function of sputum microbiota between lung adenocarcinoma patients with and without EGFR mutation. (A) Differentially abundant taxonomy between EGFR- lung adenocarcinoma and EGFR+ lung adenocarcinoma identified by LEFse; (B) Differentially abundant taxonomy and predicted metabolic function of sputum microbiota between lung adenocarcinoma patients with and without EGFR mutation. (A) Differentially abundant taxonomy between EGFR- lung adenocarcinoma and EGFR+ lung adenocarcinoma identified by LEFse; (B) Differentially abundant taxonomy between EGFR- non-smoker lung adenocarcinoma and EGFR+ non- smoker lung adenocarcinoma identified by LEFse; (C) Differentially abundant of phylum Bacteroidetes between EGFR- non-smoker lung adenocarcinoma and EGFR+ non-smoker lung adenocarcinoma; (D) Differentially abundant of genera Actinobacillus and Parvimonas between EGFR- non-smoker lung adenocarcinoma and EGFR+ non-smoker lung adenocarcinoma; (E) Differential predicted metabolic function based on MetaCyc database between EGFR- non-smoker lung adenocarcinoma and EGFR+ non- smoker lung adenocarcinoma. P＜0.05, P was calculated using Mann-Whitney test. Figure 5 Figure 5 Genera Co-occurrence network based on SparCC of patients in (A) ES group; (B) AS group. Only P value ≤ 0.05 and SparCC correlation scores ≥ 0.5 or ≤ -0.5 were included for networks inference. The genus nodes were colored based on phylum level. The size of each node was determined by the relative abundance of each genus. Genera Co-occurrence network based on SparCC of patients in (A) ES group; (B) AS group. Only P value ≤ 0.05 and SparCC correlation scores ≥ 0.5 or ≤ -0.5 were included for networks inference. The genus nodes were colored based on phylum level. The size of each node was determined by the relative abundance of each genus. Page 25/26 Page 25/26 Figure 6 Differentially abundant taxonomy and predicted metabolic function of sputum microbiota between lung adenocarcinoma patients with and without EGFR mutation. (A) Differentially abundant taxonomy between EGFR- lung adenocarcinoma and EGFR+ lung adenocarcinoma identified by LEFse; (B) Differentially abundant taxonomy between EGFR- non-smoker lung adenocarcinoma and EGFR+ non- smoker lung adenocarcinoma identified by LEFse; (C) Differentially abundant of phylum Bacteroidetes between EGFR- non-smoker lung adenocarcinoma and EGFR+ non-smoker lung adenocarcinoma; (D) Differentially abundant of genera Actinobacillus and Parvimonas between EGFR- non-smoker lung adenocarcinoma and EGFR+ non-smoker lung adenocarcinoma; (E) Differential predicted metabolic function based on MetaCyc database between EGFR- non-smoker lung adenocarcinoma and EGFR+ non- smoker lung adenocarcinoma. *P＜0.05, P was calculated using Mann-Whitney test. Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. SupplementaryFigure.pdf supplementarytable.pdf Page 26/26
https://openalex.org/W2037228554	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0088025&type=printable	English	null	Trap Configuration and Spacing Influences Parameter Estimates in Spatial Capture-Recapture Models	PloS one	2,014	cc-by	8,976	Abstract Funding: The New York State Department of Environmental Conservation provided the f design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: cs752@cornell.edu minimizing the ratio of edge to area of the sampling grid [9], or by adding a buffer strip around the sampling array to account for movements of ‘edge’ individuals [10–13]. Conversely, SCR models directly estimate both population size and density; SCR models allow for individual-specific detection probabilities by accounting for the spatial organization of traps and by estimating the activity centers of individuals. SCR models are thus liberated from the assumption of geographic closure. Catherine C. Sun1, Angela K. Fuller2, J. Andrew Royle3 1 New York Cooperative Fish and Wildlife Research Unit, Department of Natural Resources, Cornell University, Ithaca New York, United States of America, 2 U.S. Geological Survey, New York Cooperative Fish and Wildlife Research Unit, Department of Natural Resources, Cornell University, Ithaca, New York, United States of America, 3 U.S. Geological Survey, Patuxent Wildlife Research Center, Laurel, Maryland, United States of America Abstract An increasing number of studies employ spatial capture-recapture models to estimate population size, but there has been limited research on how different spatial sampling designs and trap configurations influence parameter estimators. Spatial capture-recapture models provide an advantage over non-spatial models by explicitly accounting for heterogeneous detection probabilities among individuals that arise due to the spatial organization of individuals relative to sampling devices. We simulated black bear (Ursus americanus) populations and spatial capture-recapture data to evaluate the influence of trap configuration and trap spacing on estimates of population size and a spatial scale parameter, sigma, that relates to home range size. We varied detection probability and home range size, and considered three trap configurations common to large-mammal mark-recapture studies: regular spacing, clustered, and a temporal sequence of different cluster configurations (i.e., trap relocation). We explored trap spacing and number of traps per cluster by varying the number of traps. The clustered arrangement performed well when detection rates were low, and provides for easier field implementation than the sequential trap arrangement. However, performance differences between trap configurations diminished as home range size increased. Our simulations suggest it is important to consider trap spacing relative to home range sizes, with traps ideally spaced no more than twice the spatial scale parameter. While spatial capture-recapture models can accommodate different sampling designs and still estimate parameters with accuracy and precision, our simulations demonstrate that aspects of sampling design, namely trap configuration and spacing, must consider study area size, ranges of individual movement, and home range sizes in the study population. Citation: Sun CC, Fuller AK, Royle JA (2014) Trap Configuration and Spacing Influences Parameter Estimates in Spatial Capture-Recapture Models. PLoS ONE 9(2): e88025. doi:10.1371/journal.pone.0088025 Editor: David L. Roberts, University of Kent, United Kingdom Received September 10, 2013; Accepted January 3, 2014; Published February 5, 2014 Received September 10, 2013; Accepted January 3, 2014; Published February 5, 2014 This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Funding: The New York State Department of Environmental Conservation provided the funding that supported the work. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Trap Configuration and Spacing Influences Parameter Estimates in Spatial Capture-Recapture Models Catherine C. Sun1, Angela K. Fuller2, J. Andrew Royle3 February 2014 \| Volume 9 \| Issue 2 \| e88025 Introduction Our second objective was to identify consequences of trap spacing for a fixed study area by decreasing the number of traps while maintaining the same spatial extent of the sampling array. We conducted our simulation study using sampling design consider- ations for American black bear (Ursus americanus), but the results are generalizable to any wide-ranging animal population to which SCR models might be applicable. [15]. As a result, recommendations have been made to set at least four traps in each potential non-overlapping home range [17]. With a constant number of traps due to logistical or monetary considerations, a sampling trade-off occurs between spatial extent and trap spacing. Few simulation-based studies have been conducted on the influence of sampling design on SCR parameter estimates [15,18,19]. SCR approaches can theoretically accommodate different spatial arrangements of traps because trap locations are a formal part of the model [5] describing the probability of encounter of individuals. However, spatial organization of the trapping array is still an important consideration. Large numbers of encountered individuals (sample size n) and recaptures are necessary to estimate population parameters with accuracy and precision, and trap arrangements need to be at spatial densities and scales that permit detection of individual movement [15]. Much of the early research on sampling design was conducted in small mammal community assemblages [20–22], and design recommendations based on small mammal populations may be hard to meet and are sometimes inappropriate for large-mammal systems [12,19]. Recent work has focused specifically on sampling designs for large-mammal populations with large home ranges and ranges of movement [15,16,23,24], but the body of published research remains scant. Notably, Sollmann et al. [15] demon- strated with simulations and a study of a Michigan black bear population that previously recommended spatial extents of at least 46 the home range size of individuals may be unnecessary. The authors showed that spatial extents smaller than an average male home range and only 1.56 larger than a female’s yielded parameter estimates similar to when the full spatial extent was used. The authors cautioned that the range of movement over the sampling array is important for SCR models, and that the SCR model performed well as long as the spatial scale parameter sigma (s) was at least half the average trap spacing. Introduction Estimating population parameters such as abundance and density is crucial for understanding, managing, and conserving animal populations. Capture-mark-recapture (CMR) methods are a well-established approach in which repeated sampling with replacement of a population provides information about detection probabilities of individuals. CMR models have become increas- ingly realistic by addressing assumptions about population closure and capture probability [1–4], including the recent developments of spatial capture-recapture (SCR) models. SCR models incorpo- rate the geographic locations where individuals are detected, thereby explicitly accounting for unequal detection probabilities among individuals due to their unique spatial locations relative to sampling devices (traps, snares, etc.) [5,6]. Unequal exposure of individuals to the sampling array occurs when, for example, some individuals have home ranges at the edge of the sampling array while others are located more centrally and therefore are always exposed to the sampling array [2,4,7,8]. As a result, non-spatial capture-recapture methods estimate population size, but require various ad-hoc approaches to convert estimates of population size to estimates of density. Non-spatial approaches attempt to homogenize the unequal trap exposure with methods such as p g g p Two primary considerations of mark-recapture sampling design are the spatial extent of the trap array and the spacing between traps. An advantage of a large spatial extent is that it helps increase the expected number of unique individuals detected. For non- spatial approaches, Bondrup-Nielson [9] suggested that the spatial extent of a study area be at least four times the home range size of an individual. Large spatial extents also aim to capture the full range of movement of individuals and homogenize unequal detection rates among individuals [9,14,15]. Simultaneously, trap spacing influences rates of detection and recaptures: trap configurations with ‘‘holes’’, or traps that are too widely spaced relative to ranges of individual movement, can lead to individuals not being detected [12,16] as well as fewer recaptures of individuals at different traps (i.e., spatial recaptures), which are important for estimating home range sizes and movement ranges February 2014 \| Volume 9 \| Issue 2 \| e88025 1 February 2014 \| Volume 9 \| Issue 2 \| e88025 PLOS ONE \| www.plosone.org Sampling Designs in Capture-Recapture Models increase detection probability, more thoroughly sample large study areas, and avoid trap habituation and behavioral response [26]. Introduction This sigma parameter describes the spatial scale over which an individual is detected, and can be converted to an estimate of the 95% home range radius [14]. The authors concluded that SCR models are able to accurately and precisely estimate population parameters for a range of sampling array extents, but that more research is necessary to explore the limits of SCR abilities with respect to trap configuration and extreme sampling designs. Methods We based simulation conditions on characteristics of a black bear population study conducted in southwestern New York, USA. The simulated study area was a 2,624 km2 square centered on a 4,100 km2 landscape. To determine trap placement in the clustered and sequential trap configurations, we overlaid a grid of 64, non-overlapping, potential home ranges of 41 km2 each, based on the average female home range size estimated in northwestern Pennsylvania [27] (Figure 1). SCR Model Formulation and Implementation We used a binomial model for detection to create encounter histories for individuals. For sampling over K sampling periods, the number of encounters for an individual, i, in each of j = 1,…, J traps, yij, has a binomial distribution with a parameter for encounter probability, pij. In other words: yijBinom(K,pij) yijBinom(K,pij) Nine detection scenarios for N = 500 were created by evaluating three values of the spatial scale parameter, (s = 10, 5, and 1 km), for each of three baseline detection rates, (p0 = 0.20, 0.10, 0.05). As distance from an individual’s activity center increases, detection decreases according to a half-normal function based on the two parameters. Dashed vertical lines indicate 95% home range radii (s* ﬃﬃﬃﬃﬃﬃﬃﬃﬃ 5:99 p ). doi:10.1371/journal.pone.0088025.g002 pij~p0 e {D2 ij 2s2 , pij~p0 e ij 2s2 , where s is a spatial scale parameter determining the rate of decrease in encounter probability as a function of distance to trap Dij. Most models for encounter probability have one or more parameters that are related to home range size and movement rates of individuals about their home range. For example, the half- normal model above can be interpreted as implying a bivariate normal model for movement, where s* ﬃﬃﬃﬃﬃﬃﬃﬃﬃ 5:99 p is the 95% home range radius [14]. Detection of an individual at multiple traps provides information on s, so we use the term ‘‘spatial captures’’ to refer to the number of unique traps at which an individual was detected or captured. where s is a spatial scale parameter determining the rate of decrease in encounter probability as a function of distance to trap Dij. Most models for encounter probability have one or more parameters that are related to home range size and movement rates of individuals about their home range. For example, the half- normal model above can be interpreted as implying a bivariate normal model for movement, where s* ﬃﬃﬃﬃﬃﬃﬃﬃﬃ 5:99 p is the 95% home range radius [14]. Detection of an individual at multiple traps provides information on s, so we use the term ‘‘spatial captures’’ to refer to the number of unique traps at which an individual was detected or captured. We simulated SCR data for a population size of N = 500 over K = 10 sampling occasions. We distributed individuals over the 4,100 km2 landscape according to a random, uniform distribution, allowing for overlapping home ranges. yijBinom(K,pij) This translates to a black bear density of 12.2 bears/100 km2, which is in the middle range of bear densities across the United States (Snowy Range of southeast Wyoming = 2.54 bears/100 km2) [29], central Appala- chian Mountains in Kentucky = 8 bears/100 km2 [30], northern New York = 20 bears/100 km2 [8], north-central Pennsylva- nia = 23 bears/100 km2 [27], and Great Smoky Mountains, Tennessee $29 bears/100 km2 [31]. We created nine detection scenarios by varying the spatial scale parameter, s, and the baseline detection probability, p0 (Figure 2). We used three values of s (1, 5, and 10 km) to model a range of representative home range sizes, spanning estimates of female and male home ranges typical of bears in the northeastern United States [8,27,32,33]. We used three values of p0 (0.05, 0.10, and 0.20) to explore a realistic range for mark-recapture studies. The upper limit, p0 = 0.20 (i.e., 20%), is the minimum suggested detection probability in non- spatial mark-recapture studies [34,35], but lower probabilities have been found to be sufficient for populations larger than N. 200 [31], so we also included lower detection probabilities. Figure 2. Nine detection scenarios by varying s and p0. Nine detection scenarios for N = 500 were created by evaluating three values of the spatial scale parameter, (s = 10, 5, and 1 km), for each of three baseline detection rates, (p0 = 0.20, 0.10, 0.05). As distance from an individual’s activity center increases, detection decreases according to a half-normal function based on the two parameters. Dashed vertical lines indicate 95% home range radii (s ﬃﬃﬃﬃﬃﬃﬃﬃﬃ 5:99 p ). doi:10.1371/journal.pone.0088025.g002 change trap spacing. We calculated trap spacing for the regular trap configuration as the distance between a trap and the next closest trap, or for the clustered and sequential trap configurations, the distance between the centroids of a cluster and the next cluster. We did not consider the clustered trap configuration when J = 32 since clusters would have consisted of only 1 trap and therefore be equivalent to the regular configuration. For each of the nine detection scenarios (p0 x s), we generated 500 simulated encounter histories for each combination of trap configuration (n = 3) and trap spacing (n = 4). To estimate abundance, N, and the spatial scale parameter, s, we used a maximum likelihood approach [36,37]. yijBinom(K,pij) For the individual and trap-specific encounter probability, pij, we used the half-normal model [28], which depends on the baseline detection probability p0~ ea0 1zea0 and a function of the Euclidean distance, Dij, between individual, i, and trap, j, such that g p g g Understanding the implications of different sampling designs is crucial, especially given the amount of effort required in large mammal mark-recapture studies and the increasing application of SCR methods. Moreover, in sampling over large landscapes, it is oftentimes not possible to achieve regular coverage of the landscape with traps that are close enough together to yield sufficient data for effective parameter estimation. Therefore, strategies for distributing traps over the landscape in an efficient manner must be developed and evaluated. To improve under- standing of sampling design with respect to SCR methods, we conducted simulations to investigate the effects of different trap configurations and spacings that would be feasible in large- mammal studies. First, we evaluated potential differences among three common trap configurations: regular spacing, clustered, and a temporal sequence of different clustered configurations (i.e., trap relocation) [25]. The regular trap configuration, in which traps are set systematically across the spatial extent, served as a baseline for comparison. The clustered configuration maintained spatially representative sampling over the entire spatial extent while providing more information on the spatial scale of detection and individual movement [17,19]. The third configuration evaluated was a clustered configuration with trap relocation midway through sampling; trap relocation is a common sampling approach to Figure 1. Schematic representation of study area. A representa- tion of the 2,264 km2 study area, divided into a grid of 64 cells of 41 km2 each, and set in the center of a 4,100 km2 landscape, which is outlined in gray. doi:10.1371/journal.pone.0088025.g001 Figure 1. Schematic representation of study area. A representa- tion of the 2,264 km2 study area, divided into a grid of 64 cells of 41 km2 each, and set in the center of a 4,100 km2 landscape, which is outlined in gray. doi:10.1371/journal.pone.0088025.g001 February 2014 \| Volume 9 \| Issue 2 \| e88025 PLOS ONE \| www.plosone.org 2 Sampling Designs in Capture-Recapture Models Figure 2. Nine detection scenarios by varying s and p0. yijBinom(K,pij) We conducted the simulations using Program R [38] and custom-written scripts (Table S1 in File S1) with package ‘snowfall’ and ‘rlecuyer’ [39,40]. Estimates of N and s were compared to the simulated truth. We used estimated means, standard deviations, ranges, root mean squared error (RMSE), and mean normalized bias (MNB) to evaluate the effects of trap configuration and spacing. Objectives To evaluate the effect of sampling design on SCR parameter estimation, we applied three trap configurations: 1) regularly distributed across the study area, 2) grouped into clusters of 4 in every other non-overlapping female home range and, 3) traps relocated from one clustered configuration halfway through the sampling period to a second clustered configuration (Figure 3). To evaluate trap spacing over the study area, we increased trap spacing from 4.7 km to 9.6 km by decreasing the number of traps from J = 128 traps to 96, 64, and 32 traps over the same spatial extent in the regular trap configuration (Table 1, Figure 4). This also resulted in different effective trap spacings, trap spacings relative to each value of s, ranging from 0.47s, when s = 10 km, to 9.60s when s = 1 km (Table 2). Decreasing the number of traps resulted in a trap density of 0.049/km2 with 128 traps, 0.037/km2 with 96 traps, 0.024/km2 with 64 traps, and 0.012/ km2 with 32 traps. The upper limit of 128 traps represents what could be realistically employed over such a large study area given a sampling frequency of once per week assuming two field teams, while also maintaining a minimum of 4 trap sites per estimated female home range. However, even this upper bound of trap density falls severely short of suggestions for black bear studies of 0.17–0.50/km2 [29]. We decreased the number of traps for the clustered and sequential trap configurations, although this did not Trap Configurations The clustered trap configuration generally resulted in the most accurate estimators of abundance, ^N. The clustered trap configuration yielded the lowest RMSEs in 8 of 9 combinations of p0 (3 cases) and s (3 cases), i.e., with the exception of s = 5 km and p0 = 0.20 in which the sequential trap configuration resulted in the most accurate ^N (Table 3). The three trap configurations resulted in similarly unbiased estimators of ^N when effective trap spacing was ,4.71s, i.e., when s .1 km. But when effective trap spacing $4.71s (s = 1 km), the clustered and sequential trap configurations resulted in the lowest MNBs in the remaining 1 and 2 cases, respectively. As the number of detected individuals and captures per individual increased because of closer effective trap spacings, February 2014 \| Volume 9 \| Issue 2 \| e88025 February 2014 \| Volume 9 \| Issue 2 \| e88025 PLOS ONE \| www.plosone.org 3 Sampling Designs in Capture-Recapture Models Figure 3. Three trap configurations: regular, clustered, and sequential. Three trap configurations were evaluated, shown with J = 128 traps: (a) regular array, (b), clustered, and (c) a temporal sequence in which clustered traps of one arrangement (e.g. triangle) are moved halfway through the sampling period to new grids (e.g. squares). Gray gridlines in (b) and (c) overlay the non-overlapping grid sizes of an estimated female home range. The black outline around the traps depicts the 2,624 km2 study area; the large gray square shows the extent of the 4,100 km2 landscape. doi:10.1371/journal.pone.0088025.g003 Figure 3. Three trap configurations: regular, clustered, and sequential. Three trap configurations were evaluated, shown with J = 128 traps: (a) regular array, (b), clustered, and (c) a temporal sequence in which clustered traps of one arrangement (e.g. triangle) are moved halfway through the sampling period to new grids (e.g. squares). Gray gridlines in (b) and (c) overlay the non-overlapping grid sizes of an estimated female home range. The black outline around the traps depicts the 2,624 km2 study area; the large gray square shows the extent of the 4,100 km2 landscape. doi:10.1371/journal.pone.0088025.g003 Estimators of ^s performed similarly well across the three trap configurations (Table 4). However, precision of the estimators for any given trap configuration increased when the effective trap spacing increased with larger values of s. Trap Spacings and Traps per Cluster As trap spacing increased from 4.71 km to 9.60 km by reducing the number of traps (J = 128 to 32 traps), effective trap spacing relative to s increased (Table 2). Individuals were detected fewer times and with fewer spatial and non-spatial captures (Table S3 in File S1). As a result, estimators of ^N and ^s decreased in accuracy and precision as trap spacing increased and number of traps per cluster decreased (Table 5,6 and Tables S5–9 in File S1). For example, consider increased effective trap spacing from 4.71s to 9.60s (when s = 1 km) at p0 = 0.20: population size was increasingly overestimated as the number of detected individuals decreased 73% and the spatial captures decreased from 1.1 to 1.0 (Table S3 in File S1). ^N increased from 509 to 637, RMSE increased from 15 to 84% (regular trap configuration, Table 5), and RMSE of ^s increased from 7% to 24% (Table 6). In some cases, including all trap spacings and trap configurations when p0 = 0.05, the number of detected individuals was as low as 10 individuals (2% of total population N = 500) and some simulated datasets yielded only one capture for all detected individuals (Table S3 in File S1). These sparse data sets caused the MLE to occur on the boundary of the parameter space, and simulated data sets for which this was the case were removed from the analysis. For example, 231 such cases were discarded under the sequential trap arrangement when p0 = 0.05 (Table S4 in File S1). Table 1. Trap spacing (km) for each combination of trap configuration (regular, clustered, and sequential) and number of traps (J = 128, 96, 64, and 32). Number of traps, J 128 96 64 32 Regular 4.71 5.24 6.4 9.6 Clustered 9.06 9.06 9.06 N/A Sequential 9.06 9.06 9.06 9.06 Trap spacing (km) in the regular trap configuration was varied by decreasing the number of traps in the study area. Trap spacing did not vary when traps were in the clustered or sequential configurations because reductions only decreased the number of traps per cluster. doi:10.1371/journal.pone.0088025.t001 Trap spacing (km) in the regular trap configuration was varied by decreasing the number of traps in the study area. Trap spacing did not vary when traps were in the clustered or sequential configurations because reductions only decreased the number of traps per cluster. Trap Configurations However, when effective trap spacing was ,4.71s (i.e., when s $5 km), the mean ^N for all trap configurations at all detection probabilities were within one individual of the true N = 500, and SD and RMSE no more than 3% (Table 3). Trap Configurations Comparing estimators across regular, clustered, and sequential trap configurations when effective trap spacings were $4.71s and #0.91s (i.e., s = 1 km versus s = 10 km), SD decreased from a maximum of 28% to 1.1% while MNB also decreased from a maximum of 18% to 0.2% (Table 4). estimators of ^N improved (Table S2 in File S1). For example, consider the clustered trap configuration when p0 = 0.05: when effective trap spacing decreased from 9.06s to 0.91s due to an increase in the fixed, biologically-determined s from 1 km to 10 km, the number of detected individuals increased from 38 individuals with fewer than 2 spatial recaptures to 493 individuals (.98% of total population N = 500) with 7.8 spatial recaptures (Table S2 in File S1). When effective trap spacing was $4.71s (i.e., when s $1 km), the sparse datasets, especially low rates of spatial recaptures, resulted in unstable maximum likelihood estimators (MLEs) and a strongly right-skewed sampling distribu- tion of ^N (Table 3). Therefore, population size was consistently overestimated at all trap arrangements and detection rates. Standard deviations (SD) and root mean square errors (RMSE) were both at least 12% (Table 3). However, when effective trap spacing was ,4.71s (i.e., when s $5 km), the mean ^N for all trap configurations at all detection probabilities were within one individual of the true N = 500, and SD and RMSE no more than 3% (Table 3). estimators of ^N improved (Table S2 in File S1). For example, consider the clustered trap configuration when p0 = 0.05: when effective trap spacing decreased from 9.06s to 0.91s due to an increase in the fixed, biologically-determined s from 1 km to 10 km, the number of detected individuals increased from 38 individuals with fewer than 2 spatial recaptures to 493 individuals (.98% of total population N = 500) with 7.8 spatial recaptures (Table S2 in File S1). When effective trap spacing was $4.71s (i.e., when s $1 km), the sparse datasets, especially low rates of spatial recaptures, resulted in unstable maximum likelihood estimators (MLEs) and a strongly right-skewed sampling distribu- tion of ^N (Table 3). Therefore, population size was consistently overestimated at all trap arrangements and detection rates. Standard deviations (SD) and root mean square errors (RMSE) were both at least 12% (Table 3). Trap Spacings and Traps per Cluster doi:10.1371/journal.pone.0088025.t001 However, when effective trap spacing was #1.92s (i.e., when s = 5 and 10 km), the properties of the estimators ^N and ^s became similar across trap spacing and number of traps per cluster (Tables S5–9 in File S1). Estimators also increased in precision February 2014 \| Volume 9 \| Issue 2 \| e88025 4 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org Sampling Designs in Capture-Recapture Models Figure 4. Trap configuration and number of traps generated eleven designs. Eleven trap designs were evaluated by varying the regular, clustered, and sequential trap arrangements for J = 128, 96, and 64 traps. Only the regular and sequential arrangements were evaluated for J = 32 traps since the clustered arrangement with one trap per cluster was equivalent to the regular arrangement. Trap spacing did not change when traps were in the clustered and sequential arrangements. doi:10.1371/journal.pone.0088025.g004 Figure 4. Trap configuration and number of traps generated eleven designs. Eleven trap designs were evaluated by varying the regular, clustered, and sequential trap arrangements for J = 128, 96, and 64 traps. Only the regular and sequential arrangements were evaluated for J = 32 traps since the clustered arrangement with one trap per cluster was equivalent to the regular arrangement. Trap spacing did not change when traps were in the clustered and sequential arrangements. doi:10.1371/journal.pone.0088025.g004 SCR models depends on obtaining a sufficiently large sample size of unique individuals and spatial recaptures. Compared to the regular trap configuration, the clustered arrangement frequently yielded more total captures and spatial recaptures, and the sequential arrangement yielded more unique individuals. Al- though the sequential configuration detected more unique individuals by moving traps to new locations, the total number of recaptures was fewer compared to the clustered configuration because each trap was only available to detect individuals for half the sampling occasions. Also, as detection rates decreased, more traps per cluster were necessary to detect individuals and recaptures. The necessity of sufficient sample sizes of individuals and spatial recaptures was also highlighted by the instability of the MLE under low detection (particularly p0 = 0.05) at small values of the spatial scale parameter (s = 1 km), which resulted in param- eter estimates on the boundary of the parameter space [41,42]. and accuracy. Trap Spacings and Traps per Cluster When s = 10 km (p0 = 0.20), even as effective trap spacing increased from 0.47s to 0.91s, the number of detected individuals did not drop below 490 (98% of the true population N = 500,) until effective trap spacing decreased to 0.96s when p0 = 0.10 and 0.52s when p0 = 0.05 (Table S3 in File S1). As a result, estimators of ^N at all trap spacings were within 1 individual of the true population ( ^N = 499.4 to 499.9) and RMSE was less than 1% (Table S6 in File S1). Estimators of ^s had RMSEs of less than 0.02% (Table S9 in File S1). Discussion We demonstrated that the clustered trap configuration generally yielded the most accurate estimators of abundance, ^N. The regular trap configuration never out-performed the clustered or sequential trap arrangements in precision of abundance estimates, and in fact often resulted in fewer detected individuals, fewer total captures, and fewer spatial recaptures. Consequently, clustered and sequential trap arrangements even with fewer traps yielded estimates of abundance that were as precise or more as the regular trap configuration. Performance differences between the three trap configurations were most marked when trap spacing was large relative to home range size (Table 7). However, performance differences between trap configurations diminished as home range size increased. Non-regular, and particularly the clustered, trap configurations helped compensate for sparse trap arrays. This suggests that precise estimates over a large study area are possible, even when limited by a sparse and widely-set trap array, by arranging traps in clusters. Clusters of traps increase the expected number of spatial recaptures of individuals while the large spatial extent increases the expected number of unique individuals detected. Our simulations also suggest that it is important to prescribe trap spacing relative to home range sizes of individuals. As the spatial scale parameter, s, increased, differences between the performance of SCR estimators with different trap configurations diminished. For example, at the smallest value of s (1 km), trap SCR models are flexible to estimate population parameters with accuracy and precision for sampling designs commonly employed in studies of wide-ranging species. However, effective estimation in Table 2. Effective trap spacings for each s, scaled by dividing trap spacings (4.71, 5.24, 6.40, and 9.60 km) by s (1, 5, 10 km). s = 1 km s = 5 km s = 10 km Trap spacing (km) Trap spacing (km) Trap spacing (km) 4.71 5.24 6.40 9.60 4.71 5.24 6.40 9.60 4.71 5.24 6.40 9.60 Regular 4.71 5.24 6.40 9.60 0.94 1.05 1.28 1.92 0.47 0.52 0.64 0.96 Clustered 9.06 9.06 9.06 N/A 1.81 1.81 1.81 N/A 0.91 0.91 0.91 N/A Sequential 9.06 9.06 9.06 9.06 1.81 1.81 1.81 1.81 0.91 0.91 0.91 0.91 For example, a trap spacing of 4.71 km equals 4.71s when s = 1 km but only 0.47s when s = 10 km. Trap spacing of 9.60 km was not evaluated for the clustered trap configuration because it employs J = 32 traps and therefore is equivalent to the regular trap spacing. doi:10.1371/journal.pone.0088025.t002 Table 2. p , p p g q y Trap spacing of 9.60 km was not evaluated for the clustered trap configuration because it employs J = 32 traps and therefore is equiv doi:10.1371/journal.pone.0088025.t002 Discussion Effective trap spacings for each s, scaled by dividing trap spacings (4.71, 5.24, 6.40, and 9.60 km) by s (1, 5, 10 km). cings for each s, scaled by dividing trap spacings (4.71, 5.24, 6.40, and 9.60 km) by s (1, 5, 10 km). Table 2. Effective trap spacings for each s, scaled by dividing trap spacings (4.71, 5.24, 6.40, and 9.60 For example, a trap spacing of 4.71 km equals 4.71s when s = 1 km but only 0.47s when s = 10 km. Trap spacing of 9.60 km was not evaluated for the clustered trap configuration because it employs J = 32 traps and therefore is equivalent to the regular trap spacing. doi:10.1371/journal.pone.0088025.t002 February 2014 \| Volume 9 \| Issue 2 \| e88025 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 5 Sampling Designs in Capture-Recapture Models Table 3. Summary estimates of ^N when true population size N = 500 and J = 128 traps, under each of the three trap arrangements: regular, clustered, and sequential, where mean, standard deviation (SD), range, root mean squared error (RMSE), and mean normalized bias (MNB) are given for each scenario (p x s x configuration). Table 3. Summary estimates of ^N when true population size N = 500 and J = 128 traps, under each of the three trap arrangements: regular, clustered, and sequential, where mean, standard deviation (SD), range, root mean squared error (RMSE), and mean normalized bias (MNB) are given for each scenario (p x s x configuration). Discussion s = 1 km s = 5 km s = 10 km p0 = 0.20 Mean SD Min Max RMSE MNB Mean SD Min Max RMSE MNB Mean SD Min Max RMSE MNB Regular 509.0 75.1 323.7 843.7 75.57 0.00 499.9 6.4 482.0 518.3 6.38 0.00 499.9 0.3 498.0 500.0 0.31 0.00 Clustered 503.4 60.8 344.8 683.6 60.80 0.01 499.3 5.9 480.0 516.1 5.93 0.00 500.0 0.2 499.0 500.0 0.20 0.00 Sequential 508.4 65.7 328.0 769.6 66.20 0.00 499.8 5.6 479.2 514.1 5.58 0.00 499.9 0.2 499.0 500.0 0.24 0.00 p0 = 0.10 Regular 546.7 177.9 240.4 1696.0 183.78 20.01 499.7 9.4 471.5 525.5 9.41 0.00 499.6 1.1 495.8 501.1 1.14 0.00 Clustered 513.1 112.9 293.3 1168.3 113.58 0.02 499.7 8.5 471.3 523.3 8.53 0.00 499.5 1.0 495.3 500.7 1.13 0.00 Sequential 541.6 143.2 236.0 1164.9 148.94 20.02 499.7 8.8 472.3 524.9 8.81 0.00 499.4 1.0 496.2 500.6 1.14 0.00 p0 = 0.05 Regular 2.8E11 6.3E12 1.7E2 1.41E14 6.29E12 20.23 499.2 14.1 454.3 538.3 14.08 0.00 499.6 3.0 491.3 507.2 3.04 0.00 Clustered 3.3E4 7.2E5 156.1 1.60E7 7.14E5 0.08 499.9 13.8 447.1 541.0 13.81 0.00 499.7 2.9 490.0 505.9 2.94 0.00 Sequential 7.0E4 1.5E6 126.4 3.43E7 1.54E6 0.02 500.6 14.0 449.0 537.9 13.97 0.00 499.3 3.0 482.3 506.3 3.12 0.00 Estimates are averages of 500 simulations. doi:10.1371/journal.pone.0088025.t003 spacing in the regular configuration was 4.71 km, or .4s; but as s increased to 10 km, this same trap spacing equated to just 0.47s (Table 2). As a result, differences between trap arrangements were negligible at s = 10 km, even at the lowest detection rate (p0 = 0.05). When traps are widely spaced relative to s, fewer captures and spatial recaptures are collected. Accordingly, parameter estimates improved markedly when s increased from 1 km to 5 km and trap spacing decreased to less than 2s (Table 7). Home range diameters of black bears in the geographic region on which these simulations were based range from 5.1–25.1 km [27], coinciding with the value of s when accuracy and precision of our parameter estimates improved. This pattern in trap spacing is similar to the conclusions of Sollmann et al. [15] that recom- mended trap distances be less than 2s. Discussion Since s is a spatial scale parameter related to an individual’s home range radius, this essentially suggests that at least ,2 traps should be placed within an individual’s home range, a minimum that is smaller than the traditional recommendation for trap density of 4 traps per home range [17]. In evaluating trap spacings and configurations over a range of values for s, our simulations also demonstrate the importance of establishing a sampling design based on the smallest (usually the female) estimate of s. Doing so helps ensure detection of all individuals, even those with larger ranges of movement. In field studies, implementing the sequential trap configuration requires that twice the number of traps be set because traps are moved half-way through the sampling period, which increases the amount of associated work that setting traps entails. Our simulations suggested that the different trap configurations performed similarly when trap spacing was less than 2s, even when the sequential trap configuration detected a greater number Table 4. Summary estimates of ^s when the true population size N = 500 and J = 128 traps, under each of the three trap arrangements: regular, clustered, and sequential, where mean, standard deviation (SD), range, root mean squared error (RMSE), and mean normalized bias (MNB) are given for each scenario (p x s x configuration). Table 4. Summary estimates of ^s when the true population size N = 500 and J = 128 traps, under each of the three trap arrangements: regular, clustered, and sequential, where mean, standard deviation (SD), range, root mean squared error (RMSE), and mean normalized bias (MNB) are given for each scenario (p x s x configuration). g doi:10.1371/journal.pone.0088025.t004 Estimates are averages of 500 simulation doi:10.1371/journal.pone.0088025.t004 Estimates are averages of 500 simulations. Discussion s = 1 km s = 5 km s = 10 km p0 = 0.20 Mean SD Min Max RMSE MNB Mean SD Min Max RMSE MNB Mean SD Min Max RMSE MNB Regular 1.00 0.07 0.80 1.22 0.07 20.01 5.00 0.04 4.89 5.16 0.04 0.01 9.99 0.06 9.84 10.17 0.06 0.02 Clustered 1.00 0.07 0.81 1.27 0.07 20.01 5.00 0.04 4.85 5.11 0.04 0.01 10.00 0.05 9.81 10.16 0.05 0.01 Sequential 1.00 0.08 0.76 1.25 0.08 20.01 5.00 0.04 4.87 5.13 0.04 0.00 10.00 0.05 9.84 10.17 0.05 0.00 p0 = 0.10 Regular 1.00 0.13 0.57 1.41 0.13 20.03 5.00 0.06 4.86 5.31 0.06 0.00 9.99 0.08 9.74 10.24 0.08 0.02 Clustered 1.01 0.14 0.63 1.52 0.15 20.05 5.00 0.07 4.77 5.22 0.07 0.01 10.00 0.08 9.74 10.21 0.08 0.00 Sequential 0.99 0.14 0.67 1.45 0.14 20.03 5.00 0.07 4.83 5.20 0.07 0.00 10.00 0.07 9.80 10.26 0.07 20.01 p0 = 0.05 Regular 0.97 0.24 0.38 1.89 0.24 20.05 5.00 0.10 4.73 5.34 0.10 0.00 10.00 0.11 9.68 10.40 0.11 0.01 Clustered 1.06 0.28 0.59 1.98 0.28 20.18 4.99 0.10 4.66 5.47 0.10 0.02 10.00 0.11 9.58 10.33 0.11 0.01 Sequential 1.01 0.24 0.55 2.44 0.24 20.10 5.00 0.10 4.68 5.28 0.10 0.00 10.00 0.11 9.67 10.44 0.11 20.01 Estimates are averages of 500 simulations. doi:10.1371/journal.pone.0088025.t004 February 2014 \| Volume 9 \| Issue 2 \| e88025 PLOS ONE \| www.plosone.org Sampling Designs in Capture-Recapture Models Table 5. For s = 1 km, summary estimates of ^N in the regular trap configuration when trap spacing increased from 4.71 to 9.60 km (J = 128 to 32 traps) and N = 500. p0 = 0.20 Mean SD Min Max RMSE MNB 4.71 509.0 75.1 323.7 843.7 75.57 0.00 5.24 654.3 318.6 222.9 2059.0 353.73 20.07 6.4 591.2 270.7 219.0 2111.5 285.41 0.00 9.6 636.8 398.3 119.7 2744.5 419.88 0.08 p0 = 0.10 4.71 546.8 177.9 240.4 1696.0 183.78 20.01 5.24 654.3 318.6 222.9 2059.0 353.73 20.07 6.4 705.0 534.0 131.8 5726.3 571.49 0.04 9.6 1.3E11 2.8E12 103.0 6.3E13 2.82E12 0.08 p0 = 0.05 4.71 2.8E11 6.3E12 168.5 1.4E14 6.29E12 20.02 5.24 1.3E7 2.9E8 120.2 6.5E9 2.90E8 20.12 6.4 1.9E13 4.1E14 91.1 8.8E15 3.94E14 0.21 9.6 3.1E11 5.0E12 45.0 9.7E13 4.39E12 0.35 ,500 iterations were used for the italicized estimates, due to instability of MLE with sparse datasets. Conclusion Our simulations demonstrate that 1) gains in precision and accuracy of parameter estimates are related to both trap configuration and trap spacing, which is relative to the spatial scale parameter and home range size, and that 2) increased numbers of traps per cluster (at least up to four traps per cluster) improve precision. Our simulations reinforce the understanding that although different SCR sampling designs can provide accurate and precise estimators of population parameters, effective estimation requires datasets that include captures and spatial recaptures of a sufficient proportion of the population. These of unique individuals. Thus, clustered trap configurations and even regular trap configurations may be sufficient, and more intense sampling designs unnecessary, when traps arrays with spacing of less than 2s can be achieved. However, if trap spacing is .2s, such as when forced due to large spatial extents, non-regular trap arrangements should be favored in order to maintain the precision of estimators. In this situation, our results suggest that the clustered configuration would likely be the most efficient to employ. Table 7. RMSE values of estimators of ^N, as effective trap spacing (i.e., trap spacing/s) increased under the regular trap configuration and across all baseline detection probabilities (p0 = 0.20, 0.10, 0.05). Trap spacing (s) p0 = 0.20 p0 = 0.10 p0 = 0.05 0.47 0.3 1.1 3 0.52 0.6 1.9 4.3 0.64 1.1 2.8 6.4 0.94 6.4 9.4 14.1 0.96 3.1 6.8 12.7 1.05 7.3 10.7 17.5 1.28 8.8 13.3 23.8 1.92 14.6 23.5 49.9 4.71 75.57 183.78 6.29E+12 5.24 353.73 353.73 2.90E+08 6.4 285.41 571.49 3.94E+14 9.6 419.88 2.82E+12 4.39E+12 doi:10.1371/journal.pone.0088025.t007 Table 7. RMSE values of estimators of ^N, as effective trap spacing (i.e., trap spacing/s) increased under the regular trap configuration and across all baseline detection probabilities (p0 = 0.20, 0.10, 0.05). Table 7. RMSE values of estimators of ^N, as effective trap spacing (i.e., trap spacing/s) increased under the regular trap configuration and across all baseline detection probabilities (p0 = 0.20, 0.10, 0.05). We identified several instances of tradeoff between precision (SD) and bias (MNB) in parameter estimation. However, values of RMSE, which incorporates bias and variance, were similar to the corresponding values of SD, and estimates of bias were low. Thus, any observed tradeoffs between precision and bias were not consequential. Naturally, our simulations were not exhaustive of the parameter space. Discussion At p0 = 0.20 and trap spacing of 9.60 km, 498 iterations were used to calculate the mean estimate (2 iterations discarded). At p0 = 0.10 and trap spacing of 9.60 km, 492 iterations were used to calculate the mean estimate (8 iterations discarded). At p0 = 0.05, and trap spacings increasing from 4.71 km to 9.60 km, 497, 489, 457, and 381 iterations were used to calculate mean estimates (3, 11, 43, and 119 iterations discarded, respectively). doi:10.1371/journal.pone.0088025.t005 Table 6. For s = 1 km, summary estimates of ^s in the regular trap configuration when trap spacing increased from 4.71 to 9.60 km (J = 128 to 32 traps) and N = 500. p0 = 0.20 Mean SD Min Max RMSE MNB 4.71 1.00 0.07 0.80 1.22 0.07 20.01 5.24 0.98 0.10 0.57 1.30 0.10 0.00 6.40 0.98 0.17 0.49 1.39 0.17 20.03 9.60 0.99 0.24 0.46 1.78 0.24 20.08 p0 = 0.10 4.71 1.00 0.13 0.57 1.41 0.13 20.03 5.24 0.96 0.19 0.50 1.76 0.20 20.01 6.40 0.98 0.24 0.50 1.51 0.24 20.07 9.60 1.06 0.90 0.36 16.53 0.89 20.33 p0 = 0.05 4.71 0.97 0.24 0.38 1.89 0.24 20.05 5.24 0.93 0.36 0.36 5.56 0.36 20.04 6.40 1.02 0.32 0.39 2.90 0.31 20.13 9.60 1.45 2.44 0.26 19.77 2.16 21.26 ,500 iterations were used for the italicized estimates, due to instability of MLE with sparse datasets. See Table 5 footnote for number of iterations used for the italicized estimates. doi:10.1371/journal.pone.0088025.t006 Table 6. For s = 1 km, summary estimates of ^s in the regular trap configuration when trap spacing increased from 4.71 to 9.60 km (J = 128 to 32 traps) and N = 500. Table 6. For s = 1 km, summary estimates of ^s in the regular trap configuration when trap spacing increased from 4.71 to 9.60 km (J = 128 to 32 traps) and N = 500. See Table 5 footnote for number of iterations used for the italicized estimates. doi:10.1371/journal.pone.0088025.t006 February 2014 \| Volume 9 \| Issue 2 \| e88025 Supporting Information We thank the New York State Department of Environmental Conservation for support, C. Sutherland for assistance with implementing code, and M. Hare for helpful comments of earlier drafts of this manuscript. Any use of trade, firm, or product names is for descriptive purposes only and does not imply endorsement by the U.S. Government. File S1 Combined supporting information file contain- ing Tables S1–S9. Table S1 Custom-written R scripts for data simulation and parameter estimation. Table S2 in File S1. Summary of mean capture data across trap configuration, s, and p0 for N = 500 and J = 128 traps. Table S3 in File S1. Summary of capture data across s and p0 when trap spacing increased (4.71, 5.24, 6.40, and 9.60 km). Table S4 in File S1. For s = 1 km, summary of estimated in the clustered and sequential trap configurations when trap spacing increased from 4.71 to References 17. Otis DL, Burnham KP, White GC, Anderson DR (1978) Statistical Inference from Capture Data on Closed Animal Populations. Wildl Monogr: 3–135. doi:10.2307/3830650. 1. Pollock KH (1982) A Capture-Recapture Design Robust to Unequal Probability of Capture. J Wildl Manag 46: 752–757. doi:10.2307/3808568. 2. Kendall WL, Nichols JD, Hines JE (1997) Estimating temporary emigration using capture-recapture data with Pollock’s robust design. Ecology 78: 563–578. 18. Marques TA, Thomas L, Royle JA (2011) A hierarchical model for spatial capture–recapture data: comment. Ecology 92: 526–528. doi:10.1890/10- 1440.1. 3. Pledger S, Efford M (1998) Correction of Bias Due to Heterogeneous Capture Probability in Capture- Recapture Studies of Open Populations. Biometrics 54: 888–898. doi:10.2307/2533843. 19. Efford MG, Fewster RM (2013) Estimating population size by spatially explicit capture–recapture. Oikos 122: 918–928. doi:10.1111/j.1600- 0706.2012.20440.x. 4. Kendall WL (1999) Robustness of closed capture-recapture methods to violations of the closure assumption. Ecology 80: 2517–2525. 20. Read V, Malafant KW, Myers K (1988) A comparison of Grid and Index-line Trapping Methods for Small Mammal Surveys. Aust Wild Res 15: 673–687. 5. Royle JA, Young KV (2008) A Hierarchical Model for Spatial Capture- Recapture Data. Ecology 89: 2281–2289. doi:10.2307/27650753. 21. Parmenter RR, Yates TL, Anderson DR, Burnham KP, Dunnum JL, et al. (2003) Small-Mammal Density Estimation: A Field Comparison of Grid-Based vs. Web-Based Density Estimators. Ecol Monogr 73: 1–26. doi:10.2307/ 3100072. 6. Borchers D (2012) A non-technical overview of spatially explicit capture– recapture models. J Ornithol 152: 435–444. doi:10.1007/s10336-010-0583-z. 7. Mowat G, Strobeck C (2000) Estimating Population Size of Grizzly Bears Using Hair Capture, DNA Profiling, and Mark-Recapture Analysis. J Wildl Manag 64: 183–193. doi:10.2307/3802989. 22. Pearson DE, Ruggiero LF (2003) Transect versus Grid Trapping Arrangements for Sampling Small-Mammal Communities. Wildl Soc Bull 31: 454–459. doi:10.2307/3784324. 8. Gardner B, Royle JA, Wegan MT, Rainbolt RE, Curtis PD (2010) Estimating Black Bear Density Using DNA Data From Hair Snares. J Wildl Manag 74: 318–325. 23. Long RA, Donovan TM, Mackay P, Zielinski WJ, Buzas JS (2007) Comparing Scat Detection Dogs, Cameras, and Hair Snares for Surveying Carnivores. J Wildl Manag 71: 2018–2025. 9. Bondrup-Nielsen S (1983) Density estimation as a function of live-trapping grid and home range size. Can J Zool 61: 2361–2365. doi:10.1139/z83-313. 24. Tobler MW, Carrillo-Percastegui SE, Leite Pitman R, Mares R, Powell G (2008) An evaluation of camera traps for inventorying large- and medium-sized terrestrial rainforest mammals. Anim Conserv 11: 169–178. doi:10.1111/j.1469- 1795.2008.00169.x. 10. Conclusion For s = 10 km, summary of estimates of in the regular, clustered and sequential trap configurations when trap spacing increased from 4.71 to 9.60 km (J = 128 to 32 traps) and N = 500. (DOC) In developing sampling designs for spatial capture-recapture studies, our results suggest the following strategy for devising a sampling design: 1) determine the spatial extent of the study population, 2) determine the maximum trap spacing based on the minimum value of the spatial scale parameter, 2smin, 3) if enough traps are available to space traps less than 2s in a regular arrangement, do so, assuming it is practical to implement, 4) otherwise, consider traps in a clustered configuration with wider spacing between clusters, and more traps per cluster as expected detection rate decreases. With the increasing application of SCR methods and the effort required of mark-recapture efforts, it is important to understand the consequences of different sampling designs for large-mammal populations. Simulations provide an accessible opportunity to explore different sampling arrangements, allowing researchers to identify feasible designs that most efficiently utilize effort and resources. Author Contributions Conceived and designed the experiments: CS AKF JAR. Performed the experiments: CS. Analyzed the data: CS AKF JAR. Contributed reagents/ materials/analysis tools: CS AKF JAR. Wrote the paper: CS AKF JAR. Conceived and designed the experiments: CS AKF JAR. Performed the experiments: CS. Analyzed the data: CS AKF JAR. Contributed reagents/ materials/analysis tools: CS AKF JAR. Wrote the paper: CS AKF JAR. Conclusion Particularly, we held the spatial extent constant to mimic conditions for a predetermined study area, and defined the upper bound of number of traps (J = 128) based on limits we expect researchers would likely face. As a result, we did not explore trap clusters with .4 traps, which would have allowed larger spatial extents by setting clusters farther apart than 9.06 km. Larger spatial extents allow for more individuals to be detected, and would be applicable for populations with lower densities and/or larger ranges of movement. At the same time, spatial extents smaller than examined here would provide further insight into the minimum requirements for robust parameter estimation. Such simulations that continue to investigate the balance between spatial extent and trap spacing would be valuable for future research. February 2014 \| Volume 9 \| Issue 2 \| e88025 PLOS ONE \| www.plosone.org 7 Sampling Designs in Capture-Recapture Models results highlight the importance of understanding the spatial characteristics of a study population, such as home range sizes of different portions of the population, spatial scales of movement, as well as information about the ability to detect individuals. 9.60 km (J = 128 to 32 traps) and N = 500. Table S5 in File S1. For s = 5 km, summary estimates of in the regular, clustered, and sequential trap configurations when trap spacing increased from 4.71 to 9.60 km (J = 128 to 32 traps) and N = 500. Table S6 in File S1. For s = 10 km, summary estimates of in the regular, clustered, and sequential trap configurations when trap spacing increased from 4.71 to 9.60 km (J = 128 to 32 traps) and N = 500. Table S7 in File S1. For s = 1 km, summary of estimates of in the regular, clustered and sequential trap configurations when trap spacing increased from 4.71 to 9.60 km (J = 128 to 32 traps) and N = 500. Table S8 in File S1. For s = 5 km, summary of estimates of in the regular, clustered and sequential trap configurations when trap spacing increased from 4.71 to 9.60 km (J = 128 to 32 traps) and N = 500. Table S9 in File S1. References Karanth KU, Nichols JD (1998) Estimation of tiger densities in India using photographic captures and recaptures. Ecology 79: 2852–2862. photographic captures and recaptures. Ecology 79: 2852–2862. 11. Efford M (2004) Density Estimation in Live-Trapping Studies. Oikos 106: 598– 610. doi:10.2307/3548382. 25. Karanth KU, Nichols JD, Wildlife Conservation Society (New York NY., Geological Survey (U.S.), World Wildlife Fund (U.S.), editors (2002) Monitoring tigers and their prey: a manual for researchers, managers, and conservationists in tropical Asia. Bangalore: Centre for Wildlife Studies. 193 p. 12. Dillon A, Kelly MJ (2007) Ocelot Leopardus pardalis in Belize: the impact of trap spacing and distance moved on density estimates. Oryx 41: 469–477. doi:10.1017/S0030605307000518. 13. Foster RJ, Harmsen BJ (2012) A critique of density estimation from camera-trap data. J Wildl Manag 76: 224–236. doi:10.1002/jwmg.275. tropical Asia. Bangalore: Centre for Wildlife Studies. 193 p. 26. Boulanger J, Proctor M, Himmer S, Stenhouse G, Paetkau D, et al. (2006) An Empirical Test of DNA Mark-Recapture Sampling Strategies for Grizzly Bears. Ursus 17: 149–158. doi:10.2307/3873092. g j g 14. Royle JA, Chandler RB, Sollman R, Gardner B (2013) Spatial Capture Recapture. Elsevier Press. 577 p. 27. Alt GL, Matula GJ, Alt FW, Lindzey JS (1980) Dynamics of Home Range and Movements of Adult Black Bears in Northeastern Pennsylvania. Bears Their Biol Manag 4: 131–136. doi:10.2307/3872856. 15. Sollmann R, Gardner B, Belant JL (2012) How Does Spatial Study Design Influence Density Estimates from Spatial Capture-Recapture Models? PLoS ONE 7(4): e34575. doi:10.1371/journal.pone.0034575. 28. Buckland ST, Anderson DR, Burnham KP, Laake JL (1993) Distance sampling: estimating abundance of biological populations: xii +446 pp. 16. Wegge P, Pokheral CP, Jnawali SR (2004) Effects of trapping effort and trap shyness on estimates of tiger abundance from camera trap studies. Anim Conserv 7: 251–256. doi:10.1017/S1367943004001441. February 2014 \| Volume 9 \| Issue 2 \| e88025 PLOS ONE \| www.plosone.org 8 Sampling Designs in Capture-Recapture Models Sampling Designs in Capture-Recapture Models 29. Grogan RG, Lindzey FG (1999). Estimating population size of a low-density black bear population using capture-resight. Ursus 11: 117–122. doi: 10.2307/ 3872992. and density estimates of grizzly bears. J Wildl Manag 68: 457–469. doi:10.2193/ 0022-541X(2004)068[0457:SDABID]2.0.CO;2. and density estimates of grizzly bears. J Wildl Manag 68: 457–469. doi:10.2193/ 0022-541X(2004)068[0457:SDABID]2.0.CO;2. 36. Borchers DL, Efford MG (2008) Spatially Explicit Maximum Likelihood Methods for Capture–Recapture Studies. Biometrics 64: 377–385. doi:10.1111/ j.1541-0420.2007.00927.x. 30. Frary VJ, Duchamp J, Maehr DS, Larkin JL (2011) Density and distribution of a colonizing front of the American black bear Ursus americanus. Wildlife Biology 17: 404–416. j 37. Hastie T, Tibshirani R, Friedman JH (2001) The elements of statistical learning: data mining, inference, and prediction?: with 200 full-color illustrations. New York: Springer. 31. Settlage KE, Manen FTV, Clark JD, King TL (2008) Challenges of DNA-Based Mark-Recapture Studies of American Black Bears. J Wildl Manag 72: 1035– 1042. doi:10.2307/25097648. p g 38. R Core Team (2012) R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. ISBN 3–900051–07– 0, URL http://www.R-project.org/. 32. Fecske DM, Barry RE, Precht FL, Quigley HB, Bittner SL, et al. (2002) Habitat Use by Female Black Bears in Western Maryland. Southeast Nat 1: 77–92. doi:10.2307/3878247. 39. Knaus J (2013) snowfall: Easier cluster computing (based on snow). R package version 1.84–4. http://CRAN.R-project.org/package = snowfall. 33. Carter NH, Brown DG, Etter DR, Visser LG (2010) American black bear habitat selection in northern Lower Peninsula, Michigan, USA, using discrete- choice modeling. Ursus 21: 57–71. doi:10.2192/09GR011.1. 40. Sevcikova H, Rossini T (2012) rlecuyer: R interface to RNG with multiple streams. R package version 0.3–3. Available: http://CRAN.R-project.org/ pacage = rlecuyer. 34. White GC, Anderson DR, Burnham KP, Otis DL (1982) Capture-recapture and removal methods for sampling closed populations/. Los Alamos, N.M.: Available: http://hdl.handle.net/2027/mdp.39015013542280. 41. Harris RB, Burnham KP (2002) On estimating wildlife densities from line transect data. Current Zoology 48: 812–818. 35. Boulanger J, Mclellan BN, Woods JG, Proctor MF, Strobeck C, et al. (2004) Sampling design and bias in DNA-based capture-mark-recapture population 42. Manning CD, Raghavan P, Schu¨tze H (2008) Introduction to Information Retrieval. Cambridge University Press. 569 p. February 2014 \| Volume 9 \| Issue 2 \| e88025 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 9 9
https://openalex.org/W2954340443	https://advancesindifferenceequations.springeropen.com/track/pdf/10.1186/s13662-019-2196-z.pdf	Latin	null	On a nonlocal 1-D initial value problem for a singular fractional-order parabolic equation with Bessel operator	Advances in difference equations	2,019	cc-by	7,351	© The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, pro- vided 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. MSC: 35D35; 35L20 MSC: 35D35; 35L20 Keywords: Solvability of the problem; Weighted integral conditions; Fractional diﬀerential equation; Initial boundary value problem Keywords: Solvability of the problem; Weighted integral conditions; Fractional diﬀerential equation; Initial boundary value problem R ES EA RCH Open Access On a nonlocal 1-D initial value problem for a singular fractional-order parabolic equation with Bessel operator Said Mesloub1* and Imed Bachar1 *Correspondence: mesloub@ksu.edu.sa 1Mathematics Department, College of Science, King Saud University, Riyadh, Saudi Arabia ( 2019) 2019:254 ( 2019) 2019:254 Mesloub and Bachar Advances in Diﬀerence Equations https://doi.org/10.1186/s13662-019-2196-z Abstract In this paper, we obtain some results of the existence and uniqueness of a generalized solution for a singular fractional initial boundary value problem in the Caputo sense subject to Neumann and weighted integral conditions. We show that a priori estimate or energy inequality methods can be successfully applied to obtaining a priori estimates for the solution of initial fractional boundary problems as in the classical case. The obtained results will contribute in the development of the functional analysis method and enrich the existing nonextensive literature on the nonlocal fractional mixed problems in the Caputo sense. 1 Introduction Page 2 of 14 Mesloub and Bachar Advances in Diﬀerence Equations ( 2019) 2019:254 Mesloub and Bachar Advances in Diﬀerence Equations ( 2019) 2019:254 Mesloub and Bachar Advances in Diﬀerence Equations ( 2019) 2019:254 In the literature, there are very many papers using the functional analysis method for the proof of the well-posedness of mixed problems (having local or nonlocal boundary conditions) in the classical sense, such as [14, 15, 21], but in the fractional case, there are only few papers using the previous method to prove their well-posedness. Therefore our work can be considered as a contribution to the development of the functional analysis method used to prove the well-posedness of mixed problems with fractional order. We should like also to mention that the positivity of the fractional derivative operator helps us to obtain a priori bounds for solutions of certain classes of fractional initial and boundary value problems. This paper is organized as follows: In Sect. 2, we set and pose the problem and give diﬀerent types of fractional derivatives used in the paper. In Sect. 3, we introduce some function spaces, give some useful tools, and write the given problem in operator form. In Sect. 4, by choosing an appropriate functional diﬀerential operator multiplier we es- tablish an a priori estimate, from which we deduce the uniqueness of the solution and its dependence on the given data of the posed problem. In Sect. 5, we prove the main result concerning the solvability of the given problem. With some modiﬁcations in the classical method (energy inequality method) used for classical equations, we could show that the range of the operator generated by the studied problem is dense in the weighted Hilbert space H = L2 x(0,1) × L2 x(Q), where Q = (0,1) × (0,T), T < ∞. 1 Introduction The one-dimensional fractional-order diﬀusion heat equation has become a real model for all linear and nonlinear fractional and nonfractional partial diﬀerential equations of parabolic type [5, 8, 10, 18, 19]. Although mathematical models in two and three dimen- sions are of great signiﬁcance for applications, the majority of recent papers are devoted to the fractional-order diﬀusion equations in the one-dimensional case. Papers dealing with the multidimensional fractional diﬀusion equations are still not numerous. For frac- tional parabolic equations, we interpret physically the fractional derivative appearing in the equation as the degree of memory in the diﬀusing material [9]. Many authors have studied analytically and numerically various models of time-fractional diﬀerential equa- tions; see, for example, [2–4, 7, 13, 20]. Many physical phenomena can be modeled in terms of local and nonlocal initial bound- ary value problems where the classical time and space derivatives are present, but, unfor- tunately, many others cannot be modeled by such problems. Diﬀerent methods have been used to solve fractional diﬀusion equations. We can cite, for example, the works [11, 17]. In this paper, we apply the traditional functional analysis method, the so-called energy inequality method based mainly on some a priori bounds and on the density of the range of the operator generated by the considered problem for a fractional singular equation with Bessel operator and Caputo fractional derivative of order 0 < α < 1 (see [6]). Many physical phenomena can be modeled in terms of local and nonlocal initial bound- ary value problems where the classical time and space derivatives are present, but, unfor- tunately, many others cannot be modeled by such problems. Diﬀerent methods have been used to solve fractional diﬀusion equations. We can cite, for example, the works [11, 17]. In this paper, we apply the traditional functional analysis method, the so-called energy i lit th d b d i l i i b d d th d it f th g q p [ ] In this paper, we apply the traditional functional analysis method, the so-called energy inequality method based mainly on some a priori bounds and on the density of the range of the operator generated by the considered problem for a fractional singular equation with Bessel operator and Caputo fractional derivative of order 0 < α < 1 (see [6]). 2 Problem setting We consider the governing equation of Caputo’s time fractional order subject to initial and boundary conditions of integral and Neumann types in the domain Q = (0,1)×(0,T), T < ∞. By ∂α t θ we denote the Caputo time fractional derivative. This initial boundary value problem is nonlocal in time derivative and in one of the boundary conditions: ⎧ ⎪⎪⎨ ⎪⎪⎩ Lθ = ∂α t θ – 1 x ∂ ∂x(xθx) + Y(x,t)θ = f (x,t), 0 < x < 1,0 < t < T, l1θ = θ(x,0) = ω(x), x ∈(0,1), 1 0 xθ(x,t)dx = 0, θx(1,t) = 0,t ∈(0,T), (2.1) (2.1) The functions Y(x,t) and f (x,t) are given functions, which will be speciﬁed later. The functions Y(x,t) and f (x,t) are given functions, which will be speciﬁed later. The time fractional derivative of order 0 < α < 1 is taken in the Caputo sense. It is deﬁned for a diﬀerentiable function by ∂α t θ(x,t) = 1 Γ (1 – α) d dt t 0 θ(x,τ) – θ(x,0) (t – τ)α dτ, t > 0 (LCFD), ∂α Tθ(x,t) = 1 Γ (1 – α) d dt T t θ(x,τ) – θ(x,0) (t – τ)α dτ, t > 0 (RFFD), (2.2) (2.2) or, equivalently, or, equivalently, or, equivalently, ∂α t θ(x,t) = 1 Γ (1 – α) t 0 θ′(x,τ) (t – τ)α dτ, t > 0 (LCFD), ∂α Tθ(x,t) = 1 Γ (1 – α) T t θ′(x,τ) (t – τ)α dτ, t < T (RCFD), (2.3) (2.3) where Γ is the gamma function. where Γ is the gamma function. where Γ is the gamma function. where Γ is the gamma function. ns ( 2019) 2019:254 Page 3 of 14 Mesloub and Bachar Advances in Diﬀerence Equations ( 2019) 2019:2 Page 3 of 14 ( 2019) 2019:254 Mesloub and Bachar Advances in Diﬀerence Equations We also need to use the Riemann–Liouville integral of order 0 < α < 1 deﬁned by D–α t V(t) = 1 Γ (α) t 0 V(τ) (t – τ)1–α dτ. (2.4) (2.4) For diﬀerent properties of the Caputo fractional derivative, we refer the reader to [12, 16] and the references therein. 3 Preliminaries We need the following function spaces and tools. We denote by C2,1(Q) the set of functions that, together with their partial derivatives of orders 2 and 1 in x and t, are continuous on Q, by Cm(0,T) the space of m-fold diﬀerentiable functions, and by C∞ 0 (0,T) the space of inﬁnitely diﬀerentiable functions having their support in (0,T). We use the usual L2(0,T) space of measurable square-integrable functions on (0,T). Lemma 3.1 ([1]) For any absolutely continuous function β(s) on the interval [0,T], we have the inequality have the inequality β(s)∂α s β(s) ≥1 2∂α s β2(s), 0 < α < 1. (3.1) β(s)∂α s β(s) ≥1 2∂α s β2(s), 0 < α < 1. (3.1) Lemma 3.2 ([1]) Let a nonnegative absolutely continuous function R(s) satisfy the in- equality ∂α t R(s) ≤c1R(s) + c2(s), 0 < α < 1, (3.2) ∂α t R(s) ≤c1R(s) + c2(s), 0 < α < 1, (3.2) for almost all s ∈[0,T], where c1 is a positive constant, and c2(s) is an integrable nonnega- tive function on [0,T]. Then R(s) ≤R(0)Eα c1sα + Γ (α)Eα,α c1sα D–α t c2(s), (3.3) (3.3) where Eα(x) = ∞ n=0 xn Γ (αn + 1) and Eα,μ(x) = ∞ n=0 xn Γ (αn + μ) (3.4) (3.4) are the Mittag–Leﬄer functions. are the Mittag–Leﬄer functions. are the Mittag–Leﬄer functions. Young’s inequality with ε: For any ε > 0, we have the inequality aY ≤1 p\|εa\|p + p – 1 p Y ε p p–1 , a,b ∈R,p > 1, (3.5) (3.5) which is the generalization of the Cauchy inequality with ε: which is the generalization of the Cauchy inequality with ε: aY ≤ε 2a2 + 1 2ε Y 2, ε > 0, (3.6) aY ≤ε 2a2 + 1 2ε Y 2, ε > 0, (3.6) where a and Y are nonnegative numbers. where a and Y are nonnegative numbers. 3 Preliminaries Mesloub and Bachar Advances in Diﬀerence Equations ( 2019) 2019:254 Mesloub and Bachar Advances in Diﬀerence Equations ( 2019) 2019:254 Page 4 of 14 ( 2019) 2019:254 Poincaré-type inequalities [14]: Poincaré-type inequalities [14]: Ix(ξθ) 2 L2(0,b) ≤b3 2 ∥θ∥2 L2x(0,b), (3.7) I2 x(ξθ) 2 L2(0,b) ≤b2 2 Ix(ξθ) 2 L2(0,b), (3.8) Ix(ξθ) 2 L2(0,b) ≤b3 2 ∥θ∥2 L2x(0,b), I2 x(ξθ) 2 L2(0,b) ≤b2 2 Ix(ξθ) 2 L2(0,b), (3.8) here where Ix(U) = x 0 U(ξ,t)dξ, I2 x(U) = x 0 ξ 0 U(η,t)dη dξ. To establish the existence and uniqueness of the solution of problem (2.1), we write it in an equivalent operator form. To establish the existence and uniqueness of the solution of problem (2.1), we write it in an equivalent operator form. The solution of problem (2.1) can be regarded as the solution of the operator equation Mθ = F, (3.9) Mθ = F, (3.9) where M = (L,l1), and the operator M acts from S to H with domain of deﬁnition D(M) = ⎧ ⎪⎪⎨ ⎪⎪⎩ θ ∈L2 x(Q) : θx,θxx,∂α t θ ∈L2 x(Q), 1 0 xθ(x,t)dx = 0, t ∈(0,T), θx(1,t) = 0, t ∈(0,T), (3.10) (3.10) ere S is a Banach space of functions θ endowed by the ﬁnite norm where S is a Banach space of functions θ endowed by the ﬁnite norm ∥θ∥2 S = sup 0≤t≤T Dα–1Ix(ξθ) 2 L2(0,1) + Dα–1∥θ∥2 L2x(0,1) + T 0 ∥θx∥2 L2x(0,1) dt, (3.11) ∥θ∥2 S = sup 0≤t≤T Dα–1Ix(ξθ) 2 L2(0,1) + Dα–1∥θ∥2 L2x(0,1) + T 0 ∥θx∥2 L2x(0,1) dt, (3.11) (3.11) and H is the weighted Hilbert space L2 x(Q)×L2 x(0,1) consisting of vector-valued functions F = (f ,ω) with ﬁnite norm ∥F∥2 H = ∥ω∥2 L2x(0,1) + ∥f ∥2 L2x(Q). (3.12) ∥F∥2 H = ∥ω∥2 L2x(0,1) + ∥f ∥2 L2x(Q). (3.12) 4 A priori estimate for the solution and its consequences We establish an a priori bound for the solution of problem (2.1), from which we deduce its uniqueness. Theorem 4.1 Suppose that the function Y satisﬁes (i) Y(x,t) ≥C0, (ii) Yxx(x,t) ≤C1, (x,t) ∈Q, (4.1) (4.1) where C0 and C1 are positive constants, and f ∈L2 x(Q). Then there exists a positive constant μ such that the following a priori estimate holds: where C0 and C1 are positive constants, and f ∈L2 x(Q). Then there exists a positive constant μ such that the following a priori estimate holds: sup 0≤t≤T Dα–1Ix(ξθ) 2 L2(0,1) + Dα–1∥θ∥2 L2x(0,1) + T 0 ∥θx∥2 L2x(0,1) dt Mesloub and Bachar Advances in Diﬀerence Equations ( 2019) 2019:254 Page 5 of 14 Mesloub and Bachar Advances in Diﬀerence Equations ( 2019) 2019:254 Mesloub and Bachar Advances in Diﬀerence Equations ( 2019) 2019:254 ( 2019) 2019:254 Page 5 of 14 ≤μ ∥ω∥2 L2x(0,1) + ∥f ∥2 L2x(Q) ≤μ ∥ω∥2 L2x(0,1) + ∥f ∥2 L2x(Q) (4.2) (4.2) for all θ ∈D(M), where μ = μ(δ,σ,d) is given by for all θ ∈D(M), where μ = μ(δ,σ,d) is given by for all θ ∈D(M), where μ = μ(δ,σ,d) is given by μ = δ max 3 2,1 + Tα Γ (1 + α) , (4.3) (4.3) and δ,σ,d are respectively given by (4.29), (4.24), and (4.22). and δ,σ,d are respectively given by (4.29), (4.24), and (4.22). and δ,σ,d are respectively given by (4.29), (4.24), and (4.22). Proof Consider the identity Proof Consider the identity ∂α t θ – 1 x ∂ ∂x x∂θ ∂x + Y(x,t)θ,xθ – xI2 x(ξθ) L2(0,1) = f ,xθ – xI2 x(ξθ) L2(0,1). (4.4) (4.4) Under the boundary and initial conditions in (2.1), the terms of the left-hand side of (4.4) give Under the boundary and initial conditions in (2.1), the terms of the left-hand side of (4.4) give – ∂α t θ,xI2 x(ξθ) L2(0,1) – ∂α t θ,xI2 x(ξθ) L2(0,1) = – 1 0 x∂α t θ I2 x(ξθ) dx = – ∂α t x 0 ξθ dξ I2 x(ξθ) \|1 0 + 1 0 ∂α t Ix(ξθ) Ix(ξθ) dx 1 = – 1 0 x∂α t θ I2 x(ξθ) dx = – ∂α t x 0 ξθ dξ I2 x(ξθ) \|1 0 + 1 0 ∂α t Ix(ξθ) Ix(ξθ) dx = 1 0 ∂α t Ix(ξθ) Ix(ξθ) dx, (4.5) = 1 0 ∂α t Ix(ξθ) Ix(ξθ) dx, (4.5) (4.5) ∂ ∂x x∂θ ∂x ,I2 x(ξθ) L2(0,1) = – 1 0 x∂θ ∂xIx(ξθ)dx, (4.6) (4.6) – Y(x,t)θ,xI2 x(ξθ) L2(0,1) – Y(x,t)θ,xI2 x(ξθ) L2(0,1) – Y(x,t)θ,xI2 x(ξθ) L2(0,1) = 1 0 ∂Y(x,t) ∂x Ix(ξθ) I2 x(ξθ) dx + 1 0 Y(x,t) Ix(ξθ) 2 dx = 1 0 Y(x,t) Ix(ξθ) 2 dx – 1 2 1 0 ∂2Y(x,t) ∂x2 I2 x(ξθ) 2 dx, (4.7) ∂α t θ,xθ L2(0,1) = 1 0 xθ∂α t θ dx, (4.8) – ∂ ∂x x∂θ ∂x ,θ L2(0,1) = 1 0 xθ2 x dx, (4.9) Y(x,t)θ,xθ L2(0,1) = 1 0 xY(x,t)θ2 dx. (4.10) = 1 0 ∂Y(x,t) ∂x Ix(ξθ) I2 x(ξθ) dx + 1 0 Y(x,t) Ix(ξθ) 2 dx = 1 0 ∂Y(x,t) ∂x Ix(ξθ) I2 x(ξθ) dx + 1 0 Y(x,t) Ix(ξθ) 2 dx = 1 0 Y(x,t) Ix(ξθ) 2 dx – 1 2 1 0 ∂2Y(x,t) ∂x2 I2 x(ξθ) 2 dx, (4.7) ∂α t θ,xθ L2(0 1) = 1 xθ∂α t θ dx, (4.8) = 1 0 Y(x,t) Ix(ξθ) 2 dx – 1 2 1 0 ∂2Y(x,t) ∂x2 I2 x(ξθ) 2 dx, (4.7) – ∂ ∂x x∂θ ∂x ,θ L2(0,1) = 1 0 xθ2 x dx, (4.9) Y(x,t)θ,xθ L2(0,1) = 1 0 xY(x,t)θ2 dx. Proof Consider the identity (4.10) Substitution of equalities (4.5)–(4.10) into (4.4) yields Substitution of equalities (4.5)–(4.10) into (4.4) yields Substitution of equalities (4.5)–(4.10) into (4.4) yields 1 0 ∂α t Ix(ξθ) Ix(ξθ) dx + 1 0 xθ∂α t θ dx + 1 0 xY(x,t)θ2 dx + 1 0 Y((x,t) Ix(ξθ) 2 dx + 1 0 xθ2 x dx nce Equations ( 2019) 2019:254 Page 6 of 14 Mesloub and Bachar Advances in Diﬀerence Equations ( 2019) 2019:254 Mesloub and Bachar Advances in Diﬀerence Equations ( 2019) 2019:254 Page 6 of 14 ( 2019) 2019:254 Mesloub and Bachar Advances in Diﬀerence Equations ( 2019) 2019:254 Page 6 of 14 Page 6 of 14 Mesloub and Bachar Advances in Diﬀerence Equations = 1 2 1 0 ∂2Y(x,t) ∂x2 I2 x(ξθ) 2 dx + 1 0 x∂θ ∂xIx(ξθ)dx + Ix(ξf ),Ix(ξθ) L2(0,1) + (xθ,f )L2(0,1). (4.11) (4.11) + Ix(ξf ),Ix(ξθ) L2(0,1) + (xθ,f )L2(0,1). (4.11) To estimate the ﬁrst and second terms on the left-hand side of (4.11), we use Lemma 3.1 to obtain To estimate the ﬁrst and second terms on the left-hand side of (4.11), we use Lemma 3.1 to obtain To estimate the ﬁrst and second terms on the left-hand side of (4.11), we use Lemma 3.1 to obtain 1 0 ∂α t Ix(ξθ) Ix(ξθ) dx ≥1 2 1 0 ∂α t Ix(ξθ) 2 dx, (4.12) 1 0 xθ∂α t θ dx ≥1 2 1 0 ∂α t xθ2 dx. (4.13) (4.12) 1 0 xθ∂α t θ dx ≥1 2 1 0 ∂α t xθ2 dx. (4.13) 1 0 xθ∂α t θ dx ≥1 2 1 0 ∂α t xθ2 dx. (4.13) Conditions (4.1) and inequality (3.8) lead to Conditions (4.1) and inequality (3.8) lead to 1 0 ∂2Y(x,t) ∂x2 I2 x(ξθ) 2 dx ≤C1 1 0 I2 x(ξθ) 2 dx ≤C1 2 1 0 Ix(ξθ) 2 dx, (4.14) 1 0 Y(x,t) Ix(ξθ) 2 dx ≥C0 1 0 Ix(ξθ) 2 dx, (4.15) 1 0 xY(x,t)θ2 dx ≥C0 1 0 xθ2 dx. (4.16) 1 0 xY(x,t)θ2 dx ≥C0 1 0 xθ2 dx. Proof Consider the identity (4.16) The terms on the right-hand side of (4.11) can be estimated in the following way: The terms on the right-hand side of (4.11) can be estimated in the following way: Ix(ξf ),Ix(ξθ) L2(0,1) ≤ε1 2 1 0 Ix(ξθ) 2 dx + 1 4ε1 1 0 xf 2 dx, (4.17) 1 0 xθxIx(ξθ)dx ≤ε2 2 1 0 Ix(ξθ) 2 dx + 1 4ε2 1 0 xθ2 x dx, (4.18) (xθ,f )L2(0,1) ≤1 2 1 0 xθ2 dx + 1 2 1 0 xf 2 dx. (4.19) Ix(ξf ),Ix(ξθ) L2(0,1) ≤ε1 2 1 0 Ix(ξθ) 2 dx + 1 4ε1 1 0 xf 2 dx, (4.17) ≤ε2 2 1 0 Ix(ξθ) 2 dx + 1 4ε2 1 0 xθ2 x dx, (4.18) (xθ,f )L2(0,1) ≤1 2 1 0 xθ2 dx + 1 2 1 0 xf 2 dx. (4.19) (4.19) Combination of (4.11)–(4.19) yields Combination of (4.11)–(4.19) yields Combination of (4.11)–(4.19) yields Combination of (4.11)–(4.19) yields (4.22) (4.22) Replacing t by τ and integrating both sides of (4.21) with respect to τ from 0 to t, we obtain Dα–1 1 0 Ix(ξθ) 2 dx + Dα–1 1 0 xθ2 dx + t 0 1 0 xθ2 x dxdτ ≤σ t 0 1 0 Ix(ξθ) 2 dxdτ + t 0 1 0 xθ2 dxdτ + t 0 1 0 xf 2 dxdτ + 1 0 Ix(ξω) 2 dx + 1 0 xω2 dx , (4.23) Dα–1 1 0 Ix(ξθ) 2 dx + Dα–1 1 0 xθ2 dx + t 0 1 0 xθ2 x dxdτ ≤σ t 0 1 0 Ix(ξθ) 2 dxdτ + t 0 1 0 xθ2 dxdτ + t 0 1 0 xf 2 dxdτ 1 1 + 1 0 Ix(ξω) 2 dx + 1 0 xω2 dx , (4.23) (4.23) where σ = max d, T1–α (1 – α)Γ (1 – α) . (4.24) (4.24) Dropping the last term on the left-hand side of (4.23), applying Lemma 3.2, and setting R(t) = t 0 1 0 Ix(ξθ) 2 dxdτ + t 0 1 0 xθ2 dx, R(0) = 0, (4.25) (4.25) R(t) = 0 0 Ix(ξθ) 2 dxdτ + 0 0 xθ2 dx, R(0) = 0, (4.25) ∂α t R(t) = Dα–1Ix(ξθ) 2 dx + Dα–1 1 0 xθ2 dx, (4.26) ∂α t R(t) = Dα–1Ix(ξθ) 2 dx + Dα–1 1 0 xθ2 dx, (4.26) (4.26) obtain we obtain t 0 1 0 Ix(ξθ) 2 dxdτ + t 0 1 0 xθ2 dx ≤Γ (α)Eα,α dtα · D–α–1 t 1 0 xf 2 dx + 1 0 (Ixξω)2 dx + 1 0 xω2 dx . (4.27) t 0 1 0 Ix(ξθ) 2 dxdτ + t 0 1 0 xθ2 dx ≤Γ (α)Eα,α dtα · D–α–1 t 1 0 xf 2 dx + 1 0 (Ixξω)2 dx + 1 0 xω2 dx . + 1 0 xω2 dx . Combination of (4.11)–(4.19) yields 1 0 ∂α t Ix(ξθ) 2 dx + 1 2 1 0 ∂α t xθ2 dx + 1 0 xθ2 x dx + C0 1 0 Ix(ξθ) 2 dx + C0 1 0 xθ2 dx ≤C1 2 1 0 Ix(ξθ) 2 dx + ε1 2 1 0 Ix(ξθ) 2 dx Mesloub and Bachar Advances in Diﬀerence Equations ( 2019) 2019:254 Mesloub and Bachar Advances in Diﬀerence Equations ( 2019) 2019:254 ( 2019) 2019:254 Page 7 of 14 + 1 4ε1 1 0 xf 2 dx + ε2 2 1 0 Ix(ξθ) 2 dx + 1 4ε2 1 0 xθ2 x dx + ε3 2 1 0 xθ2 dx + 1 2ε3 1 0 xf 2 dx. (4.20) (4.20) + ε3 2 1 0 xθ2 dx + 1 2ε3 1 0 xf 2 dx. (4.20) By choosing ε1 = 2C0,ε2 = 1 2, and ε3 = 3C0 (4.20) becomes By choosing ε1 = 2C0,ε2 = 1 2, and ε3 = 3C0 (4.20) becomes 1 0 ∂α t Ix(ξθ) 2 dx + 1 0 ∂α t xθ2 dx + 1 0 xθ2 x dx ≤d 1 0 Ix(ξθ) 2 dx + 1 0 xθ2 dx + 1 0 xf 2 dx , (4.21) (4.21) where d = max( C1 2 + 1 4, C0 2 , 5C0 2 , 1 2) min( C0 2 , 1 2) . Combination of (4.11)–(4.19) yields (4.27) (4.27) On the light of (4.27) and (3.7), inequality (4.23) becomes Dα–1Ix(ξθ) 2 L2(0,1) + Dα–1∥θ∥2 L2x(0,1) + t 0 ∥θx∥2 L2x(0,1) dτ Mesloub and Bachar Advances in Diﬀerence Equations ( 2019) 2019:254 Mesloub and Bachar Advances in Diﬀerence Equations ( 2019) 2019:254 Page 8 of 14 ( 2019) 2019:254 ≤δ D–1–α t ∥f ∥2 L2x(0,1) + Ix(ξω) 2 L2(0,1) + ∥ω∥2 L2x(0,1) + t 0 ∥f ∥2 L2x(0,1) dτ ≤δ D–1–α t ∥f ∥2 L2x(0,1) + ∥ω∥2 L2x(0,1) + t 0 ∥f ∥2 L2x(0,1) dτ , (4.28) (4.28) where δ = max σΓ (α)Eα,α dtα ,σ . (4.29) δ = max σΓ (α)Eα,α dtα ,σ . (4.29) It is easy to see that It is easy to see that D–α–1 t ∥f ∥2 L2x(0,1) ≤ Tβ Γ (1 + β) T 0 ∥f ∥2 L2x(0,1) dτ. (4.30) D–α–1 t ∥f ∥2 L2x(0,1) ≤ Tβ Γ (1 + β) T 0 ∥f ∥2 L2x(0,1) dτ Inequalities (4.28) and (4.30) yield Dα–1Ix(ξθ) 2 L2(0,1) + Dα–1∥θ∥2 L2x(0,1) + t 0 ∥θx∥2 L2x(0,1) dτ ≤μ ∥ω∥2 L2x(0,1) + T 0 ∥f ∥2 L2x(0,1) dτ , (4.31) ≤μ ∥ω∥2 L2x(0,1) + T 0 ∥f ∥2 L2x(0,1) dτ , (4.31) where μ = δ max 3 2,1 + Tα Γ (1 + α) . Now since the right-hand side of (4.31) does not depend on t, the a priori estimate (4.2) follows by taking the upper bound for both sides with respect to t over [0,T]. Note that the uniqueness and continuous dependence of the solution on the data of problem (2.1) follows from the a priori bound (4.2). □ 5 Existence of solution The a priori estimate (4.2) shows that the unbounded operator M has an inverse M–1 : R(M) →S. Since R(M) is a subset of H, we can construct its closure M so that estimate (4.2) holds for this extension and R(M) coincides with the whole space H. Hence we have the following: Corollary 5.1 The operator M : S →H admits a closure (proof is similar to that in [14]. Estimate (4.2) can be then extended to Corollary 5.1 The operator M : S →H admits a closure (proof is similar to that in [14]. Estimate (4.2) can be then extended to Estimate (4.2) can be then extended to sup 0≤t≤T Dα–1Ix(ξθ) 2 L2(0,1) + Dα–1∥θ∥2 L2x(0,1) + T 0 ∥θx∥2 L2x(0,1) dt ≤μ ∥ω∥2 L2x(0,1) + ∥f ∥2 L2x(Q) (5.1) ≤μ ∥ω∥2 L2x(0,1) + ∥f ∥2 L2x(Q) (5.1) for all θ ∈D(M). Mesloub and Bachar Advances in Diﬀerence Equations ( 2019) 2019:254 Page 9 of 14 Page 9 of 14 It follows from (5.1) that the strong solution of problem (2.1) is unique, that is, Mθ = H. From estimate (5.1) we also deduce the following: Corollary 5.2 R(M) is a closed subset in H, R(M) = R(M), and M –1 = M–1. We are now ready to give the result on the existence of the solution of problem (2.1). Theorem 5.3 Suppose that the conditions of Theorem 4.1 are satisﬁed. Then for all F = (f ,ω) ∈H, there exists a unique strong solution θ = M –1F = M–1F of problem (2.1). Theorem 5.3 Suppose that the conditions of Theorem 4.1 are satisﬁed. Then for all F = (f ,ω) ∈H, there exists a unique strong solution θ = M –1F = M–1F of problem (2.1). Proof Estimate (5.1) asserts that if a strong solution of (2.1) exits, then it is unique and de- pends continuously on the data. Corollary 5.2 says that to prove that problem (2.1) admits a strong solution for any F = (f ,ω) ∈H, it suﬃces to show that the closure of the range of the operator M is dense in H. To establish the existence of the strong solution of problem (2.1), we use a density argument, that is, we show that the range R(M) of the operator M, is dense in the space H for every element θ in the Banach space S. For this, we consider the following particular case of density. □ □ Theorem 5.4 Suppose that the conditions of Theorem 4.1 are satisﬁed. Suppose that for all functions θ ∈D(M) such that l1θ = θ(x,0) = 0 and for some function ψ ∈L2(Q), we have T 0 (Lθ,ψ)L2x(0,1) dt = 0. T 0 (Lθ,ψ)L2x(0,1) dt = 0. (5.2) (5.2) Then ψ vanishes a.e. in Q. Then ψ vanishes a.e. in Q. Then ψ vanishes a.e. in Q. Proof Identity (5.2) is equivalent to Proof Identity (5.2) is equivalent to Proof Identity (5.2) is equivalent to T 0 ∂α t θ – 1 x ∂ ∂x x∂θ ∂x + Y(x,t)θ,ψ L2x(0,1) dt = 0. (5.3) (5.3) Assume that a function γ (x,t) satisﬁes the conditions boundary and initial conditions in (2.1) and that γ , γx, and ∂ ∂x(x t 0 γ (x,s)ds) ∈L2(Qt). We then set θ(x,t) = t 0 γ (x,s)ds. (5.4) θ(x,t) = t 0 γ (x,s)ds. (5.4) uation (5.3) then becomes Equation (5.3) then becomes Equation (5.3) then becomes T 0 ∂α t t 0 γ (x,s)ds – 1 x ∂ ∂x t 0 xγx(x,s)ds + Y(x,t) t 0 γ (x,s)ds ,ψ)L2x(0,1) dt T 0 ∂α t t 0 γ (x,s)ds – 1 x ∂ ∂x t 0 xγx(x,s)ds + Y(x,t) t 0 γ (x,s)ds ,ψ)L2x(0,1) dt = 0. (5.5) (5.5) = 0. We now introduce the function We now introduce the function ψ(x,t) = t 0 γ (x,s)ds – I2 x ξ t 0 γ (ξ,s)ds . (5.6) ψ(x,t) = t 0 γ (x,s)ds – I2 x ξ t 0 γ (ξ,s)ds . (5.6) Mesloub and Bachar Advances in Diﬀerence Equations ( 2019) 2019:254 Mesloub and Bachar Advances in Diﬀerence Equations ( 2019) 2019:254 Page 10 of 14 Mesloub and Bachar Advances in Diﬀerence Equations Equation (5.5) then reduces to Equation (5.5) then reduces to T 0 ∂α t t 0 γ (x,s)ds ,x t 0 γ (x,s)ds – xI2 x ξ t 0 γ (ξ,s)ds L2(0,1) dt + T 0 ∂ ∂x t 0 xγx(x,s)ds ,– t 0 γ (x,s)ds + I2 x ξ t 0 γ (ξ,s)ds L2(0,1) dt – T 0 Y(x,t) t 0 γ (x,s)ds ,–x t 0 γ (x,s)ds + xI2 x ξ t 0 γ (ξ,s)ds L2(0,1) dt = 0. (5.7) (5.7) Recall that the function γ satisﬁes boundary conditions in (2.1). Then ψ vanishes a.e. in Q. Then computing the inner products in (5.7), we have – ∂α t t 0 γ (x,s)ds ,xI2 x ξ t 0 γ (ξ,s)ds L2(0,1) = ∂α t Ix ξ t 0 γ (ξ,s)ds ,Ix ξ t 0 γ (ξ,s)ds L2(0,1) ≥1 2 1 0 ∂α t Ix ξ t 0 γ (ξ,s)ds 2 dx – ∂α t t 0 γ (x,s)ds ,xI2 x ξ t 0 γ (ξ,s)ds L2(0,1) = ∂α t Ix ξ t 0 γ (ξ,s)ds ,Ix ξ t 0 γ (ξ,s)ds L2(0,1) = 1 2∂α t Ix t 0 ξγ (ξ,s)ds 2 L2(0,1) , (5.8) (5.8) (5.9) (5.10) (5.11) Page 11 of 14 Mesloub and Bachar Advances in Diﬀerence Equations ( 2019) 2019:254 = Y(x,t) t 0 γ (x,s)ds 2 L2x(0,1) . (5.12) ∂α t t 0 γ (x,s)ds ,x t 0 γ (x,s)ds L2(0,1) ≥1 2 1 0 x∂α t t 0 γ (x,s)ds 2 dx = 1 2∂α t t 0 γ (x,s)ds 2 L2x(0,1) . (5.13) (5.12) (5.13) Using (5.8)–(5.13) and the Cauchy ε-inequality, replacing t by τ, and integrating with re- spect to τ over (0,t), we see that Using (5.8)–(5.13) and the Cauchy ε-inequality, replacing t by τ, and integrating with re- spect to τ over (0,t), we see that 1 2Dα–1 t Ix t 0 ξγ (ξ,s)ds 2 L2(0,1) + 1 2Dα–1 t t 0 γ (x,s)ds 2 L2x(0,1) + t 0 1 0 Y(x,τ) Ix ξ τ 0 γ (ξ,s)ds 2 dxdτ + t 0 τ 0 γx(x,s)ds 2 L2x(0,1) dτ + t 0 Y(x,τ) τ 0 γ (x,s)ds 2 L2x(0,1) dτ ≤1 ε t 0 1 0 Ix ξ τ 0 γ (ξ,s)ds 2 dxdτ + ε 2 t 0 τ 0 γx(x,s)ds 2 L2x(0,1) dτ + t 0 1 0 ∂2Y(x,τ) ∂x2 I2 x ξ τ 0 γ (ξ,s)ds 2 dxdτ. Then ψ vanishes a.e. in Q. (5.14) (5.14) Choosing ε = 1 and using inequalities (3.6) and (3.7) and conditions (4.1), we rewrite in- equality (5.14) as Dα–1 t Ix t 0 ξγ (ξ,s)ds 2 L2(0,1) + Dα–1 t t 0 γ (x,s)ds 2 L2x(0,1) + 2C0 t 0 Ix τ 0 ξγ (x,s)ds 2 L2(0,1) dτ + t 0 τ 0 γx(x,s)ds 2 L2x(0,1) dτ + 2C0 t 0 τ 0 γ (x,s)ds 2 L2x(0,1) dτ ≤ t 0 τ 0 ξγ (x,s)ds 2 L2x(0,1) dτ + C1 t 0 Ix τ 0 γ (x,s)ds 2 L2x(0,1) dτ. (5.15) (5.15) Discarding the last three terms on the left-hand side of (5.15), we obtain Dα–1 t Ix t 0 ξγ (ξ,s)ds 2 L2(0,1) + Dα–1 t t 0 γ (x,s)ds 2 L2x(0,1) ≤V t 0 Ix τ 0 γ (ξ,s)ds 2 L2(0,1) dτ + t 0 τ 0 γ (ξ,s)ds 2 L2x(0,1) dτ , (5.16) (5.16) Page 12 of 14 ( 2019) 2019:254 Mesloub and Bachar Advances in Diﬀerence Equations ( 2019) 2019:254 where V = max(1,C1). V = max(1,C1). V = max(1,C1). We can now apply Lemma 3.2 to (5.16) by taking We can now apply Lemma 3.2 to (5.16) by taking We can now apply Lemma 3.2 to (5.16) by taking ϕ(t) = t 0 Ix τ 0 γ (ξ,s)ds 2 L2(0,1) dτ + t 0 τ 0 γ (ξ,s)ds 2 L2x(0,1) dτ, ϕ(0) = 0, (5.17) ϕ(t) = t 0 Ix τ 0 γ (ξ,s)ds 2 L2(0,1) dτ + t 0 τ 0 γ (ξ,s)ds 2 L2x(0,1) dτ, and ∂α t ϕ(t) = Dα–1 t Ix t 0 ξγ (ξ,s)ds 2 L2(0,1) + Dα–1 t t 0 γ (x,s)ds 2 L2x(0,1) . (5.18) Then t 0 Ix τ 0 γ (ξ,s)ds 2 L2(0,1) dτ + t 0 τ 0 γ (ξ,s)ds 2 L2x(0,1) dτ ≤ϕ(0)Eα Vtα + Γ (α)Eα,α Vtα D–α t (0) = 0. (5.19) t 0 Ix τ 0 γ (ξ,s)ds 2 L2(0,1) dτ + t 0 τ 0 γ (ξ,s)ds 2 L2x(0,1) dτ (5.19) Inequality (5.19) implies that T 0 Ix τ 0 γ (ξ,s)ds 2 L2(0,1) dτ + T 0 τ 0 γ (ξ,s)ds 2 L2x(0,1) dτ ≤0. Then ψ vanishes a.e. in Q. Mesloub and Bachar Advances in Diﬀerence Equations ( 2019) 2019:254 Mesloub and Bachar Advances in Diﬀerence Equations ( 2019) 2019:254 ( 2019) 2019:254 Page 13 of 14 Mesloub and Bachar Advances in Diﬀerence Equations Example In the considered problem (2.1), we set Example In the considered problem (2.1), we set Y(x,t) = xn + 1 tν + 1 , n ∈N,ν > 0, (5.24) Y(x,t) = xn + 1 tν + 1 , n ∈N,ν > 0, (5.24) Y(x,t) = xn + 1 tν + 1 , n ∈N,ν > 0, (5.24) Y(x,t) = xn + 1 tν + 1 , n ∈N,ν > 0, and f (x,t) = λ(x)t1–α (1 – α)Γ (1 – α) + 2ρ(x)t2–α (1 – α)(2 – α)Γ (1 – α) + 12 5 x–1 – 2 (t + 1) – t2 + xn + 1 tν + 1 λ(t + 1) + ρt2 , (5.25) f (x,t) = λ(x)t1–α (1 – α)Γ (1 – α) + 2ρ(x)t2–α (1 – α)(2 – α)Γ (1 – α) + 12 5 x–1 – 2 (t + 1) – t2 + xn + 1 tν + 1 λ(t + 1) + ρt2 , (5.25) here λ(x) = 6x2 – 12x + 5 5 , ρ(x) = 2x2 – 4lnx – 3 8 . (5.26) h f i Y i ﬁ i (4 1) i h C 1 d C ( 1)(Tν 1) Th f (x,t) = λ(x)t1–α (1 – α)Γ (1 – α) + 2ρ(x)t2–α (1 – α)(2 – α)Γ (1 – α) + 12 5 x–1 – 2 (t + 1) f (x,t) = λ(x)t1–α (1 – α)Γ (1 – α) + 2ρ(x)t2–α (1 – α)(2 – α)Γ (1 – α) + 12 5 x–1 – 2 (t + 1) – t2 + xn + 1 tν + 1 λ(t + 1) + ρt2 , – t2 + xn + 1 tν + 1 λ(t + 1) + ρt2 , (5.25) (5.25) where λ(x) = 6x2 – 12x + 5 5 , ρ(x) = 2x2 – 4lnx – 3 8 . (5.26) (5.26) The function Y satisﬁes assumptions (4.1) with C0 = 1 and C1 = n(n – 1)(Tν + 1). Then ψ vanishes a.e. in Q. The inclusion f ∈L2 x(Q) holds, and we can easily verify that the function θ(x,t) = 6x2 – 12x + 5 5 (t + 1) + 2x2 – 4lnx – 3 8 t2 (5.27) (5.27) satisﬁes the fractional diﬀerential equation in (2.1) and the initial and boundary conditions with the initial condition ω(x) = 6x2–12x+5 5 , which satisﬁes the compatibility conditions ωx(1) = 0, 1 0 xω dx = 0. ωx(1) = 0, 1 0 xω dx = 0. (5.28) (5.28) Moreover, θ ∈L2 x(Q), and Moreover, θ ∈L2 x(Q), and ∂α t θ = λ(x) (1 – α)Γ (1 – α)t1–α + 2ρ(x) (1 – α)(2 – α)Γ (1 – α)t2–α ∈L2 x(Q). (5.29) (5.29) All conditions in Theorem 4.1 and Theorem 5.3 are satisﬁed, and the existence and uniqueness of a solution of problem (2.1) follows. All conditions in Theorem 4.1 and Theorem 5.3 are satisﬁed, and the existence and uniqueness of a solution of problem (2.1) follows. All conditions in Theorem 4.1 and Theorem 5.3 are satisﬁed, and the existence and uniqueness of a solution of problem (2.1) follows. 6 Conclusion The existence and uniqueness of a generalized solution for a singular fractional initial boundary value problem in the Caputo sense subject to Neumann and weighted integral conditions are established. It is found that the method of energy inequalities is successfully applied to obtaining a priori estimates for the solution of the initial fractional boundary value problem as in the classical case. The obtained results will contribute to the develop- ment of the functional analysis method and enrich the existing nonextensive literature on the nonlocal fractional mixed problems in the Caputo sense. Acknowledgements Th h i h h Acknowledgements The authors wish to thank anonymous referees for their comments and valuable suggestions. Then ψ vanishes a.e. in Q. (5.20) (5.20) Then from (5.20) it follows that the function ψ = t 0 γ (x,s)ds – I2 x(ξ t 0 γ (ξ,s)ds) is zero a.e. in Q. To complete the proof of Theorem 5.3, assume that for (Ψ ,ω1) ∈R(M)⊥, we have T 0 (Lθ,Ψ )L2x(0,1) ds + (l1θ,ω1)L2x(0,1) = 0. (5.21) T 0 (Lθ,Ψ )L2x(0,1) ds + (l1θ,ω1)L2x(0,1) = 0. (5.21) (5.21) Then we should show that Ψ = 0,ω1 = 0. If we put θ ∈D(M) satisfying condition l1θ = θ(x,0) = 0 into (5.20), we get Then we should show that Ψ = 0,ω1 = 0. If we put θ ∈D(M) satisfying condition l1θ = θ(x,0) = 0 into (5.20), we get T 0 (Lθ,Ψ )L2x(0,1) ds = 0, θ ∈D(M). (5.22) (5.22) By Theorem 5.4 equation (5.22) implies that Ψ vanishes a.e. in Q. Then (5.21) becomes By Theorem 5.4 equation (5.22) implies that Ψ vanishes a.e. in Q. Then (5.21) becomes (l1θ,ω1)L2x(0,1) = 0, θ ∈D(M). (5.23) (l1θ,ω1)L2x(0,1) = 0, θ ∈D(M). (5.23) Since the range of the trace operator l1 is dense in L2 x(0,1), from (5.22) we conclude that ω1 = 0, and Theorem 5.3 is proved. □ We present the following example to illustrate our main results. We present the following example to illustrate our main results. Authors’ contributions Authors contributions Both authors contributed equally to the writing of this paper. Both authors read and approved the ﬁnal manuscript. References 1. Alikhanov, A.A.: A priori estimates for solutions of boundary value problems for fractional-order equations. Diﬀer. Equ. 46(5), 660–666 (2010) 2. Béla, J.S., Izsák, F.: A ﬁnite diﬀerence method for fractional diﬀusion equations with Neumann boundary conditio Open Math. 13, 581–600 (2015) p 3. Beshtokov, M.K.H.: To boundary-value problems for degenerating pseudoparabo Gerasimov–Caputo fractional derivative. Russ. Math. 62(10), 1–14 (2018). https://doi.org/10.3103/S1066369X18100018 3. Beshtokov, M.K.H.: To boundary-value problems for degenerating pseudoparabolic equations with Gerasimov–Caputo fractional derivative. Russ. Math. 62(10), 1–14 (2018). https://doi org/10 3103/S1066369X18100018 p g 4. Beshtokov, M.K.H.: Local and nonlocal boundary value problems for degenerating and nondegenerating pseudoparabolic equations with a Riemann–Liouville fractional derivative. Diﬀer. Equ. 54(6), 758–774 (2018). https://doi org/10 1134/S0012266118060058 p g 5. Cannon, J.R.: The One-Dimensional Heat Equation. Cambridge University Press, Cambridge (1984) 5. Cannon, J.R.: The One-Dimensional Heat Equation. Cambridge 6. Caputo, M.: Elasticitae Dissipazione, Zanichelli, Bologna (1969) 6. Caputo, M.: Elasticitae Dissipazione, Zanichelli, Bologna (1969) p p g 7. El-Sayed, A.M.A., Gaber, M.: The Adomian decomposition method for solving partial diﬀerential equations of 7. El-Sayed, A.M.A., Gaber, M.: The Adomian decomposition method for solving partial diﬀ fractional order inﬁnite domains. Phys. Lett. A 359, 175–182 (2006) y , , , p g p q fractional order inﬁnite domains. Phys. Lett. A 359, 175–182 (2006) y 8. Friedman, A.: Partial Diﬀerential Equations of Parabolic Type. Prentice-Hall, Englewood Cliﬀs (1964) 8. Friedman, A.: Partial Diﬀerential Equations of Parabolic Type. Prentice-Hall, Englewood Cliﬀs (1964 9. Gorenﬂo, R., Mainardi, F., Moretti, D., Paradisi, P.: Time fractional diﬀusion: a discrete random walk approach. Nonlinear Dyn. 29, 129–143 (2002) y 10. Huy Tuan, N., Tran Bao, N., Tatar, S.: Recovery of the solute concentration and dispersion ﬂux in an inhomogeneous y 10. Huy Tuan, N., Tran Bao, N., Tatar, S.: Recovery of the solute concentration and dispersion ﬂux in an inhomogeneous time fractional diﬀusion equation J Comput Appl Math 342 96–118 (2018) 10. Huy Tuan, N., Tran Bao, N., Tatar, S.: Recovery of the solute concentration and dispersion ﬂux in an in time fractional diﬀusion equation. J. Comput. Appl. Math. 342, 96–118 (2018) y y time fractional diﬀusion equation. J. Comput. Appl. Math. 342, 96–118 (2018) 11. Jafari, H., Daftardar-Gejji, V.: Solving linear and non-linear fra decomposition. Appl. Math. Comput. 180, 488–497 (2006) 11. Jafari, H., Daftardar-Gejji, V.: Solving linear and non-linear fractional diﬀusion and wave equations by Adomian decomposition. Appl. Math. Comput. 180, 488–497 (2006) 11. Funding Th h Funding The authors would like to extend their sincere appreciation to the Deanship of Scientiﬁc Research at King Saud University for its funding this Research group NO (RG-1435-043). Mesloub and Bachar Advances in Diﬀerence Equations ( 2019) 2019:254 Page 14 of 14 ( 2019) 2019:254 Mesloub and Bachar Advances in Diﬀerence Equations Competing interests The authors declare that they have no competing interests. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional aﬃliations. Received: 17 January 2019 Accepted: 14 June 2019 References Jafari, H., Daftardar-Gejji, V.: Solving linear and non-linear fra decomposition. Appl. Math. Comput. 180, 488–497 (2006) 12. Kilbas, A.A., Srivastava, H.M., Trujillo, J.J.: Theory and Applications of Fractional Diﬀerential Equations. Elsevier, Amsterdam (2006) 13. Liu, F., Anh, V., Turner, I., Zhuang, P.: Time fractional advection dispersion equation. J. Appl. Math. Comput. 13, 233–245 (2003) 14. Mesloub, S.: A nonlinear nonlocal mixed problem for a second order parabolic equation. J. Math. Anal. Appl. 316, 189–209 (2006) 14. Mesloub, S.: A nonlinear nonlocal mixed problem for a second order parabolic equation. J. Math. Anal. Appl. 316, 189–209 (2006) 15. Mesloub, S., Bouziani, A.: On a class of singular hyperbolic equations with a weighted integral condition. Int. J. Math Math. Sci. 22(3), 511–519 (1999) 16. Podlubny, I.: Fractional Diﬀerential Equations. Academic Press, San Diego (1999) 17. Schneider, W.R., Wyss, W.: Fractional diﬀusion and wave equations. J. Math. Phys. 30, 34–144 (1989) 18. Wei, T., Li, Y.S.: Identifying a diﬀusion coeﬃcient in a time-fractional diﬀusion equation. Math. Comput. Simul. 151, 77–95 (2018) 19. Widder, D.V.: The Heat Equation. Academic Press, New York (1975) 20. Xianjuan, L., Chuanju, X.: A space-time spectral method for the time fractional diﬀusion equation. SIAM J. Numer. Anal. 47(3), 2108–2131 (2009) 20. Xianjuan, L., Chuanju, X.: A space-time spectral method for the time fractional diﬀusion equation. SIAM J. Numer. Anal. 47(3), 2108–2131 (2009) 21. Yurchuk, N.I.: Mixed problem with an integral condition for certain parabolic equations. Diﬀer. Uravn. 22(12), 2117–2126 (1986) 21. Yurchuk, N.I.: Mixed problem with an integral condition for certain parabolic equations. Diﬀer. Uravn. 22(12), 2117–2126 (1986)
https://openalex.org/W4293235925	https://www.frontiersin.org/articles/10.3389/fpsyg.2022.868793/pdf	English	null	The relationship between attribution of blame and the perception of resistance in relation to victims of sexual violence	Frontiers in psychology	2,022	cc-by	9,362	TYPE Original Research PUBLISHED 25 August 2022 DOI 10.3389/fpsyg.2022.868793 TYPE Original Research PUBLISHED 25 August 2022 DOI 10.3389/fpsyg.2022.868793 TYPE Original Research PUBLISHED 25 August 2022 DOI 10.3389/fpsyg.2022.868793 OPEN ACCESS 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. attribution of blame, resistance, rape, group sexual offending, scenarios, victims, sexual violence OPEN ACCESS OPEN ACCESS EDITED BY Lisa Chiara Fellin, University of Bergamo, Italy REVIEWED BY Adrienne Baldwin-White, University of Georgia, United States Sieun An, Eastern New Mexico University, United States CORRESPONDENCE Jesús de la Torre Laso jesustl@usal.es SPECIALTY SECTION This article was submitted to Personality and Social Psychology, a section of the journal Frontiers in Psychology RECEIVED 03 February 2022 ACCEPTED 27 July 2022 PUBLISHED 25 August 2022 OPEN ACCESS EDITED BY Lisa Chiara Fellin, University of Bergamo, Italy REVIEWED BY Adrienne Baldwin-White, University of Georgia, United States Sieun An, Eastern New Mexico University, United States Jesús de la Torre Laso 1 and Juan M. Rodríguez-Díaz 2,3 1 Department of Social Psychology and Antropology, University of Salamanca, Salamanca, Spain, 2 Department of Statistics, Faculty of Science, University of Salamanca, Salamanca, Spain, 3 Institute of Fundamental Physics and Mathematics (IUFFyM), Faculty of Science, University of Salamanca, Salamanca, Spain Several studies have examined victim blaming in rape scenarios. However, there is limited research on the analysis of the perception of blame when two or more perpetrators are involved. The present article explores the perception of blame in cases involving rape based on the level of resistance shown by the victim and the presence of one or more perpetrators. A study was carried out involving 351 university students who responded to a survey after reading a hypothetical assault scenario. Six situations were established where the victim showed either low or high resistance, depending on whether the resistance was verbal or physical and verbal, and in the presence of one or two male perpetrators. It is expected that perpetrators are more culpable when acting in groups and that less resistance from the victim leads to greater attribution of blame. The results confirm that more blame is attributed to the perpetrators when they act in groups than when they act alone. Likewise, women consider the victim generally exerts greater resistance and this variable influences the attribution of greater blame. CITATION de la Torre Laso J and Rodríguez-Díaz JM (2022) The relationship between attribution of blame and the perception of resistance in relation to victims of sexual violence. Front. Psychol. 13:868793. d i 10 3389/f 2022 868793 COPYRIGHT © 2022 de la Torre Laso and Rodríguez-Díaz. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). Introduction The crime of sexual assault committed in a group is little known, although it is an internationally experienced reality. Studies have suggested that between 10% and 33% of sexual assaults have been committed by multiple perpetrators (Franklin, 2004). In South Africa, the country with the highest rates of multiple sexual assaults in the world, it would account for between one-third and one-half of all reported rapes (Artz and Kunisaki, 2003). In the United States, referrals range from 2% to 26% (Horvath and Kelly, 2009), in the United Kingdom they account for ~11%–19% of all sexual assault cases (Harkins and Dixon, 2013) and in South Korea it is estimated that 7.7% of sexual assault crimes involve two or more offenders (Park and Kim, 2016). Substance use is also an influential element in the attribution of blame for an episode of sexual violence for both victims and perpetrators (Horvath, 2006); and in general, victims who consume alcohol are judged more harshly and are perceived by observers as more responsible for the attack than women who do not consume alcohol (Richardson and Campbell, 1982; Alicke, 2000). However, regardless of any other factors, most people tend to attribute greater blame for the incident towards the perpetrator rather than the victim (Strömwall et al., 2013; Persson and Dhingra, 2020), and place minimal blame on the victim. Studies on sexual violence have identified the importance of studying victims’ attribution of blame in order to determine the labels associated with women who have been sexually assaulted (Setia et al., 2020) and to prevent and avoid these situations (Rogers et al., 2009; Lim, 2017; Adolfsson et al., 2020). Victim resistance can affect the victim’s perception of blame for the sexual offence. Victim resistance relates to expressions of consent or refusal of sexual activity and, in particular, the form of verbal or non-verbal expression (Wignall et al., 2020). The absence of resistance has sometimes been associated with consent, but other studies have shown that this behavior, in the form of tonic immobility, is an involuntary and temporary state of motor inhibition, characteristic of sexual violence (Möller et al., 2017). In general terms, attribution of blame is understood as the preconception of blame and responsibility that is placed on a person in a negative event (Shaver, 1985). Frequently the victim is blamed in sexual crimes (Gerber et al., 2004; Randall, 2010). Introduction Sexual violence affects 100 of 1,000 of women every year. In Spain it is estimated that 8.9% of women have suffered sexual violence at some point in their lives by their current partner and 6.5% by someone with whom they do not have or have not had a relationship (Ministerio de Igualdad, 2019). In other countries, data show higher figures, as for example in the United Kingdom, where it is estimated that 20% of women have suffered a sexual assault (Wignall et al., 2020). However, a very small proportion of these situations are reported or end up in legal proceedings, as most of the episodes of sexual violence go unreported. This lack of Frontiers in Psychology 01 frontiersin.org de la Torre Laso and Rodríguez-Díaz 10.3389/fpsyg.2022.868793 10.3389/fpsyg.2022.868793 Another of the basic concepts associated with attribution of blame has to do with rape myths, first explained by Burt (1980). This concept captures those persistent and pervasive beliefs and attitudes that serve to exonerate the perpetrator and blame the victim for the rape. Four categories have been established: blaming the victim; excusing the perpetrator; beliefs that rape is not very serious; and that only certain types of women are raped (Gerger et al., 2013). Greater acceptance of rape myth is related to the crime being perceived as being less serious and greater victim blaming (Grubb and Turner, 2012). Some researchers have found that men are more likely than women to accept rape myths, and attribute greater victim blaming (Strömwall et al., 2014; Russell and Hand, 2017) although others have found a lack of differences between men and women (e.g., Abrams et al., 2003).l reporting has given rise to the so-called black figure of crime (Datta and Bales, 2013). Among the circumstances in which victims do not report a sexual crime is the shame they experience in acknowledging they having been a victim; their state of confusion, guilt or shock after the assault; the fear of the offender and the perceived consequences of filing a complaint; the lack of witnesses or the difficulty in identifying the aggressors; the fear of not being believed; and distrust in institutions with regard to criminal proceedings (Drury et al., 2020). Another factor that discourages victims from reporting is the fear of being blamed for the crime, therefore, avoiding revictimization (Gutiérrez de Piñeres Botero et al., 2009). Introduction Attribution of blame is sometimes modulated by personal factors, such as the gender of the person assessing blame, also the gender of the perpetrator, his or her relationship with the victim, as well as situational factors such as rape myth acceptance of the person assessing blame, substance use, or the level of victim resistance (Grubb and Turner, 2012; Ayala et al., 2018). Previous studies have shown that in the case of verbal and physical resistance, is more likely to be considered rape when the woman only resists verbally (Shotland and Goodstein, 1983; Black and McCloskey, 2013). Likewise, an expression of physical resistance has been associated with a lack of consent in relationships (Ullman, 2007a) and, therefore, the absence of resistance may be perceived with increased victim blaming, and decreased perpetrator accountability (Davies et al., 2008). It has been found that men assessing blame tend to blame the victim to a greater extent than women (Grubb and Harrower, 2009), which is explained because women tend to have higher levels of empathy towards rape victims and therefore tend to attribute greater credibility (Jimenez and Abreu, 2003). There are gender differences in the interpretation about certain relationship cues (such as having eye contact with another person, touching, flirting or going home with someone), and as Jozkowski et al. (2018) assert, men perceive these cues as consent to sexual activity, whereas women tend to see them as indicators of sexual interest, but not consent. This study aims to examine the circumstances under which victim blaming is attributed to rape. Victim blaming of rape has been shown to be associated not only with lower reporting rates, but also with a significant increase in the period required for psychological recovery (Ullman, 1996; Kline et al., 2021). Frontiers in Psychology Resistance, attribution of blame, and sexual offences committed by multiple offenders When it has been analyzed whether sexual offences are committed by acquaintances or strangers, research presents contradictory results (Grubb and Harrower, 2009); although rape victims who know their attacker are blamed to a greater extent than victims of stranger rape (Strömwall et al., 2013; Persson and Dhingra, 2020). In the last decade, the phenomenon of sexual offences jointly committed by more than two people or in groups has been Frontiers in Psychology 02 frontiersin.org frontiersin.org de la Torre Laso and Rodríguez-Díaz 10.3389/fpsyg.2022.868793 analyzed (Morgan et al., 2012; Harkins and Dixon, 2013; among others). Attempts have been made to describe the circumstances of these offences (e.g., Horvath and Kelly, 2009; Quarshie et al., 2018), as well as the differences between assaults committed by a single perpetrator and by multiple perpetrators (Bijleveld and Hendriks, 2003; da Silva et al., 2015; Park and Kim, 2016). These studies focus on examining offender behavior (Chambers et al., 2010) or the impact of such crimes on the victims (Woodhams et al., 2007; Morgan et al., 2015). violence when the assault was carried out by a group of offenders (Ullman, 2007b). On the other hand, there are few references that have analyzed the differences between the blame or responsibility of sexual offenders depending on whether they actively participate or remain as observers. According to Adolfsson et al. (2020), subjects who remain passive in a rape are also attributed a degree of blame, although this view may be conditioned by legislation and the punishment given to non-active participants. In terms of the level of victim resistance, few studies have examined victim resistance in sexual assaults perpetrated by two or more people. However, those that do exist have found that victims of these crimes tend to show reduced levels of resistance (Porter and Alison, 2006), compared to assaults committed by a single subject (Wright and West, 1981; Hauffe and Porter, 2009). Wijkman et al. (2014) analyzed the cases of 25 women who had been sexually assaulted by groups of offenders and found that 31% of victims physically resisted the assault, while 12% expressed verbal resistance. According to Woodhams and Cooke (2013), depending on the level of violence shown by the perpetrators, the victim usually cognitively evaluates the situation and responds in such a way, increasing or decreasing resistance, in order to overcome or survive the assault. Resistance, attribution of blame, and sexual offences committed by multiple offenders H2: There will be less attribution of blame to the participant who does not actively participate in the sexual assault and only observes the sexual assault. H3: Perceptions of victim resistance is expected to be lower in events involving more perpetrators. H4: Less perceived resistance by the victim leads to higher attribution of blame. H4: Less perceived resistance by the victim leads to higher attribution of blame. Studies on third party observer effects establish some relationship between the degree of victim resistance and victim blame. A recent study found that a victim’s attribution of blame in a hypothetical rape scenario increased for single-subject versus gang rapes, and that the blame increased as the degree of resistance decreased (Lim, 2017). Frontiers in Psychology Resistance, attribution of blame, and sexual offences committed by multiple offenders This psychological explanation serves to understand why when a woman is faced with a situation of danger to her physical and sexual integrity, she feels she must choose between survival and resistance. In the case of multiple offenders, the presence of more than one subject may deter victim resistance, reducing the need to display specific control strategies. The aim of this paper is to analyze the attribution of perceived blame in a hypothetical rape scenario, according to the different levels of resistance of the victim (verbal, physical, or both). In this study, in addition to the attribution of blame and resistance of the victim, the analysis of the role of a second aggressor who acts as an observer in the rape and does not carry out any sexual act with the victim was introduced. From a legal point of view, in Spain, the presence of a person or other persons who act in collusion with the one performing the forced sexual act is called a ‘necessary co-operator’. In these cases, the observer who is aware of the action being performed is also considered as if he or she were a direct perpetrator, even when there is no force involved, because it is considered that the intimidation exercised by such a figure weakens or annuls the victim’s will to be able to resist (Serrano, 2019). Taking into account the objectives, the following hypotheses were put forward: H1: Perpetrators are expected to be more perceived at fault when they act in groups. H1: Perpetrators are expected to be more perceived at fault when they act in groups. Most studies on attribution of blame in rape cases have typically used a scenario-based experimental method, where groups of observers read a hypothetical rape scenario with defined characteristics regarding the situation and the victim and then are asked to make judgments about these scenarios using quantitative rating scales (Sims et al., 2007; Adolfsson et al., 2020; Sjöberg and Sarwar, 2020). This approach is considered a good way to examine attribution of blame in rape offences because it minimizes the intrinsic difficulties of such studies and overcomes the associated practical, ethical, and sampling issues (Van der Bruggen and Grubb, 2014). H2: There will be less attribution of blame to the participant who does not actively participate in the sexual assault and only observes the sexual assault. Design Initially, a 2 × 2 design scenario was considered, using ‘Number of perpetrators’ (One-Two) and ‘Level of resistance’ (Low = verbal–High = physical) as independent factors. But eventually, the factor ‘origin of the invitation’ (Man–Woman) was added and crossed with ‘Level of resistance’ for the situations where there were two perpetrators, in order to assess whether the invitation of the woman affected the scenarios of sexual violence with several perpetrators. Thus six different In contrast, researchers Adolfsson et al. (2020) examined the relationship between attribution of blame and the use of force in sexual assaults involving multiple perpetrators, and found higher levels of blame for victims of sexual crimes committed by a group of men compared to rape committed by a single perpetrator. Also, previous studies have previously detected greater negative social reactions to victims of sexual 03 frontiersin.org 10.3389/fpsyg.2022.868793 10.3389/fpsyg.2022.868793 de la Torre Laso and Rodríguez-Díaz scenarios were finally studied (Annex 1), with the following scheme (Figure 1): FIGURE 1 Scenarios. the man and the woman, although this condition was only taken into account in the scenarios in which there were two men. Other studies have shown scenarios involving duos or groups of three people (Adolfsson et al., 2020). However, since hardly any significant differences are observed when considering duos and larger groups, in this study, only the condition of two perpetrators has been presented as a group aggression. Participants The study was conducted on a student population enrolled in the first 2 years of a bachelor’s degree in Criminology at the University of Salamanca. All the participants were Spanish. A total of 359 questionnaires were received from the participants, but eight were discarded because the verification questions had not been appropriately answered. Therefore, only the responses of 351 participants (290 women and 61 men) were taken into account. The average of the participants age was 20.9 years (M = 20.9; SD = 3.07; range 18–50). A five-point scale (1 = no resistance, 5 = high resistance) was used to assess the difference between the victim’s level of resistance and the level of responsibility of both the victim and the aggressor. The last question examined the degree to which the scenario could be considered a rape (To what extent do you think a rape has taken place?). Four questions were added to the questionnaires presenting a scenario involving two perpetrators (the second person with a passive role), and all questions involving the single aggressor (Oliver) were repeated to include the second aggressor (Dario), for the purpose of comparison. Questionnaire To examine the attribution of blame, the questions used in previous studies (Krahé et al., 2008; Sleath and Bull, 2010; Lim, 2017) were adapted. As a result, a 12-item questionnaire was designed where the participant had to rate their level of agreement regarding each question based on a 7-point Likert scale (1 = none; 7 = totally). There were six questions related to the victim and six about the aggressor. For the questions indicating attribution of blame (for example: How much do you think Luna is to blame for what happened?) a high score reflected greater blame on the part of the victim and less attribution of blame on the part of the aggressor. The questions also addressed the explicit consent expressed by the victim (To what extent do you think Luna gave her consent?). The internal consistency of the 12 questions used was calculated with the 351 participants and is acceptable, with Cronbach’s alpha = 0.86. scenarios were finally studied (Annex 1), with the following scheme (Figure 1): Materials A total of six vignettes or scenarios were developed, similar to those used in other research studies (e.g., Rusinko et al., 2010; Lim, 2017). The scenarios in this study depicted a hypothetical situation of a date between a woman (Luna) and a man (Oliver) after meeting through an online dating application (Appex 1). The scenarios were manipulated based on the degree of resistance shown by the victim, and the number of perpetrators. This factor was dichotomized between high or low resistance. Low resistance was considered as the woman verbally refusing to have sex and high resistance when she expressed her refusal to have sex verbally and physically. Likewise, the word rape was not shown in the text so as to avoid a possible bias in subsequent classifications, in line with what has been established in other studies (Strömwall et al., 2014). Frontiers in Psychology Questionnaire verificationi Two questionnaire verification questions were presented in this study. The first question asked about the number of men participating in the scenario and whether had been aware of what was going to occur. The second question asked whether the participant could identify with the individual who decided to go to the other person’s house. All questionnaires in which these two questions were not accurately answered were eliminated. Blame attribution The participants being invited to take part in a study on how people perceive and attribute responsibility based on an event involving a date between two young people who end up having sex. Table 1 shows the differences among the variables studied with regard to the level of blame attributed towards the victim. With respect to gender, Table 2 shows that men attribute more blame to the victim (M = 1.69; SD = 1.16) than women (M = 1.37; SD = 0.92). The questionnaire was distributed to the participants via a form constructed using Google Forms. For its completion, participants were required to provide informed consent by ticking the option: ‘Yes, I agree to participate’ at the bottom of the first page before continuing with the questions. The participants were informed that the questionnaire was completely anonymous in order to control for the effects of social bias. When analyzing the scenarios according to the number of perpetrators involved, the victim’s attribution of blame is greater in the scenarios where one perpetrator participate (M = 1.52; SD = 1.19) than those involving two perpetrators (M = 1.38; SD = 0.85). However, the difference is not significative, which does not allow us to confirm H1. Participants were randomly assigned one of the six scenarios and the distribution per scenario was as follows: 53 participants in scenario 1; 62 in scenario 2, 60 in scenario 3, 60 in scenario 4, 60 in scenario 3 and 56 in scenario 3. Were asked to read the situation they had been presented with and answer the questions. The corresponding statistical analyses were carried out using the program spss v25. Perpetrator 1 is considered the perpetrator in the one-man scenario and the one who had sex with the victim in the two-man scenario; perpetrator 2 is the one who acted as an observer in the scene of the two-man scenario. Overall, the results showed that attribution of blame was high for both perpetrators (Figure 2); however, perpetrator 2 was statistically attributed less blame than perpetrator 1 both for Men and Women (t (38) = 3.195, p = 0.003, and t (196) = 2.259, p = 0.025, respectively), confirming H2 hypothesis. Demographic information All participants were asked to indicate their age and gender. And at the end of the questionnaire, two questions were asked to determine whether the participant had ever been a victim of the scenario presented or knew someone who had experienced a similar situation. In the scenarios, the initiative to go home with someone, where the rest of the scene takes place, was differentiated between 04 frontiersin.org de la Torre Laso and Rodríguez-Díaz 10.3389/fpsyg.2022.868793 Blame attribution The participants were randomly assigned to one of the six scenarios: 53 were assigned to scenario 1 (15.1% male and 84.9% female), 62 to scenario 2 (22.6% male and 77.4% female), 60 to scenario 3 (16.7% male and 83.3% female), 60 to scenario 4 (11.7% male and 88.3% female), 60 to scenario 5 (20% male and 80% female), 56 to scenario 6 (17.9% male and 82.1% female). It can be seen that female participants attributed greater blame to the perpetrator who had sex with the woman (M = 6.75, SD = 0.87) versus the perpetrator who remained passive or perpetrator 2 (M = 6.64, SD = 0.89). In addition, female participants attributed significantly greater blame than males, (t (47.156) =2.751, p = 0.008), in the case of the perpetrator who does not have sex with the victim. We were interested in determining whether the attribution of blame could vary according to whether one had experienced a similar situation directly or indirectly. A total of 65 participants had experienced a similar situation, but only three of them were men, thus only women were considered when studying this variable. The results show that the experience of direct victimization attributed less victim blaming (M = 1.27, SD =0.91) than those who did not have that direct experience (M = 1.40, SD = 0.92), although this difference was not significative (p = 0.343). TABLE 1 Different scenarios with variables. TABLE 1 Different scenarios with variables. Scenarios A B C E1 − − − E2 − + − E3 + − − E4 + + − E5 + − + E6 + + + A = Number of aggressors: One (−), Two (+); B = Level of resistance: Low (−), High (+); C = Invitation to go home for a drink: Man (−), Woman (+). TABLE 2 Attribution of blame. However, when considering the participants who knew someone else who had experienced a similar situation (165 participants, 87.27% of whom were female), the proportions by gender were Levene’s test for equality of variances t-Test for equality of means Variable Levene’s test for equality of variances t-Test for equality of means F Sig t df Sig. Blame attribution Participant’s gender 8.690 0.003* 1.997 77 0.049* Number of perpetrators 5.23 0.023* 1.14 172 0.258 Participants who had been invited to go home with someone 0.648 0.422 0.286 0.234 0.775 Participants who had experienced a similar situation 4.60 0.033* −1.475 103 0.143 Participants who knew someone who had experienced the same situation 6.73 0.010* −1.889 287.47 0.60 Women participants who had experienced a similar situation 1.91 0.168 0.950 288 0.343 Women participants who knew someone who had experienced the same situation 3.459 0.064** 1.463 256 0.145 p < 0.05; p < 0.1. Variable 05 de la Torre Laso and Rodríguez-Díaz 10.3389/fpsyg.2022.868793 FIGURE 2 Attribution of blame according to gender. TABLE 3 Resistance levels. Variable Levene’s test for equality of variances t-Test for equality of means F Sig t df Sig. Low/high resistance 16.613 <0.001 −12.100 349 <0.001* Participant’s gender 4.654 0.032* −2.341 81 0.022* Number of perpetrators 0.205 0.651 0.891 349 0.373 Participants who had experienced a similar situation 0.386 0.535 −1.701 349 0.090 Participants who knew someone who had experienced the same situation 1.732 0.189 1.723 308 0.086 Women participants who had experienced a similar situation 0.000 0.994 2.158 288 0.032* Women participants who knew someone who had experienced the same situation 4.859 0.028* −1.863 230 0.064** p < 0.05; p < 0.1. similar to those of the participants in the study, and thus men were i resistance) show lower levels than the scenarios of high resistance i FIGURE 2 Attribution of blame according to gender. TABLE 3 Resistance levels. Variable Levene’s test for equality of variances t-Test for equality of means F Sig t df Sig. Low/high resistance 16.613 <0.001 −12.100 349 <0.001* Participant’s gender 4.654 0.032* −2.341 81 0.022* Number of perpetrators 0.205 0.651 0.891 349 0.373 Participants who had experienced a similar situation 0.386 0.535 −1.701 349 0.090 Participants who knew someone who had experienced the same situation 1.732 0.189 1.723 308 0.086 Women participants who had experienced a similar situation 0.000 0.994 2.158 288 0.032* Women participants who knew someone who had experienced the same situation 4.859 0.028* −1.863 230 0.064** p < 0.05; p < 0.1. FIGURE 2 Attribution of blame according to gender. TABLE 3 Resistance levels. Variable Levene’s test for equality of variances t-Test for equality of means F Sig t df Sig. Low/high resistance 16.613 <0.001 −12.100 349 <0.001* Participant’s gender 4.654 0.032* −2.341 81 0.022* Number of perpetrators 0.205 0.651 0.891 349 0.373 Participants who had experienced a similar situation 0.386 0.535 −1.701 349 0.090 Participants who knew someone who had experienced the same situation 1.732 0.189 1.723 308 0.086 Women participants who had experienced a similar situation 0.000 0.994 2.158 288 0.032* Women participants who knew someone who had experienced the same situation 4.859 0.028* −1.863 230 0.064** p < 0.05; *p < 0.1. resistance) show lower levels than the scenarios of high resistance (physical and verbal resistance), confirming that this variable had been adequately measured. Variable Thus, there were significant differences between the scenarios where the resistance was verbal (M = 3.34; SD = 0.95) compared to the scenarios where the victim expressed physical and verbal opposition (M = 4.41; SD =0 0.701).h similar to those of the participants in the study, and thus men were not removed for the study. In this case the results show a significative difference at the 10% significance level (p = 0.06) between the perception of blame of those who know someone who had gone through a similar situation (M = 1.33, SD = 0.91), compared to those who did not (M = 1.54, SD = 1.04). The participants’ perception of resistance was high, and women felt that the victim had shown more resistance than men F (4,654; 349) = −2.538, p < 0.05. Frontiers in Psychology Resistance levels For example, men tend to blame the victim to a greater extent than women, similar to observations found in other research (Alleyne et al., 2014). Likewise, in all the variables analyzed, there is greater variability in the opinions of men, which seems to indicate they do not have as clear an opinion as women. It is likely that differences in sex create gender bias whereby individuals identify with members of their in-group and have less favorable attitudes towards members of their out-group. In this case women are more likely to empathize more with the victims, which equates to less attribution of blame. The fourth hypothesis, which proposed the relationship between attribution of blame and victim resistance, was assessed by means of a chi-square test. After dichotomizing both variables, the contrast was significant χ2 = 9.089, p = 0.002 thus supporting the relationship between these two factors. Furthermore, the fact that male participants attribute higher levels of blame to the victims and lower levels of blame to the perpetrators, compared to female participants, may have some practical implications for young people’s perceptions of rape victims and the acceptance of rape myths (Grubb and Turner, 2012), despite the fact that some authors consider that higher educational attainment results in a lower tendency to accept rape stereotypes (Idisis et al., 2007). Further studies could include examining whether other personal characteristics influence participants’ perceptions such as age, criminal history, or relationship to the victim, or whether socio-cultural factors may contribute to the assessment of blameworthiness, as Gravelin et al. (2019) argue. Both male and female participants gave high scores with regard to whether the scenarios could be considered as rape (men: M = 6.25; women M = 6.57), with significative differences between them (t (349) = −2.289, p = 0.023). However, when this variable is analyzed in the context of the participant having suffered a similar situation or knowing someone who had, no significative differences were detected (p = 0.115 and p = 0.649, respectively). As for the scenarios in which two perpetrators participate, the levels of blame are high for both. However, the participants (both men and women) attribute greater blame to the aggressor who has physical contact with the victim as opposed to the one who does not or remains in a passive role, confirming H2. Resistance levels In general, women attribute higher levels of resistance towards the victim (M = 3.94, SD = 0.96) than men (M = 3.59, SD = 1.09). Conversely, no significant differences were observed between the Table 3 shows the victim’s levels of resistance observed according to the variables analyzed. The scenarios of low resistance (or verbal 06 Frontiers in Psychology frontiersin.org de la Torre Laso and Rodríguez-Díaz 10.3389/fpsyg.2022.868793 scenarios involving 1 perpetrator (M = 3.95; SD = 0.99) or 2 (M = 3.85; SD = 0.99), thus H3 cannot be checked. Lim's (2017) research, but contrary to other similar research (Woodhams and Cooke, 2013; Adolfsson et al., 2020). Observers may perceive that responsibility is blurred in groups (Adolfsson and Strömwall, 2017). Upon analyzing whether the participants had had a similar experience, the score for attribution of resistance was lower (M = 3.69; SD = 0.92) than that for participants who had not experienced the same scenario (M = 3.92; SD = 1.00), p < 0.1. By contrast, victim resistance was scored higher (M = 3.94; SD =0.98) when the participant knew of someone who had experienced a similar situation than when they did not (M = 3.74; SD = 1.01), as well significative at the 10% significance level.i Also, the perpetrators’ attribution of blame was much higher than that of the victim, as other research has found (e.g., Strömwall et al., 2014; Sjöberg and Sarwar, 2020). These results show that in rape cases the attribution of blame is polarized towards the perpetrators, thus emphasizing the importance of continuing such measurements in future research. In a similar manner, we examined whether significant differences existed regarding the perception of victim resistance among women participants based on whether they had themselves experienced the same scenario or knew of someone who had. In these cases, the women who had first-hand experiences attributed a lower level of resistance to the victim (M = 3.71, SD = 0.91) than those who knew of someone who had had a similar experience (M = 4.01, SD = 0.91). However, less resistance was perceived among the women who had been directly victimized than those who had not personally experienced a similar situation, p < 0.05. The individual characteristics of the participants (those filling in the questionnaire) explain much of blame attribution. Resistance levels As for this type of perpetrator, women tend to blame them more than men, as shown by the significant result. Another finding of this work is the high level of blame attributed to the perpetrator who does not act physically against the victim, which indicates that he/she is also perceived as a perpetrator. On the other hand, the lower level of blame perceived regarding the aggressor who does not exert physical contact with the victim may give a false sense of lower criminal responsibility. In Spain this aggressor could be considered as a perpetrator of the crime by being treated as an accomplice or “necessary co-operator” (Barrajón, 2019), or may be attributed a crime of omission of the duty to assist (Faraldo Cabana, 2020).h Frontiers in Psychology frontiersin.org Discussion The aim of this study was to examine the relationship between attribution of blame towards the perpetrators and towards the victim, within fictional rape scenarios, based on the level victim resistance, the degree of perpetrator involvement, and the origin of the invitation to go to a more private place. In general, the data obtained support most of the hypotheses put forward. As expected, more blame is attributed to the perpetrators when they act in a group (in this case, as a duo), as opposed to when a man acts alone; thus, supporting H1 although the difference is not significative. This conclusion has been made previously in studies highlighting the importance of the presence of more than one perpetrator in attributing responsibility for sexual violence (Woodhams and Cooke, 2013; Lim, 2017; Adolfsson et al., 2020). Contrary to expectations, more blame was attributed to the perpetrator acting alone, as opposed to perpetrators acting in a group (in this case, as a duo), in line with The direct or indirect experience of victimization was found to be influential in attributing less blame towards the victim. Those who had directly or indirectly experienced a similar situation attributed 07 frontiersin.org de la Torre Laso and Rodríguez-Díaz 10.3389/fpsyg.2022.868793 less blame to the victim than those who had not experienced it or did not know anyone who had been raped. It is plausible that direct victimization produces an empathy effect towards the victim and that indirect experience also influences perceptions, confirming the theory of the discrepancy that exists between the supposed “objective risk” of being a victim and the “subjective perceptions” of being a victim (Villalba Olivella, 2017). behavior, in terms of norms derived from male/female stereotypes, can help to understand the tolerance of sexual violence against women. Although the scenarios did not include the word “rape,” the participants considered the events described as rape, especially among women who had experienced a similar situation. This outcome indicates that a non-consensual sexual experience is associated with an act of aggression. As for the levels of resistance, although the scenarios showed levels far from low or high resistance, the average score of the victim in the scenarios selected as low resistance (3.34 on a scale of 5) were higher than expected. Limitations There are a few limitations to this study. First, the study did not have a culturally diverse sample due to its location, and the applicability of these results is unclear. Cultural and regional differences are an important factor to be examined in future research to better generalize the results. This study was carried out on the basis of the questionnaires filled in by a sample of university students who are supposed to have a high cultural level. Therefore, more studies should be carried out using participants with different cultural levels, and over a wider age range. In addition, the younger population seems to be sensitized to the subject, owing to the fact that in recent years in Spain there have been different incidents of sexual aggressions committed in groups. While attribution of blame and perception of resistance are complex concepts with a social component that are constructed according to the cultural roles within a given period, the results may lack variability. In this study, no distinction was made with regard to how blame is understood, and resistance was analyzed in terms of verbal and physical rejection. Thus, perhaps the distinction between low and high resistance has not been sufficient. Likewise, the simple account of blame attribution laid out in each scenario did not provide additional details that may have influenced how the event was perceived such as the absence of alcohol or the relationship between the aggressor and the victim. Consequently, providing a more complete description may have allowed other conclusions to be drawn. No significative differences in the level of resistance were observed for scenarios in which one or two perpetrators acted, contrary to research that has reported that larger groups are associated with lower resistance (Woodhams and Cooke, 2013). The reason may be that the scenarios used for the groups are based on a small group of two perpetrators. Therefore, it would be necessary to confirm this relationship using larger perpetrator groups. The hypothesis that predicted lower victim resistance leads to higher attribution of blame has been confirmed, a finding that is in line with previous research (Black and McCloskey, 2013; Lim, 2017). Therefore, it could be concluded that the victim’s level of resistance may influence the victim’s attribution of blame. Limitations In addition, direct experience of victimization is key to perceiving lower victim resistance, maybe feeling by own experience that the victim could have exerted more resistance than that shown in the scenery. Discussion One explanation may be that the participants, mostly young people, are very aware of the rejection that a rape victim may experience and consider a verbal expression as a sign of lack of consent and, therefore, of resistance. Likewise, social movements such as #MeToo (Dio Bleichmar, 2018) or initiatives to change what is considered consent in sexual relationships (Bertolín- Guillén, 2021) may contribute to young people’s greater awareness of roles in these situations. Females attributed higher levels of resistance to the victim than males, confirming the different perceptions that males have of females in terms of signs or messages expressing explicit agreement or refusal in sexual relationships (Burkett and Hamilton, 2012; Jozkowski et al., 2018). Further research could confirm whether certain messages (e.g., accepting an invitation to go home with someone) are perceived by men and women differently. It could also be investigated whether the lack of resistance corresponds to the so-called tonic immobility (Gbahabo and Duma, 2021), which explains this lack of reaction in the victim.if Frontiers in Psychology References Abrams, D., Viki, G. T., Masser, B., and Bohner, G. (2003). Perceptions of stranger and acquaintance rape: the role of benevolent and hostile sexism in victim blame and rape proclivity. J. Pers. Soc. Psychol. 84, 111–125. doi: 10.1037/0022-3514.84.1.111 Abrams, D., Viki, G. T., Masser, B., and Bohner, G. (2003). Perceptions of stranger and acquaintance rape: the role of benevolent and hostile sexism in victim blame and rape proclivity. J. Pers. Soc. Psychol. 84, 111–125. doi: 10.1037/0022-3514.84.1.111 Ayala, E. E., Kotary, B., and Hetz, M. (2018). Blame attributions of victims and perpetrators: effects of victim gender, perpetrator gender, and relationship. J. Interpers. Violence 33, 94–116. doi: 10.1177/0886260515599160 Barrajón, N. P. (2019). Estudio temas jurídicos y doctrinales de la Sentencia No 344/2019 del caso de «La Manada», comparativa entre la Sentencia de la Audiencia Provincial y la del Tribunal Supremo. Diario La Ley 9497:2 Adolfsson, K. and Strömwall, L. A. (2017). Situational variables or beliefs? A multifaceted approach to understanding blame attributions. Psychol, Crime Law 23, 527–552. doi: 10.1080/1068316X.2017. 1290236 Bertolín-Guillén, J. M. (2021). El consentimiento sexual de los menores de edad en españa: consideraciones clínicas y jurisprudenciales. Revista Internacional de Doctrina y Jurisprudencia 24, 1–14. doi: 10.25115/ridj. v0i24.5372 Adolfsson, K., Strömwall, L. A., and Landström, S. (2020). Blame attributions in multiple perpetrator rape cases: the impact of sympathy, consent, force, and beliefs. J. Interpers. Violence 35, 5336–5364. doi: 10.1177/ 0886260517721171 Bijleveld, C., and Hendriks, J. (2003). Juvenile sex offenders: differences between group and solo offenders. Psychol. Crime Law 9, 237–245. doi: 10.1080/1068316021000030568 Bijleveld, C., and Hendriks, J. (2003). Juvenile sex offenders: differences between group and solo offenders. Psychol. Crime Law 9, 237–245. doi: 10.1080/1068316021000030568 Alicke, M. D. (2000). Culpable control and the psychology of blame. Psychol. Bull. 126, 556–574. doi: 10.1037/0033-2909.126.4.556 Black, K. A., and McCloskey, K. A. (2013). Predicting date rape perceptions: the effects of gender, gender role attitudes, and victim resistance. Violence Against Women 19, 949–967. doi: 10.1177/1077801213499244 Black, K. A., and McCloskey, K. A. (2013). Predicting date rape perceptions: the effects of gender, gender role attitudes, and victim resistance. Violence Against Women 19, 949–967. doi: 10.1177/1077801213499244 Alleyne, E., Gannon, T. A., Ciardha, C. Ó., and Wood, J. L. (2014). Community males show multiple-perpetrator rape proclivity: development and preliminary validation of an interest scale. Sex. Abus. 26, 82–104. doi: 10.1177/1079063213480819 Burkett, M., and Hamilton, K. (2012). Postfeminist sexual agency: young women’s negotiations of sexual consent. Sexualities 15, 815–833. Data availability statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding author. Funding The study has shown that there is an influence between levels of victim resistance and attribution of blame. Lower resistance is associated with greater blame. Currently, the literature is in line with these results, so it is necessary to design information and education strategies to have a better understanding of sexual assault. This research was supported by the Spanish Ministry of Science and Innovation and Junta de Castilla y León (Projects, PID2021-125211OB-I00’ and ‘SA105P20’ respectively). Supplementary material JT contributed to developing the theoretical framework, data analysis, data collection, and the writing and overall writing of the manuscript. JR-D contributed to writing and data analysis of the The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpsyg.2022.868793/ full#supplementary-material Conclusion This study suggests that the blame attributed to victims and perpetrators in a rape scenario varies according to the gender of the participant filling in the questionnaire and the level of perpetrator involvement. Overall, participants attributed less blame to victims and more blame to perpetrators. Female participants attributed lower victim- blaming and higher perpetrator-blaming, regardless of their levels of involvement. However, men attributed different levels of blame between the perpetrators, with less blame being attributed to the passive perpetrator. On the other hand, the direct or indirect experience of having experienced In the same way, the participants considered that the victim could not have avoided the incident, despite her resistance, a finding that confirms the belief in the inevitability of rape and how it contributes to the acceptance of rape myths (Burt, 1980). Likewise, considering that the victim could not have avoided the incident has consequences for the victims, as the severity of the emotional impact is related to the symptoms experienced (Sarasua et al., 2012; Echeburúa et al., 2013). Intervention through education and the elimination of victim stereotypes can contribute to preventing sexual violence. The links established between aggression against women and socially approved patterns of 08 frontiersin.org de la Torre Laso and Rodríguez-Díaz 10.3389/fpsyg.2022.868793 manuscript. JT and JR-D contributed to editing and organization of the manuscript. All authors contributed to the article and approved the submitted version. a similar situation contributes to less blame being placed on the victim. In addition, this study suggests that levels of resistance also vary according to the gender of the participant, with women perceiving the victim exerted more resistance. Ethics statement 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. Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. The patients/ participants provided their written informed consent to participate in this study. References R., and Harrower, J. (2009). Understanding attribution of blame in cases of rape: an analysis of participant gender, type of rape and perceived similarity to the victim. J. Sex. Aggress. 15, 63–81. doi: 10.1080/13552600802641649 Rogers, P., Titterington, L., and Davies, M. (2009). Attributions of blame and credibility in a hypothetical child sexual abuse case: roles of victim disability, victim resistance and respondent gender. Int. J. Disabil. Dev. Educ. 56, 205–228. doi: 10.1080/10349120903102189 Grubb, A., and Turner, E. (2012). Attribution of blame in rape cases: a review of the impact of rape myth acceptance, gender role conformity and substance use on victim blaming. Aggress. Violent Behav. 17, 443–452. doi: 10.1016/j. avb.2012.06.002 Rusinko, H. M., Bradley, A. R., and Miller, J. (2010). Assertiveness and attributions of blame toward victims of sexual assault. J. Aggress. Maltreat. Trauma 19, 357–371. doi: 10.1080/10926771003788961 Gutiérrez de Piñeres Botero, C., Coronel, E., and Andrés Pérez, C. (2009). Revisión teórica del concepto de victimización secundaria. Liberabit 15, 49–58. Russell, K. J., and Hand, C. J. (2017). Rape myth acceptance, victim blame attribution and just world beliefs: a rapid evidence assessment. Aggress. Violent Behav. 37, 153–160. doi: 10.1016/j.avb.2017.10.008 Harkins, L., and Dixon, L. (2013). “A multi-Factorial Approach to Understanding multiple Perpetrator sexual Offending,” in Crime and Crime Reduction. eds. J. L. Wood and T. A. Gannon (East Sussex, UK: Routledge), 75–95. Sarasua, B., Zubizarreta, I., de Corral, P., and Echeburúa, E. (2012). Factores de vulnerabilidad y de protección del impacto emocional en mujeres adultas víctimas de agresiones sexuales. Terapia psicológica 30, 7–18. doi: 10.4067/ S0718-48082012000300002 Hauffe, S., and Porter, L. (2009). An interpersonal comparison of lone and group rape offences. Psychol. Crime Law 15, 469–491. doi: 10.1080/10683160802409339 Horvath, M. (2006). Drug-Assisted rape: An Investigation Unpublished PhD University of Surrey. Serrano, A. (2019). Patrones y procesos delictivos. La naturaleza y características del delito en la sociedad contemporáne. Madrid: Dyckinson. Horvath, M. A. H., and Kelly, L. (2009). Multiple perpetrator rape: naming an offence and initial research findings. J. Sex. Aggress. 15, 83–96. doi: 10.1080/13552600802653818 Setia, A., Marks, M., and An, S. (2020). Double standards in perceived traits of women labeled victims versus survivors. Sexuality Culture 24, 1562–1578. doi: 10.1007/s12119-020-09712-w Idisis, Y., Ben-David, S., and Ben-Nachum, E. (2007). Attribution of blame to rape victims among therapists and non-therapists. Behav. Sci. Law 25, 103–120. doi: 10.1002/bsl.721 Shaver, K. G. (1985). The Attribution of Blame: Causality, Responsibility, and Blameworthiness. New York: Springer. References Medical care following multiple perpetrator sexual assault: a retrospective review. Int. J. STD AIDS 26, 86–92. doi: 10.1177/0956462414530886 Echeburúa, E., Sarasua, B., Zubizarreta, I., and de Corral, P. (2013). Tratamiento psicológico de mujeres víctimas de agresiones sexuales recientes y no recientes en la vida adulta. Psicol. Conduct. 21:249. Park, J., and Kim, S. (2016). Group size does matter: differences among sexual assaults committed by lone, double, and groups of three or more perpetrators. J. Sex. Aggress. 22, 342–354. doi: 10.1080/13552600.2016. 1144801 Faraldo Cabana, P. (2020). La intervención de dos o más personas en las agresiones sexuales. Estado de la cuestión. Revista de Derecho Penal y Criminología 381, 381–420. doi: 10.5944/rdpc.22.2019.26493 Franklin, K. (2004). Enacting masculinity: antigay violence and group rape as participatory theater. Sex. Res. Soc. Policy 1, 25–40. doi: 10.1525/ srsp.2004.1.2.25 Persson, S., and Dhingra, K. (2020). Attributions of blame in stranger and acquaintance rape: a multilevel meta-analysis and systematic review. Trauma Violence Abuse 23, 795–809. doi: 10.1177/1524838020977146 Gbahabo, D. D., and Duma, S. E. (2021). « I just became like a log of wood… I was paralyzed all over my body»: Women’s lived experiences of tonic immobility following rape. Heliyon 7:e07471. doi: 10.1016/j.heliyon.2021.e07471 Porter, L. E., and Alison, L. J. (2006). Examining group rape: a descriptive analysis of offender and victim behaviour. Eur. J. Criminol. 3, 357–381. doi: 10.1177/1477370806065586 Gerber, G. L., Cronin, J. M., and Steigman, H. J. (2004). Attributions of blame in sexual assault to perpetrators and victims of Both genders 1. J. Appl. Soc. Psychol. 34, 2149–2165. doi: 10.1111/j.1559-1816.2004.tb02694.x Quarshie, E. N.-B., Davies, P. A., Badasu, M. I. A., Tagoe, T., Otoo, P. A., and Afriyie, P. O. (2018). Multiple perpetrator rape in Ghana: offenders, victims and offence characteristics. J. Sex. Aggress. 24, 125–141. doi: 10.1080/13552600. 2017.1378024 Gerger, H., Kley, H., Bohner, G., and Siebler, F. (2013). Acceptance of Modern Myths About Sexual Aggression (AMMSA) scale. Measurement Instrument Database for the Social Science. Randall, M. (2010). Sexual assault law, credibility, and “ideal victims”: consent, resistance, and victim blaming. Can. J. Women Law 22, 397–433. doi: 10.3138/ cjwl.22.2.397 Gravelin, C. R., Biernat, M., and Bucher, C. E. (2019). Blaming the victim of acquaintance rape: Individual, situational, and sociocultural factors. Frontiers Psychol. 9:2422. doi: 10.3389/fpsyg.2018.02422 Richardson, D., and Campbell, J. L. (1982). Alcohol and rape: the effect of alcohol on attributions of blame for rape. Personal. Soc. Psychol. Bull. 8, 468–476. doi: 10.1177/0146167282083013 Grubb, A. References doi: 10.1177/1363460712454076 Artz, L., and Kunisaki, K. (2003). “Rape During Armed Conflict and Reflections on the “Uncivil War” on Women in South Africa,” in Paper Presented the Conference of the South Arican. Cape Town, South Africa: Institute for Justice and Reconciliation. Burt, M. R. (1980). Cultural myths and supports for rape. J. Pers. Soc. Psychol. 38, 217–230. doi: 10.1037/0022-3514.38.2.217 Burt, M. R. (1980). Cultural myths and supports for rape. J. Pers. Soc. Psychol. 38, 217–230. doi: 10.1037/0022-3514.38.2.217 09 Frontiers in Psychology frontiersin.org frontiersin.org de la Torre Laso and Rodríguez-Díaz 10.3389/fpsyg.2022.868793 10.3389/fpsyg.2022.868793 10.3389/fpsyg.2022.868793 Chambers, J. C., Horvath, M. A., and Kelly, L. (2010). A typology of multiple-perpetrator rape. Crim. Justice Behav. 37, 1114–1139. doi: 10.1177/0093854810377971 Lim, Y. J. G. (2017). Multiple perpetrator sexual assault: the relationship between the number of perpetrators, blame attribution, and victim resistance. Thesis Presented in Partial Fulfillment of the Requirements for the Masters in Forensic Mental Health Counseling John Jay College of Criminal Justice City University of New York. Available at: https://academicworks.cuny.edu/cgi/viewcontent. cgi?article=1051&context=jj etds da Silva, T., Woodhams, J., and Harkins, L. (2015). Multiple perpetrator rape: a critical review of existing explanatory theories. Aggress. Violent Behav. 25, 150–158. doi: 10.1016/j.avb.2015.07.017 Ministerio de Igualdad. (2019). Macroencuesta de Violencia contra la Mujer. Gobierno de España. Ministerio de Igualdad. Secretaría de Estado de Igualdad y contra la Violencia de Género. Delegación del Gobierno contra la Violencia de Género. Datta, M. N., and Bales, K. (2013). Slavery in Europe: part 1, estimating the dark figure. Hum. Rts. Q. 35, 817–829. doi: 10.1353/hrq.2013.0051 Davies, M., Rogers, P., and Bates, J.-A. (2008). Blame toward male rape victims in a hypothetical sexual assault as a function of victim sexuality and degree of resistance. J. Homosex. 55, 533–544. doi: 10.1080/00918360802345339 Möller, A., Söndergaard, H. P., and Helström, L. (2017). Tonic immobility during sexual assault–a common reaction predicting post-traumatic stress disorder and severe depression. Acta Obstet. Gynecol. Scand. 96, 932–938. doi: 10.1111/ aogs.13174 Dio Bleichmar, E. (2018). Cuando las gotas forman un torrente. El movimiento# MeToo. Aperturas Psicoanalíticas 57. Morgan, L., Brittain, B., and Welch, J. (2012). Multiple perpetrator sexual assault: how does it differ From assault by a single perpetrator? J. Interpers. Violence 27, 2415–2436. doi: 10.1177/0886260511433514 Drury, A. J., Elbert, M. J., and DeLisi, M. (2020). The dark figure of sexual offending: a replication and extension. Behav. Sci. Law 38, 559–570. doi: 10.1002/ bsl.2488 Morgan, L., Brittain, B., and Welch, J. (2015). References Shotland, R. L., and Goodstein, L. (1983). Just because she doesn’t want to doesn’t mean it’s rape: an experimentally based causal model of the perception of rape in a dating situation. Soc. Psychol. Q. 46, 220–232. doi: 10.2307/ 3033793 Jimenez, J. A., and Abreu, J. M. (2003). Race and sex effects on attitudinal perceptions of acquaintance rape. J. Couns. Psychol. 50, 252–256. doi: 10.1037/0022-0167.50.2.252 Jozkowski, K. N., Manning, J., and Hunt, M. (2018). Sexual consent in and out of the bedroom: disjunctive views of heterosexual college students. Women's Stud. Commun. 41, 117–139. doi: 10.1080/07491409.2018.1470121 Sims, C. M., Noel, N. E., and Maisto, S. A. (2007). Rape blame as a function of alcohol presence and resistance type. Addict. Behav. 32, 2766–2775. doi: 10.1016/j. addbeh.2007.04.013 Kline, N. K., Berke, D. S., Rhodes, C. A., Steenkamp, M. M., and Litz, B. T. (2021). Self-blame and PTSD following sexual assault: a longitudinal analysis. J. Interpers. Violence 36, NP3153–NP3168. doi: 10.1177/0886260518770652 Sjöberg, M., and Sarwar, F. (2020). Who gets blamed for rapes: effects of immigration status on the attribution of blame toward victims and perpetrators. J. Interpers. Violence 35, 2446–2463. doi: 10.1177/0886260517703371 Krahé, B., Temkin, J., Bieneck, S., and Berger, A. (2008). Prospective lawyers’ rape stereotypes and schematic decision making about rape cases. Psychol. Crime Law 14, 461–479. doi: 10.1080/10683160801932380 Sleath, E., and Bull, R. (2010). Male rape victim and perpetrator blaming. J. Interpers. Violence 25, 969–988. doi: 10.1177/0886260509340534 Sleath, E., and Bull, R. (2010). Male rape victim and perpetrator blaming. J. Interpers. Violence 25, 969–988. doi: 10.1177/0886260509340534 10 Frontiers in Psychology frontiersin.org frontiersin.org de la Torre Laso and Rodríguez-Díaz 10.3389/fpsyg.2022.868793 Villalba Olivella, J. (2017). Relación entre variables en el miedo al delito. RDUNED: revista de derecho UNED 20, 657–689. doi: 10.5944/ rduned.20.2017.19482 Strömwall, L. A., Alfredsson, H., and Landström, S. (2013). Blame attributions and rape: effects of belief in a just world and relationship level. Leg. Criminol. Psychol. 18, 254–261. doi: 10.1111/j.2044-8333.2012. 02044.x Villalba Olivella, J. (2017). Relación entre variables en el miedo al delito. RDUNED: revista de derecho UNED 20, 657–689. doi: 10.5944/ rduned.20.2017.19482 Wignall, L., Stirling, J., and Scoats, R. (2020). UK University students’ perceptions and negotiations of sexual consent. Psychol. Sexuality 13, 474–486. doi: 10.1080/19419899.2020.1859601 Wignall, L., Stirling, J., and Scoats, R. (2020). UK University students’ perceptions and negotiations of sexual consent. Psychol. Sexuality 13, 474–486. doi: 10.1080/19419899.2020.1859601 Strömwall, L. A., Landström, S., and Alfredsson, H. (2014). References Perpetrator characteristics and blame attributions in a stranger rape situation. Eur. J. Psychol. Appl. Legal Context 6, 63–67. doi: 10.1016/j.ejpal.2014.06.002 Wijkman, M., Bijleveld, C., and Hendriks, J. (2014). Juvenile female sex offenders: offender and offence characteristics. Eur. J. Criminol. 11, 23–38. doi: 10.1177/1477370813479077 Ullman, S. E. (1996). Correlates and consequences of adult sexual assault disclosure. J. Interpers. Violence 11, 554–571. doi: 10.1177/0886260960110 04007 Woodhams, J., and Cooke, C. (2013). Suspect aggression and victim resistance in multiple perpetrator rapes. Arch. Sex. Behav. 42, 1509–1516. doi: 10.1007/ s10508-013-0136-7 Ullman, S. E. (2007a). A 10-year update of “review and critique of empirical studies of rape avoidance”. Crim. Justice Behav. 34, 411–429. doi: 10.1177/0093854806297117 Ullman, S. E. (2007b). Comparing gang and individual rapes in a community sample of urban women. Violence Vict. 22, 43–51. doi: 10.1891/ vv-v22i1a003 Woodhams, J., Gillett, R., and Grant, T. (2007). Understanding the factors that affect the severity of juvenile stranger sex offenses: the effect of victim characteristics and number of suspects. J. Interpers. Violence 22, 218–237. doi: 10.1177/0886260506295349 Van der Bruggen, M., and Grubb, A. (2014). A review of the literature relating to rape victim blaming: an analysis of the impact of observer and victim characteristics on attribution of blame in rape cases. Aggress. Violent Behav. 19, 523–531. doi: 10.1016/j.avb.2014.07.008 Wright, R., and West, D. J. (1981). Rape—a comparison of group offences and lone assaults. Med. Sci. Law 21, 25–30. doi: 10.1177/002580248102100106 Wright, R., and West, D. J. (1981). Rape—a comparison of group offences and lone assaults. Med. Sci. Law 21, 25–30. doi: 10.1177/002580248102100106 Frontiers in Psychology 11 frontiersin.org Frontiers in Psychology
https://openalex.org/W3015640218	https://www.researchsquare.com/article/rs-15049/latest.pdf	English	null	Clinical Characteristics and Predictors of Mortality in Critically Ill Influenza Adult Patients	Journal of clinical medicine	2,020	cc-by	7,292	Clinical characteristics and predictors of mortality in critically ill influenza adult patients Jui-Chi Hsu Chang Gung Memorial Hospital Kaohsiung Branch Ing-Kit Lee Jui-Chi Hsu Chang Gung Memorial Hospital Kaohsiung Branch Ing-Kit Lee Chang Gung Memorial Hospital Kaohsiung Branch Wen-Chi Huang Chang Gung Memorial Hospital Kaohsiung Branch Yi-Chun Chen Chang Gung Memorial Hospital Kaohsiung Branch Ching-Yen Tsai Chang Gung Memorial Hospital Kaohsiung Branch Chang Gung Memorial Hospital Kaohsiung Branch Wen-Chi Huang Chang Gung Memorial Hospital Kaohsiung Branch Yi-Chun Chen Chang Gung Memorial Hospital Kaohsiung Branch Ching-Yen Tsai Chang Gung Memorial Hospital Kaohsiung Branch Chang Gung Memorial Hospital Kaohsiung Branch Wen-Chi Huang Chang Gung Memorial Hospital Kaohsiung Branch Yi-Chun Chen Chang Gung Memorial Hospital Kaohsiung Branch Ching-Yen Tsai Chang Gung Memorial Hospital Kaohsiung Branch Research Research Keywords: Severe influenza, Pneumonia, Acute respiratory distress syndrome, Lactate, Mortality Posted Date: February 25th, 2020 DOI: https://doi.org/10.21203/rs.2.24490/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at Journal of Clinical Medicine on April 9th, 2020. See the published version at https://doi.org/10.3390/jcm9041073. Page 1/21 Abstract Background Severe influenza is associated with high morbidity and mortality. The aim of this study was to investigate the factors affecting the clinical outcomes of critically ill influenza patients. Methods In this retrospective study, we enrolled critically ill adult patients with influenza at the Kaohsiung Chang Gung Memorial Hospital in Taiwan. We evaluated the demographic, clinical, and laboratory findings and examined whether any of these measurements correlated with mortality. We then created an event-based algorithm as a simple predictive tool using 2 variables with statistically significant associations with mortality. Results Between 2015 and 2018, 102 critically ill influenza patients (median age, 62 years) were assessed; among them, 41 (40.1%) patients died. Of the 94 patients who received oseltamivir therapy, 68 (72.3%) began taking oseltamivir 48 hours after the onset of illness. Of the 102 patients, the major influenza-associated complications were respiratory failure (97%), pneumonia (94.1%), acute kidney injury (65.7%), adult respiratory distress syndrome (ARDS) (51%), gastrointestinal bleeding (35.3%), and bacteremia (16.7%). In the multivariate regression model, high lactate levels, ARDS, acute kidney injury, and gastrointestinal bleeding were independent predictors of mortality in critically ill influenza patients. The optimal lactate level cutoff for predicting mortality was 33 mg/dL with an area under curve of 0.728. We constructed an event-associated algorithm that included lactate and ARDS. Fifteen (75%) of 20 patients with lactate levels ≥33 mg/dL and ARDS died, compared with only 1 (7.7%) of 13 patients with normal lactate levels and without ARDS. Conclusions We identified clinical and laboratory predictors of mortality at hospital admission that could aid in the care of critically ill influenza patients. Identification of these prognostic markers could be improved to prioritize key examinations that might be useful in determining patient outcomes. Study Design and Patients We retrospectively studied all critically ill adult patients (aged ≥ 18 years) with laboratory-confirmed influenza infection admitted between 2015 and 2018, at the KSCGMH, a 2,700-bed primary care and tertiary referral medical center in Taiwan. Children (< 18 years old), those with mild influenza-like symptoms (fever, cough, or sore throat), and those who recovered fully without any complications were excluded from the analysis. Confirmation of influenza virus infection required a positive finding in the respiratory specimen (nasopharyngeal swab and/or pharyngeal swab) by one or more of the following methods: rapid influenza diagnostic test, isolation of the virus in tissue-cell culture, or reverse- transcriptase–polymerase chain reaction (RT-PCR) (QiAamp Viral RNA Mini Kit; TAIGEN Bioscience Corporation, Taiwan). Background Influenza is an acute viral respiratory infection caused by different types of influenza viruses: influenza A, B, and C [1]. Influenza A subtypes H1N1, H3N2, and influenza B are the most common causes of human influenza [1]. The illness is usually mild and characterized by a sudden onset of fever, cough, sore throat, runny nose, headache, myalgia, and malaise [1]. However, the virus can cause severe illness or even death, especially in high-risk individuals such as young children, the elderly, patients with certain comorbid chronic diseases, and immunocompromised patients [2, 3]. Annually, the World Health Organization estimated that global influenza epidemics result in 3 to 5 million cases of severe illness and 290,000 to 650,000 deaths [4, 5]. In 2009, a swine-origin influenza A (pandemic 2009 A/H1N1; pdm09 A/H1) emerged and rapidly caused a global pandemic [6]. Remarkably, from April 12, 2009 to April 10, 2010, there were 60.8 million cases and 12,469 deaths in the United States due to pdm09 A/H1 [7]. Severe complicated influenza has a significantly high mortality and morbidity [8, 9]. Secondary bacterial pneumonia and acute respiratory distress syndrome (ARDS) are some of the common pulmonary Page 2/21 Page 2/21 complications of influenza, often followed by grave outcomes [10]. In addition to pulmonary complications, extra-pulmonary complications such as meningoencephalitis, myocarditis, and rhabdomyolysis have also been reported to be associated with either influenza A or B [11, 12, 13]. While early antiviral therapy may reduce complications of influenza [14, 15, 16], the majority of patients tend to delay seeking medical care and timely diagnosis, leading to the subsequent development of influenza- associated complications, particularly in the elderly and those with comorbid illnesses. Hence, key clinical data are crucial pieces of information that can help clinicians deliver the necessary management to critically ill influenza patients in a timely manner. In the present study, we reviewed the entire clinical course and laboratory data of critically ill patients with influenza and then explored the predictors of death at hospital admission. Data Collection and Definitions A standardized form for clinical data collection was designed. The data were mainly retrieved from the hospital’s electronic medical records and were supplemented by a secondary manual search. The following data were collected: demographic characteristics, underlying medical conditions, clinical signs and symptoms, antiviral treatment course (oseltamivir or peramivir therapy), results of laboratory tests and radiography findings at the time of presentation and during the entire clinical course, in-hospital complications, and fatality. Critically ill influenza patients were defined as those admitted to an intensive care unit, those who developed acute hypoxemic respiratory failure (arterial partial pressure of oxygen [PaO2] < 60 mmHg, fraction of inspired oxygen [FIO2] ≥ 60%, or tachypnea > 30/min) or complications of lower respiratory tract infection and/or multi-organ failure, those requiring mechanical ventilation, or those receiving intravenous infusion of inotropic or vasopressor medication during hospitalization. ARDS was defined as acute respiratory distress characterized by bilateral pulmonary consolidation and severe hypoxemia (PaO2/FiO2 ratio < 300 mm Hg with positive end-expiratory pressure ≥ 5 cm H2O) in the absence of Page 3/21 Page 3/21 Page 3/21 evidence for cardiogenic pulmonary edema [17]. Fulminant hepatitis meant alanine aminotransferase levels (ALT) greater than 1000 U/L. Acute kidney injury was defined as a rapid increase in the serum creatinine level to > 0.5 mg/dL compared with that at presentation. Rhabdomyolysis was defined as a five-fold increase in the serum concentrations of creatine phosphokinase above the upper limit of the normal range (reference value, 13–130 U/L), with > 95% creatine phosphokinase-muscle fraction. Meningoencephalitis was defined as an altered mental status, fulfilling at least 2 of the following criteria: 1) fever, 2) seizure, 3) focal neurologic signs, 4) abnormality of cerebrospinal fluid, 5) neuroimaging suggestive of encephalitis, and 6) abnormal findings on electroencephalography consistent with encephalitis [18]. A galactomannan cutoff optical density index of > 0.5 was used to define positivity for serum and bronchoalveolar lavage samples [19, 20]. Mortality was defined as death occurring during the hospital stay for influenza. Statistical Analysis To analyze the predictors of mortality among critically ill influenza patients, we initially compared demographic, clinical characteristics, and laboratory findings as well as complications of survivors and non-survivors using Fisher’s exact test for categorical variables, and Mann-Whitney U test for numerical variables. The differences were considered significant at P < 0.05. Significant variables in the univariate analyses were entered into a multivariate logistic regression model to identify independent predictors of mortality in critically ill influenza patients. We used receiver operating characteristic curves (ROC) to select cutoff points for independent numerical predictors according to visual assessment of the highest sensitivity and specificity. We then created an event-based algorithm as a simple predictive tool using 2 variables with statistically significant associations with mortality (P value less than or equal to 0.001) in the multivariate model. Data were entered and analyzed using the Statistical Package for the Social Sciences statistical software (version 19.0; SPSS Inc., Chicago, IL). A total of 98 (96%) patients had available RT-PCR data; among them, influenza A virus was detected in 71 (72.4%) patients (47.9% for pdm09 A/H1 and 11.3% for H3N2) and influenza B virus, in 23 (23.5%). The clinical characteristics of the included patients are summarized in Tables 1 and 2. Patient Characteristics In total, 102 critically ill patients comprising 62 men and 40 women, with a median age of 62 years (range: 24–93 years; 44 [43.1%] patients aged ≥ 65 years) with laboratory-confirmed influenza virus infection were assessed. The median time from illness onset to hospital presentation was 3 days (range: 1–14 days). Major underlying diseases found in these patients included hypertension (54.9%), type 2 diabetes mellitus (44.1%), and chronic kidney disease (15.7%). The 3 most common signs and symptoms were dyspnea (85.3%), cough (83.3%), and fever (79.4%). Among the 102 patients, 3 did not receive antiviral therapy (either oseltamivir or peramivir). Of the 99 patients who received antiviral therapy, 94 patients received oseltamivir and 10 received peramivir; 5 patients received oseltamivir and peramivir during their hospitalization. Of the 94 patients who received oseltamivir therapy, 68 (72.3%) began taking oseltamivir 48 hours after the onset of illness. Among the 10 patients who received peramivir, 60% received antiviral therapy 48 hours after the onset of symptoms. Page 4/21 Page 4/21 A total of 98 (96%) patients had available RT-PCR data; among them, influenza A virus was detected in 71 (72.4%) patients (47.9% for pdm09 A/H1 and 11.3% for H3N2) and influenza B virus, in 23 (23.5%). The clinical characteristics of the included patients are summarized in Tables 1 and 2. A total of 98 (96%) patients had available RT-PCR data; among them, influenza A virus was detected in 71 (72.4%) patients (47.9% for pdm09 A/H1 and 11.3% for H3N2) and influenza B virus, in 23 (23.5%). The clinical characteristics of the included patients are summarized in Tables 1 and 2. Patient Characteristics Page 5/21 Table 1 Characteristics and diagnostic methods of patients with severe influenza Variable Overall (n = 102) Survivors (n = 61) Non-survivors (n = 41) P value Demographic and clinical features Age, years, median (range) 62 (24–93) 65 (24–92) 61 (24–93) 0.186 Age group, N (%) 0.068 20–49 year 20 (19.6) 11 (18) 9 (22) 50–64 38 (37.3) 19 (31.1) 19 (46.3) ≧ 65 year 44 (43.1) 31 (50.8) 13 (31.7) Female gender, N (%) 40 (39) 23 (37.7) 17 (41.5) 0.836 Underlying condition, N (%) Bronchial asthma 7 (6.9) 4 (6.6) 3 (7.3) > 0.99 Hypertension 56 (54.9) 38 (62.3) 18 (43.9) 0.073 Type 2 diabetes mellitus 45 (44.1) 28 (45.9) 17 (41.5) 0.689 Chronic kidney disease 16 (15.7) 13 (21.3) 3 (7.3) 0.094 End stage renal disease 9 (8.8) 5 (8.2) 4 (9.8) > 0.99 Chronic obstructive pulmonary disease 8 (7.8) 7 (11.5) 1 (2.4) 0.139 Times from illness onset to hospital presentation, days, median (range) 3 (1–14) 2 (1–10) 3 (1–14) 0.414 Times from illness onset to fatality, day, median (range) ‒ ‒ 18.5 (2–53) ‒ Hospital length of stay, days, median (range) 23 (1-107) 32 (2-107) 14 (1–53) < 0.001 Stay in intensive care unit, N (%) 98 (96.1%) 60 (98.3%) 38 (92.7%) 0.300 Use antibiotic at presentation, N (%) 100 (98) 60 (98.4) 40 (97.6) > 0.99 Use oseltamivir, N (%) 94 (92.2) 56 (91.8) 38 (92.7) > 0.99 Use oseltamivir ≧ 48 h after onset of illness, N/total N (%) 68/94 (72.3) 39/56 (69.6) 29/38 (76.3) 0.639 Use peramivir, N (%) 10 (9.8) 8 (13.1) 2 (4.9) 0.793 RT-PCR Reverse transcription-polymerase chain reaction Table 1 Table 1 diagnostic methods of patients with severe influenza Characteristics and diagnostic methods of patients with severe influenza Variable Overall (n = 102) Survivors (n = 61) Non-survivors (n = 41) P value Use peramivir ≧ 48 h after onset of illness, N/total N (%) 6/10 (60) 5/8 (62.5) 1/2 (50) > 0.99 Use statin, N (%) 21 (20.6) 14 (23) 7 (17.1) 0.610 Use metformin, N (%) 17 (16.7) 13 (21.3) 4 (9.8) 0.177 Diagnostic methods for influenza, N/total N (%) Positive of influenza rapid test 51/93 (54.8) 34/56 (60.7) 17/37 (45.9) 0.203 Positive of RT-PCR for influenza 94/98 (95.9) 59/61 (96.7) 35/37 (94.6) 0.631 Positive of throat influenza viral culture 37/95 (38.9) 23/59 (39) 14/36 (38.9) > 0.99 Influenza virus subtype, N/total N (%) Influenza A 71/98 (72.4) 43/61 (70.5) 28/37 (75.7) 0.646 Pmd 09 H1N1 34/71 (47.9) 23/43 (53.5) 11/28 (39.3) 0.332 H3N2 8/71 (11.3) 7/43 (16.3) 1/28 (3.6) 0.135 Influenza B 23/98 (23.5) 16/61 (26.2) 7/37 (18.9) 0.469 RT-PCR Reverse transcription-polymerase chain reaction Page 7/21 Table 2 Table 2 Symptom/signs of patients with severe influenza Symptom/sign at presentation Overall (n = 102) Survivors (n = 61) Non-survivors (n = 41) P value Fever 81 (79.4) 48 (78.7) 33 (80.5) > 0.99 Rhinorrhea 8 (7.8) 3 (4.9) 5 (12.2) 0.262 Cough 85 (83.3) 49 (80.3) 35 (85.4) 0.602 Sore throat 8 (7.8) 5 (8.2) 3 (7.3) > 0.99 Malaise 24 (23.5) 17 (27.9) 7 (17.1) 0.241 Muscle pain 17 (16.7) 10 (16.4) 3 (7.3) 0.233 Headache 5 (4.9) 2 (3.3) 3 (7.3) 0.389 Vomiting/nausea 6 (5.9) 5 (8.2) 1 (2.4) 0.397 Diarrhea 2 (2) 1 (1.6) 1 (2.4) > 0.99 Abdominal pain 2 (2) 1 (1.6) 1 (2.4) > 0.99 Chest pain 8 (7.8) 5 (8.2) 3 (7.3) > 0.99 Skin rash 1 (1) 1 (1.6) 0 > 0.99 Altered consciousness 16 (15.7) 11 (18.0) 5 (12.2) 0.581 Seizure 3 (2.9) 2 (3.3) 1 (2.4) > 0.99 Dyspnea 87 (85.3) 51 (83.6) 36 (87.8) 0.776 Data expressed as number (%) L b T i Symptom/signs of patients with severe influenza Laboratory Testing The median white blood cell and platelet counts on admission were 7.9 × 109 cells/L and 156.5 × 109 cells/L, respectively, although 13 patients had thrombocytopenia (platelet count, < 100 × 109 cells/L). Regarding the laboratory data during hospitalization, the median white blood cell and platelet counts and creatinine, ALT, creatine kinase, myoglobin, C-reactive protein, and lactate concentrations were 16.8 × 109 cells/L, 101.5 × 109 cells/L, 2.4 mg/dL, 91 U/L (96 patients with data available), 322 U/L (35 patients with data available), 521.4 ng/mL (37 patients with data available), 213.6 mg/L (100 patients with data available), and 28.15 mg/dL (90 patients with available data), respectively. Five (21.7%) of the 23 patients with available data showed serum galactomannan index of > 0.5. Six patients underwent bronchoscopy, and 2 had bronchoalveolar lavage fluid galactomannan index of 5.09 and 5.57 with fatal outcomes. Urinary Streptococcus antigen was detected in 3 of the 41 patients with available data. The laboratory characteristics of the included patients are shown in Table 3. Laboratory Testing Page 8/21 Page 8/21 Table 3 Table 3 Laboratory characteristics of patients with severe influenza Variable Overall (n = 102) Survivors (n = 61) Non-survivors (n = 41) P Laboratory data at presentation WBC, (× 109 cells/L), median (range) 7.9 (0.5–178) 8.7 (2.1–27.9) 7 (0.5–178) 0.921 Platelet count, (× 109 cells/L), median (range) 156.5 (4-641) 156 (60–641) 158 (4-365) 0.407 Hemoglobin, (g/dl), median (range) 12.3 (7.2–18) 12.3 (7.5–18) 12.3 (7.2– 16.5) 0.309 Hemotocrit, (%), median (range) 36.8 (21.7– 51.7) 37 (23.2–51.7) 36.3 (21.7– 50.2) 0.785 BUN, (mg/dL), median (range) 21.5 (6-110) 22 (6-110) 21 (6–99) 0.521 Creatinine,(mg/dL), median (range) 1 (0.49-17) 1.3 (0.5–17) 1.3 (0.6–12.5) 0.771 AST, (IU/L), median (range) 57(14-3854) (n = 98) 53 (14-2366) (n = 59) 64 (15-3854) (n = 39) 0.619 ALT, (IU/L), median (range) 32 (6-2936) (n = 96) 28 (6-2936) (n = 60) 36 (8-899) (n = 36) 0.691 CRP, (mg/L), median (range) 127 (0.4–1377) (n = 100) 71.4 (0.4–380) (n = 59) 155.1 (1.3– 1377) 0.079 Creatine kinase, (U/L), median (range) 322 (10-14620) (n = 35) 332.5 (10- 11678) (n = 20) 290 (43-14620) (n = 15) 0.542 LDH, (U/L), median (range) 503.5 (174– 8951) (n = 28) 401.5 (174– 8951) (n = 16) 672 (286– 5665) (n = 12) 0.174 Myoglobin, ( ng/mL), median (range) 218.6 (0.06– 4980) (n = 37) 104.2 (0.06– 4980) (n = 19) 452.8 (85.5– 4342) (n = 18) 0.010 Troponin-I, (ng/ml), median (range) 0.06 (0.01-80) (n = 95) 0.06 (0.01– 15.9) (n = 56) 0.05 (0.01-80) (n = 39) 0.447 CK-MB, (ng/mL), median (range) 3.8 (0.4–302) (n = 84) 2.9 (0.4–68.1) (n = 49) 6.1 (0.7–302) (n = 35) 0.013 Lactate, (mg/dL), median (range) 15.9 (6.2–190) (n = 90) 15.35 (7.7- 134.6) (n = 54) 17.65 (6.2– 190) (n = 36) 0.840 ALT Alanine aminotransferase, AST Aspartate aminotransferase, BUN blood urea nitrogen, CRP C- reactive protein, LDH Lactate dehydrogenase, CK-MB Creatine kinase-MB isoenzyme, BAL Bronchial alveolar lavage, Ag Antigen Laboratory characteristics of patients with severe influenza ALT Alanine aminotransferase, AST Aspartate aminotransferase, BUN blood urea nitrogen, CRP C- reactive protein, LDH Lactate dehydrogenase, CK-MB Creatine kinase-MB isoenzyme, BAL Bronchial alveolar lavage, Ag Antigen ALT Alanine aminotransferase, AST Aspartate aminotransferase, BUN blood urea nitrogen, CRP C- reactive protein, LDH Lactate dehydrogenase, CK-MB Creatine kinase-MB isoenzyme, BAL Bronchial alveolar lavage, Ag Antigen Page 9/21 Variable Overall (n = 102) Survivors (n = 61) Non-survivors (n = 41) P Laboratory data during hospitalization Highest WBC, (× 109 cells/L), median (range) 16.8 (1.9-187.6) 15.8 (5.2–36.1) 19.6 (1.9- 187.6) 0.037 Nadir platelet count, (× 109 cells/L), median (range) 101.5 (4-311) 113 (16–311) 68 (4-271) 0.005 Highest BUN, (mg/dL), median (range) 55.5 (6-263) 41(11–263) 71 (6-226) 0.055 Highest creatinine, (mg/dL), median (range) 2.4 (0.58–18.6) 1.6 (0.6–18.6) 3.8 (0.6–12.2) 0.016 Highest AST, (IU/L), median (range) 105 (17-15224) (n = 98) 95 (19-14670) (n = 59) 153 (17-15224) (n = 39) 0.033 Highest ALT, (IU/L), median (range) 91 (14-4602) (n = 96) 89 (14-4602) (n = 60) 93 (15-3876) (n = 36) 0.226 Highest CRP, (mg/L), median (range) 213.6 (2-2641) (n = 100) 186 (2-423.3) (n = 59) 258.6 (5.97– 2641) (n = 41) 0.019 Highest creatine kinase, (U/L), median (range) 322 (10-14620) (n = 35) 322.5 (10- 11678) (n = 20) 290 (43-14620) (n = 15) 0.657 Highest LDH, (U/L), median (range) 586.5(174– 8951) (n = 28) 401.5(174– 8951) (n = 16) 1051 (286– 566) ( n = 12) 0.059 Highest myoglobin, ( ng/mL), median (range) 521.4 (0.06- 145077.2) (n = 37) 104.2 (0.06- 6813.5) (n = 19) 1487.6 (95.3- 145077.2) (n = 18) 0.002 Highest Troponin I, (ng/ml), median (range) 0.2 (0.01-80) (n = 95) 0.1 (0.01–45.6 ) (n = 56) 0.2 (0.01-80) (n = 39) 0.736 Highest CK-MB, (mg/dL), median (range) 5.4 (0.4-788.2) (n = 84) 4.3 (0.4-133.1) (n = 49) 9.2 (0.9-788.2) (n = 35) 0.007 Highest lactate,(mg/dL), median (range) 28.15 (7.7- 202.6) (n = 90) 24.2 (7.7- 134.6) (n = 54) 38.75 (8.8- 202.6) (n = 36) < 0.001 The time interval from presentation to measurement of highest lactate, day, median (range) 4 (1–51) (n = 90) 3 (1–51) (n = 54) 4 (1–30) (n = 36) 0.606 Positive of BAL galactomannan test, N/total N (%) (reference > 2/6 (33.3) 0/1 (0) 2/5 (40) > 0.99 ALT Alanine aminotransferase, AST Aspartate aminotransferase, BUN blood urea nitrogen, CRP C- reactive protein, LDH Lactate dehydrogenase, CK-MB Creatine kinase-MB isoenzyme, BAL Bronchial alveolar lavage, Ag Antigen ALT Alanine aminotransferase, AST Aspartate aminotransferase, BUN blood urea nitrogen, CRP C- reactive protein, LDH Lactate dehydrogenase, CK-MB Creatine kinase-MB isoenzyme, BAL Bronchial alveolar lavage, Ag Antigen Page 10/21 Page 10/21 Variable Overall (n = 102) Survivors (n = 61) Non-survivors (n = 41) P 0.5 index) Positive of serum galactomannan test, N/total N (%) (reference > 0.5 index) 5/23 (21.7) 2/11 (18.2) 3/12 (25) > 0.99 Positive of urine legionella Ag, N/total N (%) 0/60 0/36 0/24 - Positive of urine streptococcus Ag, N/total N (%) 3/41 (7.3) 3/25 (12) 0/16 (0) 0.268 ALT Alanine aminotransferase, AST Aspartate aminotransferase, BUN blood urea nitrogen, CRP C- reactive protein, LDH Lactate dehydrogenase, CK-MB Creatine kinase-MB isoenzyme, BAL Bronchial alveolar lavage, Ag Antigen C li i ALT Alanine aminotransferase, AST Aspartate aminotransferase, BUN blood urea nitrogen, CRP C- reactive protein, LDH Lactate dehydrogenase, CK-MB Creatine kinase-MB isoenzyme, BAL Bronchial alveolar lavage, Ag Antigen Complications Table 4 shows the in-hospital complications of the patients during the entire clinical course. Among the 102 patients (an individual might have had more than one complication), respiratory failure developed in 99 (97%), pneumonia in 96 (94.1%), acute kidney injury in 67 (65.7%), ARDS in 52 (51%), gastrointestinal bleeding in 36 (35.3%), bacteremia in 17 (16.7%), pneumothorax in 13 (12.7%), rhabdomyolysis in 12 (11.8%), fulminant hepatitis in 9 (8.8%), pulmonary edema in 3 (2.9%), meningoencephalitis and fungemia in 2 patients (2%), and intracranial hemorrhage in 1 (1%). The median time from illness onset to respiratory failure was 4 days (range, 1–27 days). Eighteen patients with severe influenza-associated ARDS were treated with extracorporeal membrane oxygenation (ECMO), of whom 15 (83.3%) patients had influenza A virus and 3 (16.7%) had influenza B virus infection. Of the 18 patients with influenza- associated ARDS who received ECMO treatment, 13 (72.2%) died. Among the 17 bacteremia patients, 5 (29.4%) patients had bacteremia (Staphylococcus aureus in 3, Pseudomonas aeruginosa in one, and Actinomyces oris and Streptococcus salivarius in one) within 48 hours after hospitalization. Staphylococcus aureus (35.3%) (16.7% were methicillin resistant), Enterococcus species (17.6%), and viridians streptococci (17.6%) were the most frequently isolated bacteria from blood cultures. Staphylococcus aureus (35.3%) (16.7% were methicillin resistant), Enterococcus species (17.6%), and viridians streptococci (17.6%) were the most frequently isolated bacteria from blood cultures. Comparison of Survivors and Non-survivors Compared with survivors, non-survivors had a significantly shorter length of hospital stay. Upon hospital admission, elevation of myoglobin and creatine kinase-MB isoenzyme levels was significantly associated with mortality. Moreover, a significantly higher white blood cell count and high creatinine, aspartate aminotransferase, C-reactive protein, myoglobin, creatine kinase-MB isoenzyme, and lactate concentrations, in addition to lower platelet count during the course of hospitalization, were reported in non-survivors. Non-survivors received ECMO treatment and had a significantly higher incidence of acute kidney injury, pneumothorax, ARDS, gastrointestinal bleeding than survivors. Multivariate analysis showed that high lactate level (adjusted odds ratio [aOR] : 1.041; 95% confidence interval [CI]: 1.019– 1.064; P < 0.001), ARDS (aOR: 10.098; 95% CI: 2.505–40.714; P = 0.001), acute kidney injury (aOR: 9.019; 95% CI: 1.804–45.089; P = 0.007), and gastrointestinal bleeding (aOR: 3.828; 95% CI: 1.180–12.415; P = 0.025) were independent predictors of mortality in critically ill influenza patients. Complications Page 11/21 Table 4 In-hospital complications of patients with severe influenza In-hospital complications of patients with severe influenza Variable Overall (n = 102) Survivors (n = 61) Non-survivors (n = 41) P Acute respiratory failure 99 (97) 58 (95.1) 41 (100) > 0.99 Time from onset illness to respiratory failure, day, median (range) 4 (1–27) (n = 99) 3.5 (1–16) (n = 58) 4 (1–27) 0.451 Time from hospital presentation to respiratory failure, day, median (range) 1 (1–21) (n = 99) 1 (1–13) (n = 58) 1 (1–21) 0.921 Acute kidney injury 67 (65.7) 34 (55.7) 33 (80.5) 0.011 Pneumothorax 13 (12.7) 4 (6.6) 9 (22) 0.033 Acute respiratory distress syndrome 52 (51) 25 (41) 27 (65.9) 0.016 Pneumonia 96 (94.1) 57 (93.4) 39 (95.1) > 0.99 Pulmonary edema 3 (2.9) 3 (4.9) 0 0.272 Meningoencephalitis 2 (2) 1 (1.6) 1 (2.4 ) > 0.99 Intracranial hemorrhage 1 (1) 0 1 (2.4) 0.402 Gastrointestinal bleeding 36 (35.3) 15 (24.6) 21 (51.2) 0.011 Fulminant hepatitis 9 (8.8) 6 (9.8) 3 (7.3) 0.737 Rhabdomyolysis 12 (11.8) 5 (8.2) 7 (17.1) 0.216 Bacteremia 17 (16.7) 11 (64.7) 6 (35.3) > 0.99 Bacteremia onset ≧ 48 h after presentation 12 (11.7) 7 (11.4) 5 (12.1) > 0.99 Fungemia 2 (2) 0 2 (4.9) 0.159 ECMO support 18 (17.6) 5 (8.2) 13 (31.7) 0.003 Data expressed as number (%). ECMO Extra-corporeal membrane oxygenation Page 12/21 Table 5 Multivariate analysis of independent risk factors associated with fatality in patients with severe influenza Odds ratio 95% confidence interval P High blood lactate levels 1.041 1.019–1.064 < 0.001 Adult respiratory distress syndrome 10.098 2.505–40.714 0.001 Acute kidney injury 9.019 1.804–45.089 0.007 Gastrointestinal bleeding 3.828 1.180-12.415 0.025 Table 5 Table 5 Outcomes Of the 102 patients, 41 (median age, 61 [range, 24–93] years) died, with an overall mortality rate of 40.1%. The middle-age (50–64 years) group had the highest mortality rate. The median duration of illness before death was 18.5 (range, 2–53) days. Nine patients (21.9%) died within 7 days after symptom onset. Hypertension (43.9%) and type 2 diabetes mellitus (41.5%) were the 2 most common underlying conditions in 41 deceased patients. Of 37 deceased patients for whom the RT-PCR data were available, 75.7% had influenza A virus and 18.9% had influenza B virus infection. Of the 38 deceased patients who received oseltamivir treatment, 76.3% of patients received oseltamivir more than 48 hours after onset of the illness. Among the 41 deceased patients, pneumonia developed in 39 (95.1%) patients acute kidney injury in 33 (80.5%), ARDS in 27 (65.9%), gastrointestinal bleeding in 21 (51.2%), and bacteremia in 6 (35.3%) patients (Table 4). Discussion In a study involving 444 adult patients with influenza in hospitals in the United States, the mortality rate was 20.9% [21]. Furthermore, a mortality rate of 20.6% was reported by Francisco et al. in their study of 2059 patients admitted to intensive care units for influenza infection [22]. In our study, a mortality rate as high as 40% was found in 102 critically ill adult patients with influenza. However, which variables can predict poor patient outcomes after influenza virus infection remain to be elucidated. In the present study, our dataset included clinical signs and symptoms and laboratory results at presentation and the entire course of hospitalization as well as complications during the clinical course. We determined which demographic, clinical, and laboratory findings were associated with death that could help clinicians deliver timely sufficient treatment to critically ill influenza patients. Our results underscore that high blood lactate levels, ARDS, acute kidney injury, and gastrointestinal bleeding were independent risk factors of mortality in critically ill influenza patients. High blood lactate levels indicate tissue hypoxia due to increased lactate generation via anaerobic glycolysis [23]. High blood lactate levels have been correlated with poor outcomes in patients with bacterial sepsis and septic shock [24]. In the present study, high blood lactate levels were found to be significantly and independently associated with fatal outcomes in critically ill influenza patients. In addition, non-survivors had a significantly higher prevalence of acute kidney injury and gastrointestinal bleeding, and received ECMO treatment in our series. Importantly, acute kidney injury and gastrointestinal bleeding have been shown to be independent risk factors of mortality. We believe that these complications are caused by clinicians’ lack of awareness of early detection of organ hypoperfusion. As patients in early phases of hypoperfusion do not always show obvious clinical signs, blood lactate level may be an important marker for this disorder. Thus, timely recognition of organ hypoperfusion and initiation of effective volume replacement to reverse tissue hypoxia are critical steps in preventing mortality and morbidity. Notably, the median time interval from patient arrival to measurement of highest blood lactate was 4 days in non-survivors in our series. Further, the median time from illness onset to fatality was 18.5 days. This finding indicates that blood lactate levels can be a useful early marker assisting clinicians in predicting the outcomes in critically ill influenza patients. ARDS is a lethal complication of influenza infection [25]. Ortiz et al. Event-based Algorithm The median lactate values (reference value < 19 mg/dL) and median time from presentation to the highest lactate level among survivors and non-survivors were 24.2 mg/dL and 38.7 mg/dL and 3 days and 4 days, respectively. We selected the lactate variable in multivariate analyses and plotted the ROC Page 13/21 Page 13/21 curve to identify the optimal cutoff value for predicting mortality. The optimal cutoff of lactate level for predicting mortality was 33 mg/dL with an area under curve of 0.728, and the sensitivity and specificity of this cutoff were estimated at 63.9% and 74.1%, respectively. We then created an event-associated algorithm that included lactate level and ARDS – the 2 variables that were independently and significantly associated with death in multivariate analyses (Fig. 1). Fifteen (75.0%) of the 20 patients with lactate levels of 33 mg/dL or above and with ARDS died, compared with only one (7.7%) of 13 patients with lactate levels below 19 mg/dL and without ARDS (P < 0.001) and 1 (10%) of 10 patients with lactate levels between 19 mg/dL and 33 mg/dL and without ARDS (P < 0.001). Discussion estimated that the incidence of influenza-associated acute respiratory failure was 2.7 events per 100,000 person-years [26]. In a study of Page 14/21 Page 14/21 58 patients with ARDS, 28 (48.2%) were due to influenza virus infection, and 32.1% of the patients with influenza-associated ARDS received ECMO treatment [27]. Davies et al. reported that the incidence of pdm09 A/H1-associated ARDS sufficient to warrant consideration of ECMO was estimated at 2.6 cases per million population [28]. ARDS is an independent risk factor for hospital mortality in critically ill influenza patients, and the mortality rate can be as high as 52% [29]. The present study results are consistent with previous findings wherein 97% of critically ill influenza patients developed acute respiratory failure with a median time of 4 days between illness onset and respiratory failure, and 51% of them subsequently developed ARDS during their clinical course. In addition, approximately one-third of the patients with influenza-associated ARDS required ECMO for profound hypoxemic respiratory failure. Our study highlights that severe oxygenation failure occurred rapidly after hospital admission, and that clinicians should not delay delivering appropriate rescue therapies as well as deploying intensive care unit resources to meet this treatment requirement, particularly during the influenza epidemic. In the present study, we established a simple event-associated algorithm including blood lactate level and ARDS for timely detection of critically ill influenza patients who are at greater risk of mortality. Notably, critically ill influenza patients without ARDS but with a blood lactate concentration of 33 mg/dL had an in-hospital mortality of 47.1%, and more importantly, the mortality rate increased to 75% for those with high blood lactate (≥ 33 mg/dL) and developed ARDS. In contrast, only 7.7% of critically ill influenza patients without ARDS and with normal blood lactate levels died. Considering the high mortality rate among critically ill influenza patients, this event-based algorithm could aid in the timely decision-making process and provision of prompt intensive care for patients with potentially fatal outcomes, particularly in resource-limited areas; some key laboratory tests such as blood lactate might be of greater value than others when allocating limited healthcare resources. Previous studies have shown that early administration of an antiviral agent is associated with a shorter duration and reduced severity of illness [14, 15, 16]. Greater benefits were shown with early treatment initiated within 2 days after the onset of illness [30, 31, 32]. Discussion In our study, the median time from illness onset to hospital presentation was 3 days and more than two-thirds of the patients received delayed (48 h after illness onset) antiviral treatment. Although the provision of antiviral therapy between survivors and non-survivors did not differ significantly in our series, the importance of early treatment with antivirals in critically ill influenza patients cannot be overemphasized. In our study, bacteremia was detected in 17 critically ill adult patients. Importantly, 5 of them acquired bacteremia within 48 h after hospitalization, and in 3 cases, the infection was caused by Staphylococcus aureus. A report of the 2009–2010 influenza pandemic among critically ill children revealed that nearly 5% of the patients had bacteremia within 72 hours and Staphylococcus aureus was the most frequently isolated bacterium, which contributed to the death rate in the current pandemic [33]. In a study of 32 influenza-positive patients (including pediatric and adult patients), poor outcomes were found among patients who were co-infected with influenza viruses and Staphylococcus aureus [34]. Although we were unable to conclude whether or not initiating timely additional antimicrobial treatment in critically ill influenza patients led to better clinical outcomes, our findings and previous reports underscore that Page 15/21 Page 15/21 Staphylococcus aureus remains the most important cause of bacterial coinfection in pediatric and adult influenza patients. Invasive pulmonary Aspergillus as a coinfection in patients with severe influenza has been described [35, 36, 37]. In a cohort study involving 7 intensive care units over a period of 7 influenza seasons showed that influenza and the use of corticosteroids were independent risk factors for invasive aspergillosis [35]. In the present study, invasive pulmonary aspergillosis was confirmed in 2 deceased influenza patients with high galactomannan index in bronchoalveolar lavage fluid. This finding emphasizes that clinicians should be aware of the risk of invasive aspergillosis in critically ill influenza patients, particularly immunocompromised patients or those receiving corticosteroids. Further studies are needed to understand the incidence, risk factors, and clinical features of invasive pulmonary aspergillosis in influenza patients. This study has several potential limitations. First, given the retrospective nature of the study, data on vaccination status, including pneumococcus and influenza, were not collected. Second, the study population comprised adult patients; therefore, the results cannot be generalized to pediatric patients. Conclusion We identified clinical and laboratory predictors of mortality at hospital admission that could aid in the early prediction of the severe outcomes in critically ill influenza patients, as timely intensive supportive care might be lifesaving. Medical services and intensive care units can be overwhelmed during the peak of influenza epidemics, particularly in point-of-care resource-limited areas. Our findings could substantially assist with allocation of resources in the selection of the main key clinical data in primary care at the initial clinical evaluation of critically ill influenza patients. Discussion However, the strengths of this study include a detailed description of clinical and laboratory information at presentation and the entire hospitalization course critically ill patients with influenza. We highlighted the key factors associated with poor outcomes for critically ill influenza patients and established decision-making algorithms that can take advantage of simple clinical and laboratory evaluations. Abbreviations ARDS: acute respiratory distress syndrome, aOR: adjusted odds ratio; ALT: alanine aminotransferase levels; ECMO: extracorporeal membrane oxygenation; FIO2: fraction of inspired oxygen; KCGMH: Kaohsiung Chang Gung Memorial Hospital; PaO2: arterial partial pressure of oxygen; pdm09 A/H1: pandemic 2009 A/H1N1; RT-PCR: reverse-transcriptase–polymerase chain reaction; Consent for publication Not applicable. Acknowledgements Page 16/21 Competing interests The authors declare that they have no competing interests. Funding This study was supported by a grant (document no. CMRPG8H0241 to IKL) from Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Availability of data and materials All data generated or analyzed during this study are included in this published article Ethics approval and consent to participate The institutional review board of Kaohsiung Chang Gung Memorial Hospital approved the study (document no. 201901724B0). Informed consent was not required as the data were analyzed anonymously. Authors’ contributions IKL made substantial contributions to the conception, study design, data analysis and interpretation, and drafting, editing, and submitting the manuscript. JCH made substantial contributions to the data collection, data analysis and writing the manuscript. WCH, YCC and CYT contributed to the design of the study and interpreted the findings. All authors read and approved the final manuscript. References 1. Nicholson KG, Wood JM, Zambon M. Influenza. The Lancet. 2003;362(9397): 1733–1745. doi:10.1016/s0140-6736(03)14854-4. 1. Nicholson KG, Wood JM, Zambon M. Influenza. The Lancet. 2003;362(9397): 1733–1745. doi:10.1016/s0140-6736(03)14854-4. 2. Thompson WW. Influenza-associated hospitalizations in the United States. JAMA. 2004;292(11):1333–1340. doi:10.1001/jama.292.11.1333. 3. Centers for Disease Control and Prevention (CDC). Estimates of deaths associated with seasonal influenza --- United States, 1976-2007. MMWR. Morb. Mortal. Wkly. Rep. 2010, 59, 1057–1062. 4. World Health Organization (WHO). Influenza (seasonal). https://www.who.int/news-room/fact- sheets/detail/influenza-(seasonal) (accessed 12 Jan, 2020). 5. Iuliano AD, Roguski KM, Chang HH, et al. Estimates of global seasonal influenza-associated respiratory mortality: a modelling study. The Lancet. 2018;391(10127):1285–1300. doi:1016/S0140- 5. Iuliano AD, Roguski KM, Chang HH, et al. Estimates of global seasonal influenza-associated respiratory mortality: a modelling study. The Lancet. 2018;391(10127):1285–1300. doi:1016/S0140- Page 17/21 Page 17/21 6736(17)33293-2 6. Dawood FS, Jain S, Finelli L, Shaw MW, et al, Novel Swine-Origin Influenza A (H1N1) Virus Investigation Team. Emergence of a novel swine-origin influenza A (H1N1) virus in humans. New England Journal of Medicine. 2009;360(25):2605–2615. doi:10.1056/nejmoa0903810 7. Shrestha SS, Swerdlow DL, Borse RH, et al. Estimating the burden of 2009 pandemic influenza A (H1N1) in the United States (April 2009-April 2010). Clinical Infectious Disease. 2011,52 Suppl 1:S75–82. doi: 10.1093/cid/ciq012. 8. Zhou F, Li H, Gu L, Liu M, et al. Risk factors for nosocomial infection among hospitalised severe influenza A(H1N1)pdm09 patients. Respiratory medicine. 2018;134:86-91. doi:10.1016/j.rmed.2017.11.017. 9. Paddock CD, Liu L, Denison AM, et al. Myocardial injury and bacterial pneumonia contribute to the pathogenesis of fatal influenza B virus infection. The Journal of Infectious Diseases. 2012;205(6):895-905. doi:10.1093/infdis/jir861. 10. Daoud A, Laktineh A, Macrander C, Mushtaq A, Soubani AO, et al. Pulmonary complications of influenza infection: a targeted narrative review. Postgraduate Medicine. 2019;131(5):299–308. doi:10.1080/00325481.2019.1592400. 11. Kumar A, Zarychanski R, Pinto R, et al. Canadian Critical Care Trials Group H1N1 Collaborative. Critically ill patients with 2009 influenza A (H1N1) infection in Canada. JAMA. 2009;302(17):1872– 1879. doi:10.1001/jama.2009.1496. 12. Rello J, Rodríguez A, Ibañez P, et al. H1N1 SEMICYUC Working Group. Intensive care adult patients with severe respiratory failure caused by Influenza A (H1N1)v in Spain. Critical Care. 2009;13(5):R148. doi:10.1186/cc8044 13. Domínguez-Cherit G, Lapinsky SE, Macias AE, et al. Critically Ill patients with 2009 influenza A(H1N1) in Mexico. JAMA. 2009;302 (17):1880–1887. doi:10.1001/jama.2009.1536 14. Dobson J, Whitley RJ, Pocock S, Monto AS. Oseltamivir treatment for influenza in adults: a meta- analysis of randomised controlled trials. The Lancet. 2015;385(9979):1729-1737. doi:10.1016/s0140-6736(14)62449-1. 15. Doll MK, Winters N, Boikos C, Kraicer-Melamed H, Gore G, Quach C. Safety and effectiveness of neuraminidase inhibitors for influenza treatment, prophylaxis, and outbreak control: a systematic review of systematic reviews and/or meta-analyses. Journal of Antimicrobial Chemotherapy. 2017;72(11):2990-3007. doi:10.1093/jac/dkx271. 15. Doll MK, Winters N, Boikos C, Kraicer-Melamed H, Gore G, Quach C. Safety and effectiveness of neuraminidase inhibitors for influenza treatment, prophylaxis, and outbreak control: a systematic review of systematic reviews and/or meta-analyses. Journal of Antimicrobial Chemotherapy. 2017;72(11):2990-3007. doi:10.1093/jac/dkx271. 16. Fry AM, Goswami D, Nahar K, et al. Efficacy of oseltamivir treatment started within 5 days of symptom onset to reduce influenza illness duration and virus shedding in an urban setting in Bangladesh: a randomised placebo-controlled trial. The Lancet Infectious Diseases. 2014;14(2):1 118. doi:10.1016/s1473-3099(13)70267-6. 16. Fry AM, Goswami D, Nahar K, et al. 6736(17)33293-2 Efficacy of oseltamivir treatment started within 5 days of symptom onset to reduce influenza illness duration and virus shedding in an urban setting in Bangladesh: a randomised placebo-controlled trial. The Lancet Infectious Diseases. 2014;14(2):109- 118. doi:10.1016/s1473-3099(13)70267-6. 17. Ranieri VM, Rubenfeld GD, Thompson BT, et al. Acute Respiratory Distress Syndrome. JAMA. 2012;307(23): 2526–2533. doi:10.1001/jama.2012.5669. 17. Ranieri VM, Rubenfeld GD, Thompson BT, et al. Acute Respiratory Distress Syndrome. JAMA. 2012;307(23): 2526–2533. doi:10.1001/jama.2012.5669. Page 18/21 Page 18/21 18. Steiner I, Budka H, Chaudhuri A, et al. Viral meningoencephalitis: a review of diagnostic methods and guidelines for management. European Journal of Neurology. 2010;17(8). doi:10.1111/j.1468- 1331.2010.02970.x. 19. Heer KD, Gerritsen MG, Visser CE, Leeflang MM. Galactomannan detection in broncho-alveolar lavage fluid for invasive aspergillosis in immunocompromised patients. Cochrane Database of Systematic Reviews. 2019. doi:10.1002/14651858.cd012399.pub2. 20. Maertens J, Theunissen K, Verbeken E, et al. Prospective clinical evaluation of lower cut-offs for galactomannan detection in adult neutropenic cancer patients and haematological stem cell transplant recipients. British Journal of Haematology. 2004;126(6):852-860. doi:10.1111/j.1365- 2141.2004.05140.x. 21. Shah NS, Greenberg JA, Mcnulty MC, et al. Severe Influenza in 33 US Hospitals, 2013–2014: Complications and Risk Factors for Death in 507 Patients. Infection Control & Hospital Epidemiology. 2015;36(11):1251-1260. doi:10.1017/ice.2015.170. 22. Álvarez-Lerma F, Marín-Corral J, Vila C, et al. Delay in diagnosis of influenza A (H1N1)pdm09 virus infection in critically ill patients and impact on clinical outcome. Critical Care. 2016;20(1). doi:10.1186/s13054-016-1512-1. 23. Fall PJ, Szerlip HM. Lactic Acidosis: From Sour Milk to Septic Shock. Journal of Intensive Care Medicine. 2005;20(5):255-271. doi:10.1177/0885066605278644. 24. Mikkelsen ME, Miltiades AN, Gaieski DF, et al. Serum lactate is associated with mortality in severe sepsis independent of organ failure and shock. Critical Care Medicine. 2009;37(5):1670-1677. doi:10.1097/ccm.0b013e31819fcf68. 25. Short KR, Kroeze EJBV, Fouchier RAM, Kuiken T. Pathogenesis of influenza-induced acute respiratory distress syndrome. The Lancet Infectious Diseases. 2014;14(1):57-69. doi:10.1016/s1473- 3099(13)70286-x. 26. Ortiz JR, Neuzil KM, Rue TC, et al. Population-based Incidence Estimates of Influenza-associated Respiratory Failure Hospitalizations, 2003 to 2009. American Journal of Respiratory and Critical Care Medicine. 2013;188(6):710-715. doi:10.1164/rccm.201212-2341oc. 27. Yoo J-W, Ju S, Lee SJ, et al. Characteristics and Outcomes of Patients with Pulmonary Acute Respiratory Distress Syndrome Infected with Influenza versus Other Respiratory Viruses. Tuberculosis and Respiratory Diseases. 2019;82(4):328. doi:10.4046/trd.2019.0017. 28. Davies A, Jones D, Bailey M, et al. Extracorporeal Membrane Oxygenation for 2009 Influenza A(H1N1) Acute Respiratory Distress Syndrome. JAMA. 2009;302(17):1888–1895. doi:10.1001/jama.2009.1535. 29. Li G, Yilmaz M, Kojicic M, et al. 6736(17)33293-2 37. Veerdonk FLVD, Kolwijck E, Lestrade PPA, et al. Influenza-associated Aspergillosis in Critically Ill Patients. American Journal of Respiratory and Critical Care Medicine. 2017;196(4):524-527. doi:10.1164/rccm.201612-2540le. 6736(17)33293-2 Outcome of critically ill patients with influenza virus infection. Journal of Clinical Virology. 2009;46(3):275-278. doi:10.1016/j.jcv.2009.07.015. 29. Li G, Yilmaz M, Kojicic M, et al. Outcome of critically ill patients with influenza virus infection. Journal of Clinical Virology. 2009;46(3):275-278. doi:10.1016/j.jcv.2009.07.015. 30. Jain S, Kamimoto L, Bramley AM, et al. Hospitalized Patients with 2009 H1N1 Influenza in the United States, April–June 2009. New England Journal of Medicine. 2009;361(20):1935-1944. doi: 10.1056/NEJMoa0906695 Page 19/21 31. Hanshaoworakul W, Simmerman JM, Narueponjirakul U, et al. Severe Human Influenza Infections in Thailand: Oseltamivir Treatment and Risk Factors for Fatal Outcome. PLoS ONE. 2009;4(6). doi:10.1371/journal.pone.0006051. 32. Kumar A. Early versus late oseltamivir treatment in severely ill patients with 2009 pandemic influenza A (H1N1): speed is life. Journal of Antimicrobial Chemotherapy. 2011;66(5):959-963. doi:10.1093/jac/dkr090. 32. Kumar A. Early versus late oseltamivir treatment in severely ill patients with 2009 pandemic influenza A (H1N1): speed is life. Journal of Antimicrobial Chemotherapy. 2011;66(5):959-963. doi:10.1093/jac/dkr090. 33. Randolph AG, Vaughn F, Sullivan R, et al. Critically ill children during the 2009-2010 influenza pandemic in the United States. Pediatrics. 2011;128(6):e1450–e1458. doi:10.1542/peds.2011-0774 33. Randolph AG, Vaughn F, Sullivan R, et al. Critically ill children during the 2009-2010 influenza pandemic in the United States. Pediatrics. 2011;128(6):e1450–e1458. doi:10.1542/peds.2011-0774 34. Mcdanel JS, Perencevich EN, Storm J, et al. Increased Mortality Rates Associated withStaphylococcus aureusand Influenza Co-infection, Maryland and Iowa, USA. Emerging Infectious Diseases. 2016;22(7):1253-1256. doi:10.3201/eid2207.151319. 34. Mcdanel JS, Perencevich EN, Storm J, et al. Increased Mortality Rates Associated withStaphylococcus aureusand Influenza Co-infection, Maryland and Iowa, USA. Emerging Infectious Diseases. 2016;22(7):1253-1256. doi:10.3201/eid2207.151319. 35. Schauwvlieghe AFAD, Rijnders BJA, Philips N, et al. Invasive aspergillosis in patients admitted to the intensive care unit with severe influenza: a retrospective cohort study. The Lancet Respiratory Medicine. 2018;6(10):782-792. doi:10.1016/s2213-2600(18)30274-1. 35. Schauwvlieghe AFAD, Rijnders BJA, Philips N, et al. Invasive aspergillosis in patients admitted to the intensive care unit with severe influenza: a retrospective cohort study. The Lancet Respiratory Medicine. 2018;6(10):782-792. doi:10.1016/s2213-2600(18)30274-1. 36. Wauters J, Baar I, Meersseman P, et al. Invasive pulmonary aspergillosis is a frequent complication of critically ill H1N1 patients: a retrospective study. Intensive Care Medicine. 2012;38(11):1761- 1768. doi:10.1007/s00134-012-2673-2. 36. Wauters J, Baar I, Meersseman P, et al. Invasive pulmonary aspergillosis is a frequent complication of critically ill H1N1 patients: a retrospective study. Intensive Care Medicine. 2012;38(11):1761- 1768. doi:10.1007/s00134-012-2673-2. 37. Veerdonk FLVD, Kolwijck E, Lestrade PPA, et al. Influenza-associated Aspergillosis in Critically Ill Patients. American Journal of Respiratory and Critical Care Medicine. 2017;196(4):524-527. doi:10.1164/rccm.201612-2540le. Figures Page 20/21 Figure 1 Event-based algorithm Figure 1 Event-based algorithm Page 21/21
https://openalex.org/W3120521042	https://birmingham.elsevierpure.com/files/136117512/corruption_and_sme_growth_the_roles_of_institutional_networking_and_financial_slack.pdf	English	null	Corruption and SME growth: the roles of institutional networking and financial slack	Journal of institutional economics	2,021	cc-by	12,460	Corruption and SME growth Adomako, Samuel; Ahsan, Mujtaba; Amankwah-Amoah, Joseph; Danso, Albert; Kesse, Kwabena; Frimpong, Kwabena DOI: 10.1017/s1744137421000011 License: Creative Commons: Attribution (CC BY) Document Version Publisher's PDF, also known as Version of record Citation for published version (Harvard): Adomako, S, Ahsan, M, Amankwah-Amoah, J, Danso, A, Kesse, K & Frimpong, K 2021, 'Corruption and SME growth: the roles of institutional networking and financial slack', Journal of Institutional Economics, vol. 17, no. 4 pp. 607-624. https://doi.org/10.1017/s1744137421000011 Link to publication on Research at Birmingham portal General rights U l li General rights Unless a licence is specified above, all rights (including copyright and moral rights) in this document are retained by the authors and/or the copyright holders. The express permission of the copyright holder must be obtained for any use of this material other than for purposes permitted by law. •Users may freely distribute the URL that is used to identify this publication. d l d d/ i f h bli i f h i i f i i h h l f •Users may freely distribute the URL that is used to identify this publication. d l d d/ i f h bli i f h i i •Users may freely distribute the URL that is used to identify this publication. •Users may download and/or print one copy of the publication from the University of Birmingham research portal for the purpose of private study or non-commercial research y y y p •Users may download and/or print one copy of the publication from the University of Birmingham research portal for the purpose of private study or non-commercial research. y •User may use extracts from the document in line with the concept of ‘fair dealing’ under the Copyright, Designs and Patents Act 1988 (?) •Users may not further distribute the material nor use it for the purposes of commercial gain. here a licence is displayed above, please note the terms and conditions of the licence govern your use of this document Where a licence is displayed above, please note the terms an When citing, please reference the published version. Abstract In this study, we investigate the mediating effect of institutional networking on the relationship between perceived corruption and the growth of small and medium-sized enterprises (SMEs). We also examine the moderating impact of financial slack on the relationship between perceived corruption and institutional networking. We test our moderated mediation model using data from 212 SMEs operating in Ghana. The findings from the study show that perceived corruption is positively related to institutional network- ing and this relationship is amplified when levels of financial slack are greater. The findings also show that institutional networking positively mediates the relationship between perceived corruption and SME growth. Theoretical and practical implications are discussed. Key words: Ghana; growth; perceived corruption; SMEs © Millennium Economics Ltd 2021. 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. available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S1744137421000011 Downloaded from https://www.cambridge.org/core. University College Birmingham, on 29 Jun 2021 at 09:09:21, subject to the Cambridge Core terms of us Samuel Adomako1* , Mujtaba Ahsan2, Joseph Amankwah-Amoah3, Albert Danso4, Kwabena Kesse5 and Kwabena Frimpong6 1School of Management, University of Bradford, Bradford, UK, 2Fowler School of Business, San Diego State University, San Diego, CA, USA, 3Kent Business School, University of Kent, Canterbury, Kent, UK, 4Leicester Castle Business School, De Montfort University, Leicester, UK, 5Beacom School of Business, University of South Dakota, Vermillion, SD, USA and 6KFUPM Business School, King Fahd University of Petroleum and Minerals, Dhahran, Saudi Arabia Corresponding author. Email: S.Adomako@bradford.ac.uk (Received 12 January 2020; revised 1 January 2021; accepted 1 January 2021) Journal of Institutional Economics (2021), 1–18 doi:10.1017/S1744137421000011 Corruption and SME growth: the roles of institutional networking and financial slack Samuel Adomako1 , Mujtaba Ahsan2, Joseph Amankwah-Amoah3, Albert Danso Kwabena Kesse5 and Kwabena Frimpong6 Take down policy Take down policy While the University of Birmingham exercises care and attention in making items available there are rare occasions when an item has been uploaded in error or has been deemed to be commercially or otherwise sensitive. If you believe that this is the case for this document, please contact UBIRA@lists.bham.ac.uk providing details and we will remove access to the work immediately and investigate. Download date: 24. Oct. 2024 1. Introduction Interestingly, Vorley and Williams (2016) argue that entrepreneurs can adopt two strategies when operating in environ- ments with high levels of corruption: avoiding the attention of government officials and engaging in corruption. Although this research provides useful insights into the strategies that entrepreneurs could adopt, we still have a limited understanding of the agency entrepreneurs display to manage the challenges of operating in environments fraught with corruption. In particular, we lack an understanding of the actions the key decision-makers of SMEs (e.g. owners and CEOs) operating in such an environment take to ensure the survival and success of their firms. In contrast to larger firms that have the significant financial resources and work with lobbyists to create conducive environments for their businesses (Doh et al., 2003; Luiz and Stewart, 2014), SMEs have limited resources and therefore need to be strategic in how they utilise these resources (Lee et al., 1999; Terziovski, 2010). Furthermore, in SMEs, the key decision-makers play an influential role, and they are directly involved in the actions that are taken to buffer their firms against adverse impacts (Child and Hsieh, 2014; Ling et al., 2008; Sawyerr et al., 2003). Consequently, the entrepreneurial agency they demonstrate could very well be the difference between the success and failure of their firms. y y A significant amount of literature has emphasised that entrepreneurial networking is an important action that entrepreneurs can engage in to acquire critical resources and enhance the performance of their firms (for reviews see Hoang and Antoncic, 2003; Hoang and Yi, 2015). Networks enable entre- preneurs to acquire critical resources, manage uncertainties and position their venture for success (Hallen and Eisenhardt, 2012; Phillips et al., 2013). Recent findings indicate that entrepreneurs develop networks to mitigate the uncertainties they perceive in starting and operating their ventures (Engel et al., 2017; Magnani and Zucchella, 2019; Zheng et al., 2020). Given that corruption and asso- ciated uncertainties can impact the ability of entrepreneurs to capture the value and the magnitude of the value captured (Baker et al., 2005; Baumol, 1990), networking can play a critical role in ensuring that entrepreneurs can create and capture appropriate value. Although the dominant focus of entre- preneurial networking literature has been on personal (e.g. 1. Introduction Developing nations are often fraught with corruption, which affects both business and society (Barkemeyer et al., 2018; Bryant and Javalgi, 2016; Olken and Pande, 2012). Indeed, scholars argue that corruption adversely impacts economic development and shifts the benefits from society to cor- rupt individuals (Alm et al., 2016; Gründler and Potrafke, 2019; Mo, 2001; Murphy et al., 1991). As entrepreneurship plays a key role in economic development and wealth creation (Baumol, 1990), this issue has received growing attention in the entrepreneurship literature. Researchers have predomin- antly utilised macro-level data to examine the relationship between corruption and entrepreneurial activities (Anokhin and Schulze, 2009; Dreher and Gassebner, 2013; Mohamadi et al., 2017; Wellalage et al., 2019a, 2019b). Interestingly, these studies present mixed findings. Although some scholars have emphasised that corruption has a ‘sanding’ (negative) impact on entrepreneurial activ- ities (De Rosa et al., 2015; Dutta and Sobel, 2016), others have highlighted its ‘greasing’ (enabling) effect on entrepreneurial activities (Dreher and Gassebner, 2013; Fisman and Svensson, 2007). p Furthermore, recent findings indicate that the effect of corruption varies based on the type of cor- ruption as well as on the types of entrepreneurs. In their study on private manufacturing small and medium-sized enterprises (SMEs) in Vietnam, Vu et al. (2018) find that various types of corruption (bribe) have different effects on firms’ financial performance. In particular, they find that some types of bribe do not affect firm performance, whereas bribes to obtain government contracts and licences harm firm performance. In contrast, firms that pay bribes for public services may have a higher Samuel Adomako et al. 2 performance compared to firms that do not. Similarly, scholars have recently examined whether the sanding and greasing effects of corruption vary according to the type of entrepreneur (Goel and Saunoris, 2019). They find that the sanding effect of corruption varies across types of entrepreneurs1 and it switches to greasing effect for nascent entrepreneurs. Although in developed countries entrepreneurs can rely on various institutions when starting and operating their businesses, entrepreneurs operating in developing countries often need to take actions to manage the uncertainties associated with operating in such weak institutional environments (Ahsan et al., 2020; Hunt, 2015; McMullen and Shepherd, 2006). This is because weak institutional environ- ments are characterised by underdevelopment of market-supporting formal infrastructures such as regulatory and legal systems (Khanna and Palepu, 1997; Puffer et al., 2010). available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S1744137421000011 Downloaded from https://www.cambridge.org/core. University College Birmingham, on 29 Jun 2021 at 09:09:21, subject to the Cambridge Core terms of use, 1. Introduction family and friends) and business ties (suppliers and advisors), a small number of studies have examined institutional networks2 that entre- preneurs develop with government officials to attain critical resources and special privileges (Adomako et al., 2020; Ge et al., 2019; Wang et al., 2018; Zhang et al., 2016; Zhou, 2013). Utilising insights from this literature, we argue that entrepreneurs who perceive the prevalence of corruption in the environments in which they operate will develop ties with government officials (e.g. ministers and regulators) who can help them protect their investments and attain success. However, developing such institutional networks would require financial resources beyond what is necessary to smoothly operate the firm. One operationalisation that has often been used in the strategic manage- ment literature to measure the financial resources a firm has to engage in strategic activities is financial 1(1) Individuals who are in the process of setting up a business (nascent entrepreneurs); (2) individuals who own or man- age a business that has been operational for more than 3 months but less than 42 months (new business owners); (3) indi- viduals who own or manage a business that has been operational for more than 42 months (established entrepreneurs). 2Institutional networks are relationships that firms develop with public entities (Kim and Lui, 2015). Some scholars have also referred to these ties as ‘political connections’ (Wang et al., 2018). We refer to these ties as ‘institutional networks’ as the relationships that firms in our sample develop are not solely with politicians (e.g. members of parliament and regional min- isters), but also with individuals working in various state institutions such as banks, taxation and regulatory entities. 1(1) Individuals who are in the process of setting up a business (nascent entrepreneurs); (2) individuals who own or man- age a business that has been operational for more than 3 months but less than 42 months (new business owners); (3) indi- viduals who own or manage a business that has been operational for more than 42 months (established entrepreneurs). 2Institutional networks are relationships that firms develop with public entities (Kim and Lui, 2015). Some scholars have also referred to these ties as ‘political connections’ (Wang et al., 2018). We refer to these ties as ‘institutional networks’ as the relationships that firms in our sample develop are not solely with politicians (e.g. 1(1) Individuals who are in the process of setting up a business (nascent entrepreneurs); (2) individuals who own or man- age a business that has been operational for more than 3 months but less than 42 months (new business owners); (3) indi- viduals who own or manage a business that has been operational for more than 42 months (established entrepreneurs). 2Institutional networks are relationships that firms develop with public entities (Kim and Lui, 2015). Some scholars have also referred to these ties as ‘political connections’ (Wang et al., 2018). We refer to these ties as ‘institutional networks’ as the relationships that firms in our sample develop are not solely with politicians (e.g. members of parliament and regional min- isters), but also with individuals working in various state institutions such as banks, taxation and regulatory entities. 2Institutional networks are relationships that firms develop with public entities (Kim and Lui, 2015). Some scholars have also referred to these ties as ‘political connections’ (Wang et al., 2018). We refer to these ties as ‘institutional networks’ as the relationships that firms in our sample develop are not solely with politicians (e.g. members of parliament and regional min- isters), but also with individuals working in various state institutions such as banks, taxation and regulatory entities. 1. Introduction members of parliament and regional min- isters), but also with individuals working in various state institutions such as banks, taxation and regulatory entities. Journal of Institutional Economics 3 slack (Ang and Straub, 1998; Cheng and Kesner, 1997; Voss et al., 2008). We, therefore, argue that financial slack, which reflects financial resources the firm possesses above what it needs for its current operations (Kiss et al., 2018; Kraatz and Zajac, 2001), will enable the effective development of these ties. Financial slack allows a firm to engage in strategic actions without putting itself at high risk by increasing debt and financially overextending. Financial slack is especially valuable for SMEs in coun- tries where corruption is prevalent as it is challenging to access credit in such environments (Wellalage et al., 2019a). In our study, using a sample of 212 Ghanaian SMEs from the manufacturing and service sectors, we examine the mediating effects of institutional networking on the relationship between perceived corruption and firm growth. Furthermore, we examine the moderating effect of financial slack on the relationship between perceived corruption and institutional networking. The institutional environ- ment in less developed countries differs significantly from that of more developed countries (Adomako, et al., 2018; Fainshmidt et al., 2018). Entrepreneurs operating in such environments have to actively navigate the business environments and manage uncertainties. As we have already mentioned, entrepreneurs who perceive corruption to be prevalent are likely to be more active in developing ties with individuals in government institutions (institutional networking). Such ties could buffer firms from the unpredictability of the business environment as well as give them privi- leged access to resources, which would likely enable them to attain higher performance (growth). g y g p g This study makes three main contributions. First, we contribute to the emerging literature exam- ining the relationship between corruption and firm performance. Although the extant literature pro- vides interesting insights, the findings from these studies are mixed and the mechanism through which corruption affects firm performance is unclear. Second, we examine the mediating effects of institu- tional networking to shed light on the mechanism through which corruption affects the performance of SMEs. Entrepreneurs who perceive corruption in the environments in which they operate, actively engage in actions to protect their investments. Such entrepreneurs develop relationships with officials in government, regulatory and financial institutions to mitigate the uncertainties, including opportun- istic behaviours by bribee, which enhances firm growth. available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S1744137421000011 Downloaded from https://www.cambridge.org/core. University College Birmingham, on 29 Jun 2021 at 09:09:21, subject to the Cambridge Core terms of use, 1. Introduction Third, we find that financial slack moderates the perceived corruption–institutional networking relationship. Financial slack allows entrepreneurs to initiate and develop relationships with institutional ties (e.g. government and officials), without put- ting the firm at risk by financially overextending. Our paper proceeds as follows. In the next section, we develop a theoretical model focusing on the relationships among perceived corruption, institutional networking and firm growth including the mod- erating effects of financial slack. We follow this with a section describing the research methodology and key findings. The final section outlines both the theoretical and practical implications of this study. 2. Theory and hypotheses development 2.1 Institutions and corruption 2.1 Institutions and corruption Institutions affect the allocation of entrepreneurial resources in a country (Baumol, 1990; Boudreaux, 2014; Grossman and Kim, 1995), and correspondingly entrepreneurs shape the institutions in which they operate by actively responding to the institutional environment (Henrekson and Sanandaji, 2011). Weak institutional environments provide a fertile ground for corruption to flourish and this necessi- tates action from entrepreneurs to manage associated uncertainties (McMullen and Shepherd, 2006; Tonoyan et al., 2010). In the context of developing economies, there is disorganised corruption, where bureaucrats (e.g. government officials, civil servants and bank managers) collect bribes to per- form their basic government functions (Shleifer and Vishny, 1993, Vu et al., 2018). This presents chal- lenges to entrepreneurs in operating their business. Consequently, even tasks that are considered as normal activities in developed institutional environments, such as attaining bank loans or business permits, can be challenging in environments fraught with corruption. The prevalence of corruption that the entrepreneurs perceive varies based on the type of industry in which they operate and the number of times they have to interact with bureaucrats. Some industries such as the oil and gas sector Samuel Adomako et al. 4 in Ghana (Ablo, 2019) are highly regulated. When such regulatory requirements are coupled with weak institutions, entrepreneurs are more likely to encounter corruption as the institutions responsible for enforcing the laws to dissuade civil servants and political elites from engaging in corrupt beha- viours are either unwilling to enforce them or incapable of doing so. Consequently, small firms will likely need more resources to attain the approvals needed to operate a business in this sector compared to, say, a small firm manufacturing furniture.3 y g Researchers argue that corruption increases the agency and transaction costs for entrepreneurs (Anokhin and Schulze, 2009; Avnimelech and Zelekha, 2015; Jimenez et al., 2017). As corruption entails government officials acting based on their self-interest, this has the potential to distort the func- tioning of markets and market competition (Svensson, 2005). However, weak institutional environ- ments, due to their bureaucracy and lack of supporting institutions, can hinder the entrepreneurial process and make corruption an attractive option. Under such conditions, corruption can grease the entrepreneurial process to prevent inordinate delays in procuring necessary approvals and/or resources (Bardhan, 1997; Dreher and Gassebner, 2013). 2.1 Institutions and corruption Entrepreneurs could receive special and beneficial treatment by engaging in corruption, such as bank loans, permit approvals, direct contracts and regulation favours (Acquaah, 2007; Dheer, 2017; Wellalage et al., 2019a, 2019b). Furthermore, entrepreneurs in developing countries have limited alternate opportunities to earn income (Ahsan et al., 2020), which may compel them to engage in bribery and other forms of corruption (e.g. circum- venting tax and business laws) to succeed. Consequently, entrepreneurs who perceive the prevalence of corruption in their environments may come to believe that engaging in corruption is the way to do business and attain success. Researchers suggest that individuals’ perception of how prevalent corrup- tion is in their environment can lead them to engage in similar practices and justify their actions (Gorsira et al., 2018; Rose-Ackerman, 2001; Tonoyan et al., 2010). The cost of not engaging in cor- ruption could be high for individuals who choose not to participate in this practice, and they will likely be ignored in favour of those who do – that is, pay to play (Della Porta and Vannucci, 1999). Indeed, Getz and Volkema (2001) argue that firms will engage in bribery if they perceive it to be prevalent, as reluctance to do so can significantly harm their company. Although bribing could grease the entrepreneurial process and enable firms to be successful, engaging in bribery in a weak institutional environment is not without risk. Anokhin and Schulze (2009) note that the bureaucrats whom the entrepreneur bribes may not perform the service despite getting paid for it and they may demand additional payments without fear of repercussion as the entrepreneur will have few, if any, options other than relying on them in such environments. To pre- vent such opportunistic behaviour, the entrepreneur and/or the key decision-makers will likely attempt to develop good relationships with various institutional actors. By developing good relationships with institutional ties such as government officials, bank managers, etc., they can minimise agency and transaction costs. In other words, good relationships with institutional ties could increase credit avail- ability, shorten the administrative approval process, provide access to privileged information, and, more importantly, mitigate opportunistic behaviours. Indeed, D’Aveni and Kesner (1993) emphasise that such elite connections enable firms to manipulate the external environment and sway other actors to behave in a manner that is beneficial to the firm. 3This is consistent with the notion of ‘local bribery environment’ that argues bribery is industry- and region-specific (Fisman and Svensson, 2007; Hanousek and Kochanova, 2016). This suggests that firms operating in certain industries/ regions would need to rely more on institutional ties to attain resources and/or approvals, and therefore might perceive a higher prevalence of corruption. available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S1744137421000011 Downloaded from https://www.cambridge.org/core. University College Birmingham, on 29 Jun 2021 at 09:09:21, subject to the Cambridge Core terms of use, 2.2 Perceived corruption and institutional networking Institutional networks may be broadly explained as the ties, contacts or relationships that the entre- preneurs develop with actors in public organisations for the purpose of obtaining privileged services (Acquaah, 2007; Luo et al., 2008). As discussed in the previous section, entrepreneurs who perceive corruption in their industry environment are likely to engage in corruption to protect their invest- ments and maintain their firms’ competitiveness (Getz and Volkema, 2001). This is more likely to be true in a context where corruption is prevalent and the cost of doing business is high (Goel and Saunoris, 2019; Mohamadi et al., 2017). Usually, poorly defined laws and unclear regulations increase the cost of doing business and this could motivate entrepreneurs to bribe bureaucrats to circumvent the burdensome process and sustain their firms’ competitiveness. Although corruption could facilitate the entrepreneurial process, especially in countries with weak institutional environments (De Rosa et al., 2015; Dreher and Gassebner, 2013), such environments also promote opportunistic behaviours that the firms must safeguard against (Luo, 2005; Tonoyan et al., 2010). Entrepreneurs engaging in bribery do not have the recourse of approaching the law in the event of opportunistic behaviour by the bribee nor can they draw up formal contracts to ensure that the services are rendered as per their payment agreements (Tonoyan et al., 2010). Researchers argue that entrepre- neurs can reduce the uncertainties they experience by taking action (McMullen and Shepherd, 2006), including designing an alternate mechanism to mitigate opportunism (Rose-Ackerman, 1999). Given these challenges, entrepreneurs seek to establish relationships with institutional ties to gain privileged access to resources as well as create a favourable environment for the firms (Acquaah, 2007; Dheer, 2017). In such environments, ‘who you know’ matters considerably as the ‘right’ connections can open access to resources and eliminate obstacles that firms typically encounter (D’Aveni and Kesner, 1993; Guseva, 2007). More importantly, such ties can mitigate opportunistic behaviour and reduce uncertainties (Adomako et al., 2020) as well as ensure that the corrupt transactions are performed as agreed (Lambsdorff, 2005). These arguments lead us to suggest the following hypothesis: H1: Perceived corruption is positively related to institutional networking. 2.1 Institutions and corruption Moreover, institutional ties can potentially enhance value creation and value capture, which in turn bolster firm performance (Feng et al., 2015; Lepak et al., 2007; Semrau and Sigmund, 2012; Sun et al., 2012). However, owners/CEOs of SMEs need to have sufficient financial resources to engage institu- tional ties and develop good relationships with them, as such connections are often developed by socialising in the same circles (Barton, 1985; Davis et al., 2003; Maclean et al., 2010, 2017). The chal- lenge of accessing credit in environments where corruption is prevalent (Wellalage et al., 2019a) makes 3This is consistent with the notion of ‘local bribery environment’ that argues bribery is industry- and region-specific (Fisman and Svensson, 2007; Hanousek and Kochanova, 2016). This suggests that firms operating in certain industries/ regions would need to rely more on institutional ties to attain resources and/or approvals, and therefore might perceive a higher prevalence of corruption. Journal of Institutional Economics 5 Journal of Institutional Economics 5 financial slack even more valuable. Next, we explain the hypothesised relationships among the vari- ables in the model. available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S1744137421000011 Downloaded from https://www.cambridge.org/core. University College Birmingham, on 29 Jun 2021 at 09:09:21, subject to the Cambridge Core terms of use, 2.4 The moderating role of financial slack Financial slack is the level of liquid assets (e.g. cash on hand) available for immediate deployment by a firm (Kiss et al., 2018; Kraatz and Zajac, 2001). Voss et al. (2008) suggest that financial slack is valu- able, but generic and less rare compared to other types of resource slacks. Given that SMEs, especially those in developing countries, are resource-constrained and are often confronted by challenges, such as limited access to credit (Terziovski, 2010; Wellalage et al., 2019a), financial slack can be considered a rare and valuable resource. This is because the possession of financial slack by SMEs enables them to engage in strategic organisational activities (Ang and Straub, 1998; Cheng and Kesner, 1997; Voss et al., 2008). SMEs are not restricted in the manner in which they can deploy financial slack and they can apply it to any activity they deem as strategically important for firm survival and success. That is, they can use their excess financial resources to engage in networking activities such as becom- ing members of exclusive sporting clubs and cultural organisations which would enable them to social- ise and develop relationships with elite ties (D’Aveni and Kesner, 1993). This could give SMEs privileged access to resources and other benefits. Given the opportunity cost of not engaging in cor- ruption in an environment where it is prevalent, along with the risk of losing competitiveness (Getz and Volkema, 2001; Tonoyan et al., 2010), entrepreneurs might more likely be motivated to establish relationships with elite ties (i.e. connections with important institutional actors) to influence the action of other actors (D’Aveni and Kesner, 1993) as well as mitigate opportunistic behaviours (Lambsdorff, 2005). In other words, although many SMEs may be motivated to engage in corruption in environments with high corruption, only those who have financial slack are able to engage in networking activities necessary to connect with elite ties. Although SMEs that lack financial slack could attempt to avail capital from other sources of finances such as loans and utilise debt capital to establish relationships with insti- tutional ties, the likelihood of attaining such loans in weak institutional environments is slim (Wellalage et al., 2019a). As a result, firms that do not have financial slack may be locked out from accessing oppor- tunities that privileged networks may beget (Della Porta and Vannucci, 1999; Getz and Volkema, 2001). 2.3 Perceived corruption, networking and firm performance A critical review of the literature indicates mixed findings regarding the effects of corruption on firm outcomes. Although some studies find corruption to be detrimental (De Rosa et al., 2015; Fisman and Svensson, 2007), others demonstrate that corruption is ideal for performance (Hanousek, and Kochanova, 2016; Vial and Hanoteau, 2010). These seemingly contrasting outcomes may be due to the fact that this relationship may be more nuanced and contingent on the agency of the entrepre- neurs. We contend that institutional networks serve as a lynchpin in ensuring that entrepreneurs can capture the value when they engage in corruption. Institutional ties provide three advantages to firms in weak institutional environments (Acquaah, 2007; Tonoyan et al., 2010; Wellalage et al., 2019a). First, institutional networks can provide valuable resources that are critical for firms’ growth, especially in environments with weak institutions and less transparent bureaucracies. Second, institu- tional networks reduce the length of the approval process required in the consumption of vital ser- vices. Thus, entrepreneurs who leverage on this could enhance their firms’ speed to market. Finally, and more importantly, these ties ensure that the bribes that entrepreneurs pay to ‘grease the wheel’ are not misappropriated by opportunistic actors. This is so because entrepreneurs who can establish good relationships with institutional ties can create a trust-based network and ensure that the other actors (that is, bribees) act in a manner that is favourable to the firm (D’Aveni and Kesner, 1993). Indeed, strong bonding with ties can create an environment for corruption to flourish (Tanzi, Samuel Adomako et al. 6 1998). Findings from prior studies indicate that good relationships with network ties enhance trust, which positively affects firms’ performance (Brüderl and Preisendörfer, 1998; Lin and Lin, 2016; Mackinnon et al., 2004). Thus, we state: H2: Institutional network mediates the effect of perceived corruption on SME growth. available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S1744137421000011 Downloaded from https://www.cambridge.org/core. University College Birmingham, on 29 Jun 2021 at 09:09:21, subject t 3.2 Sample and data collection The sampling frame for this study was derived from the Ghana Revenue Authority (GRA) databases. We contacted, by telephone, 800 firms to ask them for their participation in the study. Our sample met the following criteria: (1) independent, i.e. not part of any company group; (2) owned and operated by an entrepreneur or a team of entrepreneurs; (3) manufacturer of goods or a service provider; (4) employing a maximum of 250 full-time employees; the criteria we used to classify the firms in our sample as SMEs and (5) each respondent had to have direct executive authority for their respective firm. The survey questions were in a randomised order to counter bias effects. To attenuate the threat of common method bias (Podsakoff et al., 2003), we collected the data in two waves using a hand- delivery survey strategy. First, we approached the entrepreneurs with questionnaires in person to col- lect data on the independent variables (perceived corruption, institutional networking and the control variables). After two rounds of reminders, we received a total of 272 responses, representing a 34% response rate. We detected that 17 of the questionnaires were incomplete. These were therefore discarded, leaving 255 complete responses (approximately 32% response rate). Second, we collected data from the finance managers of the firms to measure financial slack and firm growth. After making two reminders by telephone, we received a total of 228 responses. We observed that 16 of the finance managers were also the founders/entrepreneurs of the firms. Hence, we discarded those 16 questionnaires to minimise common method bias. Overall, 212 complete responses across Time 1 and Time 2 were used for the analysis. This repre- sents a 26.5% response rate (i.e. [212/800] × 100). On average, the firms employ 22 full-time employees and have been in business for 8 years since their inception. The firms operate in multiple industries, including agro-processing (40%), food and beverage processing (10%), textile and garment manufac- turing (5%), security services (24%) and financial services (21%). Thus, the majority of the sampled firms operate in the manufacturing industry (55%) whereas a few provide services (45%). 2.4 The moderating role of financial slack Thus, the ‘financial buffer’ stemming from the possession of financial slack reduces the risk to the SMEs as it protects them from the uncertain outcomes of these strategic activities (Ang and Straub, 1998; Cheng and Kesner, 1997; Voss et al., 2008). It gives SMEs the flexibility to develop good relationships with different institutional ties that could help them protect their investments and attain future success. The foregoing reasoning leads us to suggest a more positive effect of perceived corruption on institu- tional networking when financial slack is higher. Accordingly, we hypothesise that: H3: Availability of financial slack amplifies the effect of perceived corruption on institutional networking. 3. Research method 3.1 Research setting: Ghana In testing our hypotheses, we used data obtained from SMEs operating in Ghana. We focused on Ghana because its environmental context was considered appropriate. First, many owners/CEOs of SMEs in the country have ties with politicians and bureaucrats (Acquaah, 2007). Second, corruption is pervasive, making the country rank 80th out of 180 countries according to Transparency International’s 2017 Corruption Perception Index (CPI) (Transparency International, 2017). This 7 Journal of Institutional Economics Journal of Institutional Economics high prevalence of corruption in Ghana presents unique challenges for entrepreneurs (Joseph, 2019; Moo and Eyiah, 2019). This makes this context a suitable environment in which to test the proposed hypotheses regarding the nexus between corruption and firm performance. Third, Ghana has struggled to be competitive on the global stage in terms of developing vibrant markets. Moreover, the institutional environment in Ghana presents an interesting context for the study because it differs significantly from those in more developed countries (Amankwah-Amoah, 2016; Fainshmidt et al., 2018). Overall, Ghana ranks 111th out of 137 countries, according to the 2017–2018 Global Competitiveness Report (GCR). Thus, Ghana offers a typical developing country’s perspective to test our conceptual model. available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S1744137421000011 Downloaded from https://www.cambridge.org/core. University College Birmingham, on 29 Jun 2021 at 09:09:21, subject to the Cambridge Core terms of u 3.4 Tests for potential biases We performed two major tests to check for potential biases. First, we checked the possibility of non- response bias by comparing respondents and non-respondents in terms of firm age, size, industry, foun- der age and education using Pearson’s chi-square test for discreet variables (Greenwood and Nikulin, 1996). We found no significant differences between the two groups. Thus, we concluded that non- response bias did not influence our dataset (Armstrong and Overton, 1977; Rogelberg and Stanton, 2007). Second, we followed the procedure suggested by Lindell and Whitney (2001) to test for potential common method variance. Accordingly, a marker test was used to assess the correlations between the marker variable and the main constructs. The item ‘I enjoy coming up with new ideas for products’ was used as a marker variable, which is considered a measure of intrinsic interest in entrepreneurship, a variable which is theoretically unrelated to firm growth. The results indicate a non-significant correl- ation ranging from −0.01 to 0.03. The results of partial correlations were also significant, even after we had discarded the effect of common method bias. A 95% sensitivity analysis was used to verify this con- clusion. Overall, we believe that issues related to common method bias are negligible in this study. 3.5 Validity and reliability assessment The reliability and validity of the constructs were subjected to exploratory factor analysis. A confirma- tory factor analysis (CFA) was performed to refine the items using LISREL 9.30 and covariance matri- ces as input data. The results of the CFA provide adequate fit for the data: χ2 (degree of freedom) = 920.44 (590); p < 0.00; RMSEA = 0.06; NNFI = 0.95; CFI = 0.97 and (SRMR) = 0.07. Also, the factor loadings for each construct were significant at p < 0.01. This indicates the convergent validity of the measures (Bagozzi and Yi, 1988). The reliability of the constructs was assessed using composite reliability, average variance extracted (AVE), and the highest shared variance (HSV) of the constructs. All the indices were greater than the recommended threshold value of 0.70 (Bagozzi and Yi, 1988). Utilising Fornell and Larcker’s (1981) approach, the discriminant validity of each construct was assessed by investigating whether the AVE for each construct was greater than the shared variances of each pair of constructs. Discriminant validity was determined as the AVE for each construct was larger than the HSV between each pair of constructs. 3.3 Measure of constructs In line with the literature, a seven-point multi-item scale was used to measure the constr the literature, a seven-point multi-item scale was used to measure the constructs. We measured perceived corruption (α = 0.93) using a six-item scale from previous studies (Collins et al., 2016; Doh et al., 2003; Uhlenbruck et al., 2006). This scale taps the extent to which entrepre- neurs perceive corruption as pervasive within their politico-economic institutional environment. Institutional networking (α = 0.88) was assessed using a four-item scale from Acquaah (2007). This scale measures network relationships with government officials and politicians. Financial slack: The approach used by Voss et al. (2008) was utilised to assess the firms’ cash reserves at the end of the 2018 financial year to measure financial slack. Firm size was controlled by dividing cash reserves by the venture’s total expenses in the 2018 financial year. To measure firm growth, the dependent variable, the finance managers were asked in the survey to record the level of employment growth in their firms 3 years before the data collection. Using these data and following previous studies (Brouwer et al., 1993; Robson and Obeng, 2008), we included an annualised growth rate for employment in the ordinary least squares regression. Control variables: Several control variables such as firm size, firm age, industry type and founder age/education were used because they were considered important factors which might influence the Samuel Adomako et al. 8 research model. Firm size was measured with the number of full-time employees, whereas firm age was captured as the number of years the business has operated since its first sales. Industry was measured with a dummy variable, with ‘0’ indicating manufacturing industry and ‘1’ indicating otherwise. Finally, we controlled for founder age and education (‘1’ = ‘High School’, ‘2’ = ‘Higher National Diploma’, ‘3’ = ‘Bachelor’s Degree’, ‘4’ = ‘Master’s Degree’ and ‘5’ = ‘Doctoral Degree’). available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S1744137421000011 Downloaded from https://www.cambridge.org/core. University College Birmingham, on 29 Jun 2021 at 09:09:21, subject to the Cambridge Core terms of use, 4. Results The descriptive statistics for the model variables are presented in Table 1. Before conducting the main regression analyses, the continuous variables were mean-centred to alleviate the potential multicolli- nearity concerns associated with moderating hypotheses (Aiken et al., 1991). The results show that the highest variance inflation factor (VIF) is 4.19, which is well below the recommended threshold value of 10 (Neter et al., 1990). We also checked the data for potential violations of normality assumptions and outliers. The results suggested no significant violations. Thus, the data were deemed suitable for the regression analysis. The hierarchical regression was then used to test the hypotheses. Table 2 presents the regression results. In models 1–4, the dependent variable is institutional networking. Model 1 includes all the control variables. In model 2, we test the effect of perceived corruption on institutional networking. The results showed that perceived corruption has a significant positive influence on institutional network- ing (β = 0.25, p < 0.01), thus supporting hypothesis 1. When financial slack was added in model 3, the at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S1744137421000011 ded from https://www.cambridge.org/core. University College Birmingham, on 29 Jun 2021 at 09:09:21, subject to the Cambr 3 4 5 6 0.02 0.01 0.00 0.00 0.23** 0.08 0.08 0.14* 0.13* 0.17* 0.09 0.17* 0.09 0.25** 0.03 0.16* 0.24 0.20 el 8 1* 1* 5 0* 4 2 stitutional networking Models 5–8: firm growth Model 3 Model 4 Model 5 Model 6 Model 7 Mode 0.19* 0.18*** 0.09* 0.10* 0.10* 0.11 0.11* 0.11* −0.10* −0.10* −0.10* −0.11 0.05 0.05 0.04 0.04 0.05 0.05 0.06 0.07* 0.09* 0.09* 0.09* 0.10 0.10* 0.11* 0.14 0.14 0.14 0.14 0.24* 0.24* 0.19* 0.02 0.18* 0.18* 0.22* 0.22* 0.18* 0.18 0.46* 0.22* 0.22 5.71* 7.28* 2.66 4.28* 5.95* 6.77 0.23 0.31 0.15 0.19 0.25 0.27 0.06 0.08 – 0.04 0.06 0.02 2.19 1.31 2.01 1.29 2.53 2.54 Journal of Institutional Economics 11 Figure 1. Interaction effect of perceived corruption with financial slack on institutional networking. Figure 1. Interaction effect of perceived corruption with financial slack on institutional networking. effect of perceived corruption on institutional networking remained significant (β = 0.24, p < 0.01). In model 4, we examine the moderating effect of financial slack on the relationship between perceived corruption and institutional networking. 4. Results The results suggest that the interaction term is positive and significant (β = 0.46, p < 0.01), indicating that financial slack positively moderates the relationship between perceived corruption and institutional networking. To establish the direction of the two-way interaction, we followed the procedure advanced by Cohen et al. (2003) to investigate the moderating hypothesis. The graphical representation in Figure 1 shows a stronger positive relationship between perceived corruption and institutional networking when financial slack is high (versus low financial slack). Simple slope analysis also shows that the effect of perceived corruption on institutional net- working is significant when financial slack is high (t = 2.99, p < 0.01) but not when it is low (t = 0.45, ns). This indicates support for hypothesis 3. pp yp The dependent variable in models 5–8 is firm growth. The results in these models allow us to test the mediating hypothesis (H2). The mediating hypothesis was tested using the approach suggested by Baron and Kenny (1986) and refined by Zhao et al. (2010). According to this approach, mediation relationships must meet three conditions. First, the independent variable and the mediator should be related significantly. The result in model 2 shows that the association between perceived corruption and institutional networking is positive and significant (β = 0.25, p < 0.01). Second, the mediating vari- able and the dependent variable should be significantly related to each other. The result in model 7 shows that institutional networking and firm growth are significantly related to each other (β = 0.19, p < 0.01). Third, the effect of the independent variable on the dependent variable must not be significant when the mediating variable is added to the regression equation. In model 8, when per- ceived corruption was included in the regression equation, institutional networking had a positive influence on firm growth (β = 0.19, p < 0.01). However, the effect of perceived corruption on firm growth was not statistically significant (β = 0.02, ns). Overall, the results of the mediation analysis sug- gest that institutional networking mediates the effect of perceived corruption on firm growth. Thus, hypothesis 2 is supported. To gain additional insights into the mediation results, we utilised the Sobel test to investigate the statistical significance of the indirect effects of institutional networking on firm growth (MacKinnon and Dwyer, 1993; Sobel, 1982). 4. Results Using this test, we calculated the magnitude of the unstandardised indirect effect and its associated standard error. The results of this test indicate that the indirect effects of institutional networking on firm growth (z = 2.98, p < 0.01) were consistent with our prediction and statistically significant. This provides further support for full mediation (H2). vailable at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S1744137421000011 ownloaded from https://www.cambridge.org/core. University College Birmingham, on 29 Jun 2021 at 09:09:21, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S1744137421000011 Downloaded from https://www.cambridge.org/core. University College Birmingham, on 29 Jun 2021 at 09:09:21, subject t 5. Discussion and conclusion Drawing on institutional theory and corruption literature, this paper argues that perceived corruption affects firm growth through institutional networking. Accordingly, we put forward a moderated medi- ation model that examined the relationship between perceived corruption and firm growth. Using data from 212 SMEs in Ghana, the analysis revealed that the relationship between corruption and firm growth is mediated by institutional networking, and this relationship is enhanced at high levels of financial slack. In other words, the study shows that perceived corruption is positively associated with institutional networking, which in turn positively impacts SMEs’ growth. Furthermore, the research revealed that the relationship between perceived corruption and institutional networking is stronger for SMEs that possess higher financial slack. g p g Theoretically, our study makes three main contributions. First, we contribute to the emerging lit- erature examining the effect of corruption on firm performance. Given that the findings from previous studies are mixed, our study sheds additional light on this relationship by testing the effect of perceived corruption on SMEs’ firm growth. Thus, our findings complement the small number of studies that have demonstrated the positive relationship between corruption and firm performance (Hanousek, and Kochanova, 2016; Vial and Hanoteau, 2010). Second, our study provides a nuanced understand- ing of the mechanism, institutional networking, through which corruption positively affects firm per- formance. In doing so, we open up the ‘black box’ of the relationship between corruption and firm performance. In the main, our findings provide useful insights into how institutional networking med- iates the corruption–firm performance linkage. In this way, our study contributes to extant research efforts exploring the effects of social ties on firm growth/performance in emerging economies (Guo et al., 2014; Lin and Lin, 2016). Third, previous studies have considered institutional networking and financial slack in isolation. Therefore, we extend the literature by simultaneously testing the rela- tionships among these variables in a contingency model. The outcomes show that financial slack amp- lifies the relationship between perceived corruption and institutional networking. By doing so, we fill an important research void in the entrepreneurship literature regarding the ‘value’ of financial slack for SMEs in environments where it is challenging to access credit (Wellalage et al., 2019a). Our study also provides useful insights to SME founders and policy makers in developing countries. 4.1 Robustness checks The robustness of the results was established by performing several analyses. First, we undertook an alternative regression analysis utilising return on assets (ROA) (Venkatraman and Ramanujam, 1986) as our dependent variable. This measure was computed as the ratio of operating income to total assets (Lee et al., 2016; Florio and Leoni, 2017). The results using ROA replicated our initial regression Samuel Adomako et al. 12 results. Second, we used financial resource availability (Cooper et al., 1994; Wiklund and Shepherd, 2005) as a proxy for financial slack and tested its effect on the relationship between perceived corrup- tion and institutional networking (β = 0.33, p < 0.01). Finally, in the second wave of the survey, we obtained information on bribery incidence from the finance managers and used this measure (‘It is common for firms in my line of business to pay some irregular ‘additional payments/gifts’ to get things done concerning customs, taxes, licences, regulations, and services’) as an alternative measure of cor- ruption (Hanousek and Kochanova, 2016). Accordingly, we re-estimated the mediating effect of insti- tutional networking on the relationship between corruption and firm growth. The results were consistent with the findings in Table 2. Thus, overall, the results obtained from the robustness test were in line with our initial findings. Third, we performed the Durban–Wu–Hausman test to examine potential endogeneity in the model by using instrumental variables (Wooldridge, 2002). The criteria for instrumental variables consisted of those that affect the level of corruption, but which do not affect firm growth (Semadeni et al., 2014). Consistent with the recommendations of Semadeni et al. (2014), we employed multiple instrumental variables. Specifically, weak institutional environment and pressures from fam- ily members for financial support met the aforementioned selection criteria and were used as instru- mental variables. Accordingly, we regressed each variable separately on perceived corruption. We then saved the residuals and entered them into the model. The p-values were not significant for either residual, indicating that the effects of endogeneity are likely minimal in our research model. available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S1744137421000011 Downloaded from https://www.cambridge.org/core. University College Birmingham, on 29 Jun 2021 at 09:09:21, subject to the Cambridge Core terms of use, 5. Discussion and conclusion First, given the importance of institutional networking in an environment where corruption is preva- lent, SMEs would be better served by deploying resources towards developing and nourishing Journal of Institutional Economics 13 institutional networking. Such actions can help to mitigate the uncertainties associated with operating in countries with weak institutional environments and where corruption is very prevalent. The finding regarding the effect of networking suggests that entrepreneurs’ relationship with the government and bureaucratic officials can go a long way in helping SMEs gain access to resources and privileges that often are crucial for firm survival and success in such environments. As firms increasingly use insti- tutional ties to access resources, future success is likely to be predicated on their ability to develop and maintain diverse institutional ties. Thus, there is a need to develop and maintain ties across political parties and agencies to mitigate network decay or obsolescence due to changes in government and accompanying administrative changes. Relatedly, the finding concerning the moderating effect of financial slack on the link between perceived corruption and institutional networking implies that SMEs that possess financial slack will likely be in a better position to establish good relationships with diverse institutional ties. This is because it is challenging to attain capital (e.g. loans) in such environments. However, it is important to recognise that, although corruption may allow entrepre- neurs to temporarily remove bureaucratic bottlenecks that significantly increase their transaction costs, a culture of unrestricted bribery may encourage opportunistic civil servants to intentionally impose such bottlenecks to enrich themselves. This could increase the cost of doing business and adversely affect economic development.4 4We thank one of the reviewers for encouraging us to elaborate on the long-term implications of corruption on entrepre- neurial activities in weak institutional environments. available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S1744137421000011 Downloaded from https://www.cambridge.org/core. University College Birmingham, on 29 Jun 2021 at 09:09:21, subject to the Cambridge Core terms of us 6. Limitations and future research directions There are some limitations of our study, which can inform the direction for future research. First, this study focuses solely on Ghana. Therefore, researchers should be cautious of generalising our findings to other developing countries, given the variation in the political and socio-economic cultures and practices across developing countries. Consequently, it may be useful for future research to test our model with data from a more diverse and larger sample across national/cultural settings. Second, future studies could determine the direction of the hypothesised relationships in our research model by using a longitudinal method to investigate whether entrepreneurs’ perceptions of corruption and institutional network change over time and how such changes influence firm growth. Relatedly, future research could examine how firm performance, over time, influences the perception of corrup- tion and institutional networking. Fourth, recent research has highlighted the contingencies such as gender (Wellalage et al., 2019b), types of entrepreneurships (Goel and Saunoris, 2019), and types of bribes (Vu et al., 2018) that can affect the relationship between corruption and firm performance. Future research can incorporate these contingencies into our theoretical model to better understand if and how these factors affect our findings. Furthermore, researchers can also examine the impact of other forms of financial resources (e.g. loans) on the relationship between perceived corruption and institutional networking. Finally, researchers can build on our study by examining a broader set of firm outcomes. For instance, Lee and Weng (2013) find that bribery in the home country can dampen firm exports. This suggests that, although corruption could enhance firms’ performance (growth), it may also lower their competitiveness in international markets. We hope that the insights from our study will prompt new research directions on this topic, especially in developing economies. Ablo, A. D. (2019), ‘Actors, Networks and Assemblages: Local Content, Corruption and the Politics of SME’s Participation in Ghana’s oil and gas Industry’, International Development Planning Review, 41(2): 193–214. Acquaah, M. (2007), ‘Managerial Social Capital, Strategic Orientation and Organizational Performance in an Emerging Economy’, Strategic Management Journal, 28(12): 1235–1255. Adomako, S., R. A. Opoku and K. Frimpong (2018), ‘Entrepreneurs’ Improvisational Behavior and new Venture Performance: Firm-Level and Institutional Contingencies’, Journal of Business Research, 83(4): 10–18. y g g Adomako, S., R. A. Opoku and K. Frimpong (2018), ‘Entrepreneurs’ Improvisational Behavior and new Venture Performance: Firm-Level and Institutional Contingencies’, Journal of Business Research, 83(4): 10–18. Ablo, A. D. (2019), ‘Actors, Networks and Assemblages: Local Content, Corruption and the Politics of SME’s Participation in Ghana’s oil and gas Industry’, International Development Planning Review, 41(2): 193–214. Acquaah, M. (2007), ‘Managerial Social Capital, Strategic Orientation and Organizational Performance in an Emerging Economy’ Strategic Management Journal 28(12): 1235–1255 Ablo, A. D. (2019), ‘Actors, Networks and Assemblages: Local Content, Corruption and the Politics of SME’s Participation in Ghana’s oil and gas Industry’, International Development Planning Review, 41(2): 193–214. References (1990), ‘Entrepreneurship: Productive, Unproductive, and Destructive’, Journal of Political Ec 893–921. Boudreaux, C. J. (2014), ‘Jumping off of the Great Gatsby Curve: How Institutions Facilitate Entrepreneurship and Intergenerational Mobility’, Journal of Institutional Economics, 10(2): 231–255. g y f Brouwer, E., A. Kleinknecht and J. O. N. Reijnen (1993), ‘Employment Growth and Innovation at the Firm Level’, Journal of Evolutionary Economics, 3(2): 153–159. y Brüderl, J. and P. Preisendörfer (1998), ‘Network Support and the Success of Newly Founded Business’, Small Business Economics, 10(3): 213–225. Bryant, C. E. and R. G. Javalgi (2016), ‘Global Economic Integration in Developing Countries: The Role of Corruption and Human Capital Investment’, Journal of Business Ethics, 136(3): 437–450. Cheng, J. L. and I. F. Kesner (1997), ‘Organizational Slack and Response to Environmental Shifts: The Impact of Resource Allocation Patterns’, Journal of Management, 23(1): 1–18. Child, J. and L. H. Hsieh (2014), ‘Decision Mode, Information and Network Attachment in the Internationalization of SMEs: A Configurational and Contingency Analysis’, Journal of World Business, 49(4): 598–610. Cohen, J., P. Cohen, S. G. West and L. S. Aiken (2003), Applied Multiple Regression/Correlation Analysis for the Behavioral Sciences (3rd edn), New Jersey: Lawrence Erlbaum. Child, J. and L. H. Hsieh (2014), ‘Decision Mode, Information and Network Attachment in the Internationalization of SMEs: A Configurational and Contingency Analysis’, Journal of World Business, 49(4): 598–610. Cohen, J , P Cohen, S G West and L S Aiken (2003), Applied Multiple Regression/Correlation Analysis for the Behavioral Cohen, S. G. West and L. S. Aiken (2003), Applied Multiple Regression/Correlation Analysis for the Behavioral d edn), New Jersey: Lawrence Erlbaum. Collins, J. D., J. S. McMullen and C. R. Reutzel (2016), ‘Distributive Justice, Corruption, and Entrepreneurial Behaviour’, Small Business Economics, 47(4): 981–1006. ( ) Cooper, A. C., F. J. Gimeno-Gascon and C. Y. Woo (1994), ‘Initial Human and Financial Capital as Predictors of new Venture Performance’, Journal of Business Venturing, 9(5): 371–395. f g D’Aveni, R. A. and I. F. Kesner (1993), ‘Top Managerial Prestige, Power and Tender Offer Response: A Study of Elite Social Networks and Target Firm Cooperation During Takeovers’, Organization Science, 4(2): 123–151. Davis, G. F., M. Yoo and W. E. Baker (2003), ‘The Small World of the American Corporate Elite, 1982–2001’, Strategic Organization, 1(3): 301–326. Della Porta, D. and A. Vannucci (1999), Corrupt Exchanges: Actors, Resources, and Mechanisms of Political Corruption, New York: Aldine de Gruyter. De Rosa, D., N. Gooroochurn and H. References Samuel Adomako et al. 14 Adomako, S., K. Frimpong, A. Danso, J. Amankwah-Amoah, M. Uddin and K. Kesse (2020), ‘Home Country Institutional Impediments and International Expansion of Developing Country SMEs’, International Business Review, doi.org/10.1016/j. ibusrev.2020.101716. Ahsan, M., S. Adomako and K. Mole (2020), ‘Perceived Institutional Support and Small Venture Performance: The Mediating Role of Entrepreneurial Persistence’, International Small Business Journal. https://doi.org/10.1177/0266242620943194. Aiken, L. S., S. G. West and R. R. Reno (1991), Multiple Regression: Testing and Interpreting Interactions, Newbury Park, CA: Sage. Alm, J., J. Martinez-Vazquez and C. McClellan (2016), ‘Corruption and Firm Tax Evasion’, Journal of Economic Behavior & Organization, 100(124): 146–163. Amankwah-Amoah, J. (2016), ‘The Evolution of Science, Technology and Innovation Policies: A Review moah, J. (2016), ‘The Evolution of Science, Technology and Innovation Policies: A Review of the Ghanaian , Technological Forecasting and Social Change, 110(C): 134–142. Experience’, Technological Forecasting and Social Change, 110(C): 134–142. Ang, S. and D. W. Straub (1998), ‘Production and Transaction Economies and IS Outsourcing: A Study of the US Banking Industry’, MIS Quarterly, 22(4): 535–552. y y Anokhin, S. and W. S. Schulze (2009), ‘Entrepreneurship, Innovation, and Corruption’, Journal of Business Venturing, 24(5): 465–476. Armstrong, J. S. and T. Overton (1977), ‘Estimating Non-Response Bias in Mail Surveys’, Journal of Marketing Research, 14(3): 396–402. Avnimelech, G. and Y. Zelekha (2015), ‘The Impact of Corruption on Entrepreneurship’, in T. Wolf, and T. Issa (eds), International Business Ethics and Growth Opportunities, Hershey, PA: IGI Global, pp. 282–294. Bagozzi, R. P. and Y. Yi (1988), ‘On the Evaluation of Structural Equation models’, Journal of Academy of Marketing Science, 16(1): 74–94. Baker, T., E. Gedajlovic and M. Lubatkin (2005), ‘A Framework for Comparing Entrepreneurship Processes Across Nations’, Journal of International Business Studies, 36(5): 492–504. Bardhan, P. (1997), ‘Corruption and Development: A Review of Issues’, Journal of Economic Literature, 35(3 ), ‘Corruption and Development: A Review of Issues’, Journal of Economic Literature, 35(3): 1320–1346. Barkemeyer, R., L. Preuss and M. Ohana (2018), ‘Developing Country Firms and the Challenge of Corruption: Do Company Commitments Mirror the Quality of National-Level Institutions?’, Journal of Business Research, 90(C): 26–39. Baron, R. M. and D. A. Kenny (1986), ‘The Moderator-Mediator variable Distinction in Social Psychological Research: Conceptual, Strategic, and Statistical Considerations’, Journal of Personality and Social Psychology, 51(6): 1173–1182. Barton, A. H. (1985), ‘Determinants of Economic Attitudes in the American Business Elite’, American Journal of Sociology, 91(1): 54–87. Baumol, W. J. References Görg (2015), ‘Corruption and Productivity: Firm-Level Evidence’, Jahrbücher für Nationalökonomie und Statistik, 235(2): 115–138. Journal of Institutional Economics 15 Dheer, R. J. (2017), ‘Cross-national Differences in Entrepreneurial Activity: Role of Culture and Institutional Factors’, Small Business Economics, 48(4): 813–842. Rodriguez, K. Uhlenbruck, J. Collins and L. Eden (2003), ‘Coping with Corruption in Foreign Markets’, Management Perspectives, 17(3): 114–127. Dreher, A. and M. Gassebner (2013), ‘Greasing the Wheels? The Impact of Regulations and Corruption on Firm Entry’, Public Choice, 155(3–4): 413–432. Dutta, N. and R. Sobel (2016), ‘Does Corruption Ever Help Entrepreneurship?’, Small Business Economics, 47(1): 179–199. Engel, Y., M. Kaandorp and T. Elfring (2017), ‘Toward a Dynamic Process Model of Entrepreneurial Networking Under Uncertainty’, Journal of Business Venturing, 32(1): 35–51. Fainshmidt, S., W. Q. Judge, R. V. Aguilera and A. Smith (2018), ‘Varieties of Institutional Systems: A Context of Understudied Countries’, Journal of World Business, 53(3): 307–322. Feng, X., A. C. Johansson and T. Zhang (2015), ‘Mixing Business with Politics: Political Participation by Entrepreneurs in China’, Journal of Banking & Finance, 59(10): 220–235. f g Fisman, R. and J. Svensson (2007), ‘Are Corruption and Taxation Really Harmful to Growth? Firm Level Evidence’, Journal of Development Economics, 83(1): 63–75. p Florio, C. and G. Leoni (2017), ‘Enterprise Risk Management and Firm Performance: The Italian Case’, British Accounting Review, 49(1): 56–74. Fornell, C. and D. F. Larcker (1981), ‘Evaluating Structural Equation Models with Unobservable Variables and Measurement Error’, Journal of Marketing Research, 18(1): 39–50. Ge, J., M. Carney and F. Kellermanns (2019), “Who Fills Institutional Voids? Entrepreneurs’ Utilization of Political and Family Ties in Emerging Markets”, Entrepreneurship Theory and Practice, 43(6): 1124–1147. Getz, K. A. and R. J. Volkema (2001), ‘Culture, Perceived Corruption, and Economics: A Model of Predictors and Outcomes’, Business & Society, 40(1): 7–30. Goel, R. K. and J. W. Saunoris (2019), ‘International Corruption and its Impacts Across Entrepreneurship Type and Decision Economics, 40(5): 475–487. Gorsira, M., A. Denkers and W. Huisman (2018), ‘Both Sides of the Coin: Motives for Corruption among Public Officials and Business Employees’, Journal of Business Ethics, 151(1): 179–194. Greenwood, P. E. and M. S. Nikulin (1996), A Guide to Chi-Squared Testing, New York: Wiley. Grossman, H. I. and M. Kim (1995), ‘Swords or Ploughshares? A Theory of the Security of Claims to Property’, Journal of Political Economy, 103(6): 1275–1289. Gründler, K. and N. References Potrafke (2019), ‘Corruption and Economic Growth: New Empirical Evidence’, Europ Political Economy, 60(C): 101810. y Guo, H., E. Xu and M. Jacobs (2014), ‘Managerial Political Ties and Firm Performance During Institutional Transitions: An Analysis of Mediating mechanisms’, Journal of Business Research, 67(2): 116–127. y g f Guseva, A. (2007), ‘Friends and Foes: Informal Networks in the Soviet Union’, East European Quarterly, 41(3): 323–347. Hallen, B. L. and K. M. Eisenhardt (2012), ‘Catalyzing Strategies and Efficient tie Formation: How Entrepreneurial Firms Obtain Investment Ties’, Academy of Management Journal, 55(1): 35–70. Hanousek, J. and A. Kochanova (2016), ‘Bribery Environments and Firm Performance: Evidence from CE European Journal of Political Economy, 43(C): 14–28. Henrekson, M. and T. Sanandaji (2011), ‘The Interaction of Entrepreneurship and Institutions’, Journal of Institutional Economics, 7(1): 47–75. Hoang, H. and B. Antoncic (2003), ‘Network-based Research in Entrepreneurship: A Critical Review’, Journal of business venturing, 18(2): 165–187. Hoang, H. and A. Yi (2015), ‘Network-based Research in Entrepreneurship: A Decade in Review’, Foundations and Trends in Entrepreneurship, 11(1): 1–54. Hunt, R. A. (2015), ‘Contagion Entrepreneurship: Institutional Support, Strategic Incoherence, and the Social Costs of Over-Entry’, Journal of Small Business Management, 53(sup1): 5–29. Jimenez, A., J. C. Puche-Regaliza, J. A. Jiménez-Eguizábal and I. Alon (2017), ‘Political Discretion and Corruption: The Impact of Institutional Quality on Formal and Informal Entrepreneurship’, European Journal of International Management, 11(3): 280–300. g ( ) Joseph, A. O. (2019), Public Officials’ Defense of Bribery as a Culturally Acceptable Behavior in Ghana. Unpublished doctoral dissertation. Khanna, T. and K. Palepu (1997), ‘Why Focused Strategies may be Wrong for Emerging Markets’, Harvard Business Review, 75(4): 41–51. Kim, Y. and S. S. Lui (2015), ‘The Impacts of External Network and Business Group on Innovation: Do the Types of Innovation Matter?’, Journal of Business Research, 68(9): 1964–1973. Kiss, A. N., S. Fernhaber and P. P. McDougall-Covin (2018), ‘Slack, Innovation, and Export Intensity: Implications for Small- and Medium-Sized Enterprises’, Entrepreneurship Theory and Practice, 42(5): 671–697. Samuel Adomako et al. 16 Kraatz, M. S. and E. J. Zajac (2001), ‘How Organizational Resources Affect Strategic Change and Performance in Turbulent Environments: Theory and Evidence’, Organization Science, 12(5): 632–657. Lambsdorff, J. G., M. Taube and M. Schramm (2004), The new institutional economics of corruption. Routle Lee, S. H. and D. H. Weng (2013), ‘Does Bribery in the Home Country Promote or Dampen Firm Exports?’, Strategic Management Journal, 34(12): 1472–1487. Lee, K. H., B. C. References Cin and E. Y. Lee (2016), ‘Environmental Responsibility and Firm Performance: The Application of an Environmental, Social and Governance Model’, Business Strategy and the Environment, 25(1): 40–53. Lee, K. S., G. H. Lim and S. J. Tan (1999), ‘Dealing with Resource Disadvantage: Generic Strategies for SMEs’, Small Business Economics, 12(4): 299–311. Lepak, D. P., K. G. Smith and M. S. Taylor (2007), ‘Value Creation and Value Capture: A Multilevel perspectiv Management Review, 32(1): 180–194. Lin, F. J. and Y. H. Lin (2016), ‘The Effect of Network Relationship on the Performance of SMEs’, Journal of Business Research, 69(5): 1780–1784. Lindell, M. K. and D. J. Whitney (2001), ‘Accounting for Common Method Variance in Cross-Sectional Research Designs’, Journal of Applied Psychology, 86(1): 114–121. f pp y gy Ling, Y., Z. Simsek, M. H. Lubatkin and J. F. Veiga (2008), ‘The Impact of Transformational CEOs on the Performance of Small-to Medium-Sized Firms: Does Organizational Context Matter?’, Journal of Applied Psychology, 93(4): 923–934. Luiz, J. M. and C. Stewart (2014), ‘Corruption, South African Multinational Enterprises and Institutions in Africa’, Journal of Business Ethics, 124(3): 383–398. Luo, Y. (2005), ‘Transactional Characteristics, Institutional Environment and Joint Venture Contracts’, Journal of International Business Studies, 36(2): 209–230. Luo, X., M. K. Hsu and S. S. Liu (2008), ‘The Moderating Role of Institutional Networking in the Customer Orientation– Trust/Commitment–Performance Causal Chain in China’, Journal of the Academy of Marketing Science, 36(2): 202–214. MacKinnon, D. P. and J. H. Dwyer (1993), ‘Estimating Mediated Effects in Prevention Studies’, Evaluation 144–158. MacKinnon, D., K. Chapman and A. Cumbers (2004), ‘Networking, Trust and Embeddedness amongst SMEs in the Aberdeen Oil Complex’, Entrepreneurship & Regional Development, 16(2): 87–106. p p p g p Maclean, M., C. Harvey and R. Chia (2010), ‘Dominant Corporate Agents and the Power Elite in France and Britain’, Organization Studies, 31(3): 327–348. g Maclean, M., C. Harvey and G. Kling (2017), ‘Elite Business Networks and the Field of Power: A Matter of Class?’, Theory, Culture & Society, 34(5–6): 127–151. Magnani, G. and A. Zucchella (2019), ‘Coping with Uncertainty in the Internationalisation Strategy’, International Marketing Review, 36(1): 131–163. McMullen, J. S. and D. A. Shepherd (2006), ‘Entrepreneurial Action and the Role of Uncertainty in the Theory of the Entrepreneur’, Academy of Management Review, 31(1): 132–152. p y f g Mo, P. H. (2001), ‘Corruption and Economic Growth’, Journal of Comparative Economics, 29(1): 66–79. Mohamadi, A., J. Peltonen and J. References Wincent (2017), ‘Government Efficiency and Corruption: A Country-Level Study with Implications for Entrepreneurship’, Journal of Business Venturing Insights, 8(C): 50–55. Mohamadi, A., J. Peltonen and J. Wincent (2017), ‘Government Efficiency and Corruption Implications for Entrepreneurship’, Journal of Business Venturing Insights, 8(C): 50–55. Moo, F. and A. Eyiah (2019), ‘Factors Influencing the Growth of Small and Medium Construction Firms in Northern Ghana’, Journal of African Business, 21(3): 416–431. Murphy, K. M., A. Shleifer and R. W. Vishny (1991), ‘The Allocation of Talent: Implications for Growth’, Quarterly Journal of Economics, 106(2): 503–530. W. Wasserman and M. H. Kutner (1990), Applied Linear Statistical Models (3rd edn), Homewood, IL: Irwin. erman and M. H. Kutner (1990), Applied Linear Statistical Models (3rd edn), Homewood, IL: Irwin. R Pande (2012) ‘Corruption in Developing Countries’ Annual Review of Economics 4(1): 479–509 Neter, J., W. Wasserman and M. H. Kutner (1990), Applied Linear Statistical Models (3rd edn), Homewood, IL: Irwin. Olken, B. A. and R. Pande (2012), ‘Corruption in Developing Countries’, Annual Review of Economics, 4(1): 479–509. Olken, B. A. and R. Pande (2012), ‘Corruption in Developing Countries’, Annual Review of Economics, 4(1): 479–509. Phillips, N., P. Tracey and N. Karra (2013), ‘Building Entrepreneurial tie Portfolios Through Strategic Hom of Narrative Identity Work in Venture Creation and Early Growth’, Journal of Business Venturing, 28 acey and N. Karra (2013), ‘Building Entrepreneurial tie Portfolios Through Strategic Homophily: The Role s, N., P. Tracey and N. Karra (2013), ‘Building Entrepreneurial tie Portfolios Through Strategic Homophily: Th Narrative Identity Work in Venture Creation and Early Growth’ Journal of Business Venturing 28(1): 134–15 entity Work in Venture Creation and Early Growth’, Journal of Business Venturing, 28(1): 134–150. Podsakoff, P. M., S. B. MacKenzie, L. Jeong-Yeon and N. P. Podsakoff (2003), ‘Common Method Biases in Behavioral Research: A Critical Review of the Literature and Recommended Remedies’, Journal of Applied Psychology, 88(5): 879–903. f pp y gy Puffer, S. M., D. J. McCarthy and M. Boisot (2010), ‘Entrepreneurship in Russia and China: The Impact of Formal Institutional Voids’, Entrepreneurship Theory and Practice, 34(3): 441–467. p p y Robson, P. J. and B. A. Obeng (2008), ‘The Barriers to Growth in Ghana’, Small Business Economics, 30(4): 385–403. Rogelberg S and J Stanton (2007) ‘Understanding and Dealing with Organisational Survey Nonresponse’ Organisational n, P. J. and B. A. Obeng (2008), ‘The Barriers to Growth in Ghana’, Small Business Economics, 30(4): 385–40 Robson, P. J. and B. References A. Obeng (2008), ‘The Barriers to Growth in Ghana’, Small Business Economics, 30(4): 385–403. Rogelberg, S. and J. Stanton (2007), ‘Understanding and Dealing with Organisational Survey Nonresponse’, Organisational Research Methods 10(2): 195 209 Rogelberg, S. and J. Stanton (2007), ‘Understanding and Dealing with Organisational Survey Nonrespons Research Methods, 10(2): 195–209. Rose-Ackerman, S. (1999), Corruption and Government. Causes, Consequences, and Reform, Cambridge: Cambridge University Press. y Rose-Ackerman, S. (2001), ‘Trust and Honesty in Post-Socialist Societies’, Kyklos, 54(3): 415–444. Sawyerr, O. O., J. McGee and M. Peterson (2003), ‘Perceived Uncertainty and Firm Performance in SMEs: The Role of Personal Networking Activities’, International Small Business Journal, 21(3): 269–290. Journal of Institutional Economics 17 Semadeni, M., M. C. Withers and S. Trevis Certo (2014), ‘The Perils of Endogeneity and Instrumental Variables in Strategy Research: Understanding Through Simulations’, Strategic Management Journal, 35(7): 1070–1079. g g g g and S. Sigmund (2012), ‘Networking Ability and the Financial Performance of New Ventures: A Mediat s among Younger and More Mature Firms’ Strategic Entrepreneurship Journal 6(4) 335 354 Semrau, T. and S. Sigmund (2012), ‘Networking Ability and the Financial Performance of New Ventures g ( ), g y s among Younger and More Mature Firms’, Strategic Entrepreneurship Journal, 6(4): 335–354. Analysis among Younger and More Mature Firms’, Strategic Entrepreneurship Journal, 6(4): 335–354. y g g g p p Shleifer, A. and R. W. Vishny (1993), ‘Corruption’, The Quarterly Journal of Economics, 108(3): 599–617. Shleifer, A. and R. W. Vishny (1993), ‘Corruption’, The Quarterly Journal of Economics, 108(3): 599–617. Sobel, M. E. (1982), ‘Asymptotic Confidence Intervals for Indirect Effects in Structural Equations Models’, in S. Leinhart (ed.), Sociological Methodology, San Francisco: Jossey-Bass, pp. 290–312. Sobel, M. E. (1982), Asymptotic Confidence Intervals for Indirect Effects in Structural Equations Models , in S. Leinhart (ed.), Sociological Methodology, San Francisco: Jossey-Bass, pp. 290–312. al Methodology, San Francisco: Jossey-Bass, pp. 290–31 Sun, P., K. Mellahi and M. Wright (2012), ‘The Contingent Value of Corporate Political Ties’, Academy o Perspectives, 26(3): 68–82. p Svensson, J. (2005), ‘Eight Questions about Corruption’, Journal of Economic Perspectives, 19(3): 19–42. ‘Corruption Around the World: Causes, Consequences, Scope, and Cures’, Staff Papers, 45(4): 559–594. Tanzi, V. (1998), ‘Corruption Around the World: Causes, Consequences, Scope, and Cures’, Staff Papers Terziovski, M. (2010), ‘Innovation Practice and its Performance Implications in Small and Medium Enterprises (SMEs) in the Manufacturing Sector: A Resource-Based View’, Strategic Management Journal, 31(8): 892–902. Tonoyan, V., R. References Strohmeyer, M. Habib and M. Perlitz (2010), ‘Corruption and Entrepreneurship: How Formal and Informal Institutions Shape Small Firm Behavior in Transition and Mature Market Economies’, Entrepreneurship Theory and Practice, 34(5): 803–832. International (2017), Corruption Perception Index, Berlin. Available at https://www.transparency.org/country/ ssed 2 July 2019). Uhlenbruck, K., P. Rodriguez, J. Doh and L. Eden (2006), ‘The Impact of Corruption on Entry Strategy: Evidence from Telecommunication Projects in Emerging Economies’, Organization Science, 17(3): 402–414. j g g g Venkatraman, N. and V. Ramanujam (1986), ‘Measurement of Business Performance in Strategy Research: A Comparison of A h ’ A d f M R i 11(4) 801 814 Venkatraman, N. and V. Ramanujam (1986), ‘Measurement of Business Performance in Strategy Research: A Comparison of Approaches’, Academy of Management Review, 11(4): 801–814. y f g Vial, V. and J. Hanoteau (2010), ‘Corruption, Manufacturing Plant Growth, and the Asian Paradox: Indonesian Evidence’, World Development, 38(5): 693–705. Vorley, T. and N. Williams (2016), “Between Petty Corruption and Criminal Extortion: How Entrepreneurs in Bulgaria and Romania Operate within A Devil’s Circle”, International Small Business Journal, 34(6): 797–817. Voss, G. B., D. Sirdeshmukh and Z. G. Voss (2008), ‘The Effects of Slack Resources and Environmental Threat on Product Exploration and Exploitation’, Academy of Management Journal, 51(1): 147–164. tation’, Academy of Management Journal, 51(1): 147–1 Vu, H. V., T. Q. Tran, T. Van Nguyen and S. Lim (2018), ‘Corruption, Types of Corruption and Firm Financial Performance: New Evidence from A Transitional Economy’, Journal of Business Ethics, 148(4): 847–858. Wang, F., L. Xu, J. Zhang and W. Shu (2018), ‘Political Connections, Internal Control and Firm Value: Evidenc Anti-Corruption Campaign’, Journal of Business Research, 86: 53–67. Wellalage, N. H., S. Locke and H. Samujh (2019a), ‘Firm Bribery and Credit Access: Evidence from Indian SMEs’, Small Business Economics, 55(3): 283–304. Wellalage, N. H., S. Locke and H. Samujh (2019b), ‘Corruption, Gender and Credit Constraints: Evidence From South Asian SMEs’, Journal of Business Ethics, 159(1): 267–280. Wiklund, J. and D. Shepherd (2005), ‘Entrepreneurial Orientation and Small Business Performance: A Configurational Approach’, Journal of Business Venturing, 20(1): 71–91. Wooldridge, J. M. (2002), Econometric Analysis of Cross Section and Panel Data, Cambridge, MA: MIT Pres Zhang, X., X. Ma, Y. Wang, X. Li and D. Huo (2016), ‘What Drives the Internationalization of Chinese SMEs? The Joint Effects of International Entrepreneurship Characteristics, Network Ties, and Firm Ownership’, International Business Review, 25(2): 522–534. Zhao, X., J. G. References Lynch Jr and Q. Chen (2010), ‘Reconsidering Baron and Kenny: Myths and Truths About Mediation Analysis’, Journal of Consumer Research, 37(2): 197–206. Zheng, C., M. Ahsan and A. F. DeNoble (2020), ‘Entrepreneurial Networking During Early Stages of Opportunity Exploitation: Agency of Novice and Experienced new Venture Leaders’, Entrepreneurship Theory and Practice, 44(4): 671–699. Zhou, W. (2013), ‘Political Connections and Entrepreneurial Investment: Evidence from China’s Transition Economy’, Journal of Business Venturing, 28(2): 299–315. available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S1744137421000011 Downloaded from https://www.cambridge.org/core. University College Birmingham, on 29 Jun 2021 at 09:09:21, subject t Cite this article: Adomako S, Ahsan M, Amankwah-Amoah J, Danso A, Kesse K, Frimpong K (2021). Corruption and SME growth: the roles of institutional networking and financial slack. Journal of Institutional Economics 1–18. https://doi.org/ 10.1017/S1744137421000011 Appendix 1: Survey items Perceived corruption (0.93) Perceived corruption (0.93) n this industry, irregular, additional payments to various government officials are required to ‘get things don • In this industry, irregular, additional payments to various government officials are required to ‘get things done’. g y g g yp p p • Engaging in various types of corruptions is a normal part of doing business in this industry. g y g g yp p p y • Engaging in various types of corruptions is a normal part of doing business in this industry. • Firms which do not engage in corruption will be at a competitive disadvantage compared to firms that do engage these types of actions. • Firms which do not engage in corruption will be at a competitive disadvantage compared to firms that do these types of actions. Samuel Adomako et al. 18 • Corruption is one of the most important considerations when doing business in this industry. • Engaging in corruption is the way things get done in this industry. Institutional networking (α = 0.88) Institutional networking (α = 0.88) • Top managers at our firm have maintained good personal relationships with officials in various leve • Top managers at our firm have maintained good personal relationships with officials in various levels of government. • So far, our firm’s relationship with regional government officials has been in a good shape. • Top managers at our firm have maintained good personal relationships with officials in various levels of government. • So far, our firm’s relationship with regional government officials has been in a good shape. p g g p p g • So far, our firm’s relationship with regional government officials has been in a good shape. • So far, our firm’s relationship with regional government officials has been in a good shape. • So far, our firm’s relationship with regional government officials has been in a good shape. • Top managers at our firm have developed good connections with officials in regulatory and supporting organisations such as tax bureaus, state banks and commercial administration bureaus. • Top managers at our firm have developed good connections with officials in regulatory and supporting organisations such as tax bureaus, state banks and commercial administration bureaus. • Top managers at our firm have developed good connections with officials in such as tax bureaus, state banks and commercial administration bureaus. available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S1744137421000011 Downloaded from https://www.cambridge.org/core. University College Birmingham, on 29 Jun 2021 at 09:09:21, subject to the Cambridge Core terms of use, Appendix 1: Survey items Perceived corruption (0.93) • Top managers at our firm have maintained good relationships with officials in industrial and investment institutions (e.g. Investment Board, Export Promotion Council, Ghana Stock Exchange). • Top managers at our firm have maintained good relationships with officials i (e.g. Investment Board, Export Promotion Council, Ghana Stock Exchange). • Top managers at our firm have maintained good relationships with officials in industrial and investment institutions (e.g. Investment Board, Export Promotion Council, Ghana Stock Exchange). Cite this article: Adomako S, Ahsan M, Amankwah-Amoah J, Danso A, Kesse K, Frimpong K (2021). Corruption and SME growth: the roles of institutional networking and financial slack. Journal of Institutional Economics 1–18. https://doi.org/ 10.1017/S1744137421000011
https://openalex.org/W3204517372	https://www.medicalherald.ru/jour/article/download/1388/843	Russian	null	Sexual features of facial skull asymmetry according to spiral computed tomography	Medicinskij vestnik Ûga Rossii	2,021	cc-by	5,516	© Коллектив авторов, 2021 УДК: 611.7:611.92-007.246 DOI 10.21886/2219-8075-2021-12-3-78-85 © Коллектив авторов, 2021 УДК: 611.7:611.92-007.246 Д DOI 10.21886/2219-8075-2021-12-3-78-85 Е.В. Чаплыгина, О.А. Каплунова, М.Г. Шепетюк, О.П. Суханова, И.М. Блинов Ростовский государственный медицинский университет, Ростов-на-Дону, Россия Цель: определить степень выраженности асимметрии линейных размеров лицевого черепа у лиц обоего пола на спиральных компьютерных томограммах (СКТ). Материалы и методы: исследованы СКТ 104 человек обоего пола (52% женщин (n = 54), 48% мужчин (n = 50)). Измерения производили с использованием стандартной цифровой ли- нейки рабочей станции компьютерного томографа, оценивали асимметрию черепов используя «веерный» метод. По- лученные результаты обрабатывали с помощью программы «Excel». Результаты: в мужской и женской сериях СКТ по величине интервалов сигм линейных размеров лицевого черепа определили частоту встречаемости асимметрии различной степени выраженности. Заключение: в мужской серии СКТ преобладает правосторонняя асимметрия ли- нейных размеров лицевого черепа во всех «веерах», в женской – правосторонняя в верхнем и боковом «веерах», но левосторонняя асимметрия в нижнем «веере». Выявлены статистически значимые линейные размеры лицевого черепа для определения степени выраженности асимметрии у лиц обоего пола. В верхнем «веере» это расстояния от назион до зигомаксиляре (N-Zm), от назион до фронтоназале (N-Fn), преобладающие справа у обоих полов. В нижнем «веере» – расстояние от супраспинале до зигомаксиляре (Ss-Zm), в боковом – расстояние от зигомаксиляре до фронтоназале (Zm-Fn), преобладающие справа у обоих полов. Выводы: выявлена незначительная или физиологичная и средняя сте- пень выраженности асимметрии размеров лицевого черепа в зависимости пола, не требующая коррекции. Ключевые слова: лицевой череп, краниометрия, асимметрия, мультиспиральная компьютерная томография Для цитирования: Чаплыгина Е.В., Каплунова О.А., Шепетюк М.Г., Суханова О.П., Блинов И.М. Половые особен- ности асимметрии лицевого черепа по данным спиральной компьютерной томографии. Медицинский вестник Юга России. 2021;12(3):X-X. DOI 10.21886/2219-8075-2021-12-3-78-85. ( ) Контактное лицо: Ольга Антониновна Каплунова, kaplunova@bk.ru. Е.В. Чаплыгина, О.А. Каплунова, М.Г. Шепетюк, О.П. Суханова, И.М. Блинов ПОЛОВЫЕ ОСОБЕННОСТИ АСИММЕТРИИ ЛИЦЕВОГО ЧЕРЕПА ПО ДАННЫМ СПИРАЛЬНОЙ КОМПЬЮТЕРНОЙ ТОМОГРАФИИ Е.В. Чаплыгина, О.А. Каплунова, М.Г. Шепетюк, О.П. Суханова, И.М. Блинов ПОЛОВЫЕ ОСОБЕННОСТИ АСИММЕТРИИ ЛИЦЕВОГО ЧЕРЕПА ПО ДАННЫМ СПИРАЛЬНОЙ КОМПЬЮТЕРНОЙ ТОМОГРАФИИ ОРИГИНАЛЬНЫЕ СТАТЬИ © Коллектив авторов, 2021 УДК: 611.7:611.92-007.246 DOI 10.21886/2219-8075-2021-12-3-78-85 Результаты Цель исследования – определить степень выражен- ности асимметрии линейных размеров лицевого черепа у лиц обоего пола на спиральных компьютерных томо- граммах (СКТ). При распределении материала в зависимости от пола на СКТ у мужчин и женщин были определены достовер- ные различия 27 линейных размеров лицевого черепа в верхнем, нижнем и боковом «веерах» с обеих сторон (табл.1). Sexual features of facial skull asymmetry according to spiral computed tomography Блинов ПОЛОВЫЕ ОСОБЕННОСТИ АСИММЕТРИИ ЛИЦЕВОГО ЧЕРЕПА ПО ДАННЫМ СПИРАЛЬНОЙ КОМПЬЮТЕРНОЙ ТОМОГРАФИИ ОРИГИНАЛЬНЫЕ СТАТЬИ Sexual features of facial skull asymmetry according to spiral computed tomography E.V. Chaplygina, O.A. Kaplunova, M.G. Shepetyuk, O.P. Suchanova, I.M. Blinov Rostov State Medical University, Rostov-on-Don, Russia Objective: To perform a comparative analysis of the linear dimensions of the facial skull that are the most signifi cant in determining asymmetry in persons of diﬀ erent sex on spiral computed tomograms (SCT). Materials and Methods: Th e CT scan of 104 people of both sexes (women – 52%, n = 54, men – 48%, n = 50) were studied. Measurements were made using a standard digital ruler of a computer tomograph workstation, and the asymmetry of the skulls was assessed using the “fan” method. Th e results were processed using the Excel program. Results: In the male and female series of SCT, according to the size of the intervals of sigma of linear dimensions of the facial skull, the occurrence rate of asymmetry of varying severity was determined. Conclusion: In the male series of SCT, right-sided asymmetry of the linear dimensions of the facial skull prevails in all “fans”, in the female – right-sided asymmetry in the upper and lateral “fans”, but left -sided asymmetry in the lower “fan”. Statistically signifi cant linear dimensions of the facial skull were revealed to determine the severity of asymmetry in both sexes. In the upper “fan”, these are the distances from the nasion to the zygomaxilar (N-Zm), from the nasion to the frontonazale (N-Fn), prevailing on the right in both sexes. In the lower “fan”– the distance from the supraspinal to the zygomaxilar (Ss-Zm), and in the lateral “fan” – the distance from the zygomaxilar to the frontonasal (Zm-Fn), prevailing on the right in both sexes. An insignifi cant or physiological and moderate degree of asymmetry in the size of the facial skull depending on gender was revealed, which did not require correction. q Keywords: facial skull, craniometry, asymmetry, multispiral computed tomography For cit ation: Chaplygina E.V., Kaplunova O.A., Shepetyuk M.G., Suchanova O.P., Blinov I.M. Sexual features of facial skull asymmetry according to spiral computed tomography. Medical Herald of the South of Russia. 2021;12(3):X-X. DOI 10.21886/2219- 8075-2021-12-3-78-85. Corresponding author: Olga A. Kaplunova, kaplunova@bk.ru Corresponding author: Olga A. Kaplunova, kaplunova@bk.ru 78 Медицинский вестник Юга России Medical Herald of the South of Russia 2021;12(3):78-85 Медицинский вестник Юга России Medical Herald of the South of Russia 2021;12(3):78-85 Е.В. Чаплыгина, О.А. Каплунова, М.Г. Шепетюк, О.П. Суханова, И.М. Медицинский вестник Юга России Medical Herald of the South of Russia 2021;12(3):78-85 Введение величине черепного указателя [12]. На СКТ для оценки асимметрии с помощью «веерного» метода [13, 14] в верх- нем, нижнем и боковом «веерах» определяли расстояния от стандартных точек назион, субспинале, зигомаксиля- ре до нестандартных точек с каждой стороны. Данный метод включает исследование 27 линейных размеров с каждой стороны лицевого черепа. Ф Ф акт асимметрии лица у людей общепризнан [1], а одной из причин данной асимметрии является неравномерность выраженности элементов черепа. На протяжении многих лет традици- онно основным методом для анализа морфологии лице- вого черепа и выявления черепно-лицевых деформаций была рентгенография, однако на рентгенограммах труд- но оценить асимметрию лицевого черепа из-за множе- ства перекрывающихся анатомических структур [2]. Компьютерная краниометрия предоставляет огромное поле для исследования, так как томографические иссле- дования высокого разрешения становятся стандартами обследования в настоящее время [3]. Результаты кра- ниометрии мацерированных черепов, рентгенограмм и спиральных компьютерных томограмм показывают, что СКТ может быть полезной альтернативой обычной рентгенографии [4]. Результаты измерений, полученные на СКТ, признаны точными и сопоставимыми с резуль- татами антропометрического анализа мацерированного черепа [5, 6, 7], что расширяет клиническое применение СКТ [8, 9]. Обработку статистического материала проводили с использованием пакета прикладных программы «Excel», рекомендованного для статистического анализа медико- биологических данных. Для каждого исследуемого па- раметра рассчитывали выборочную среднюю величину (M), стандартную ошибку средней (m). Достоверность различий средних величин независимых выборок оцени- вали с помощью параметрического критерия Стьюдента при нормальном распределении исходных данных. Раз- личия между группами показателей считали значимыми при р < 0,05. При определении коэффициента вариации (kv=δ/M – отношение величины стандартного отклонения линей- ных размеров к средней арифметической данных линей- ных размеров), согласно рекомендации Е.Ю. Ефимовой с соавт. [15], принимали значения kv < 10 % за слабую сте- пень выраженности, 10 % < kv < 25% – за среднюю и kv > 25 % – за высокую степень выраженности. По методу И.В. Гайворонского с соавт. [10] в выборках определяли среднее значение линейных размеров лицевого черепа справа и слева, вычисляли степень различий между ними в сигмах и степень выраженности асимметрии лицевого черепа в пределах 1, 2 или 3 сигм. Известно, что индивидуальную форму лица опреде- ляет физиологичная асимметрия лицевого черепа [10]. Вместе с тем асимметрия костных структур черепа со- временных людей, особенно в половом аспекте, мало из- учена [4, 11]. В связи с этим проведение сравнительного анализа краниометрических показателей людей обоего пола, проживающих в Ростовской области, является сво- евременным и актуальным. Материалы и методы Блинов ПОЛОВЫЕ ОСОБЕННОСТИ АСИММЕТРИИ ЛИЦЕВОГО ЧЕРЕПА ПО ДАННЫМ СПИРАЛЬНОЙ КОМПЬЮТЕРНОЙ ТОМОГРАФИИ ОРИГИНАЛЬНЫЕ СТАТЬИ Е.В. Чаплыгина, О.А. Каплунова, М.Г. Шепетюк, О.П. Суханова, И.М. Материалы и методы Исследованы СКТ 104 людей обоего пола (52% жен- щин (n = 54), 48% мужчин (n = 50)). СКТ были выпол- нены на базе отделения магнитно-резонансной и рентге- новской компьютерной томографии РостГМУ по поводу предполагаемой сосудистой патологии головного мозга. У мужчин на СКТ преобладает правосторонняя асим- метрия линейных размеров лицевого черепа во всех «вее- рах», в женской – правосторонняя в верхнем и боковом «веерах», но левосторонняя в нижнем «веере». Коэффициент вариации исследуемых линейных раз- меров лицевого черепа в подавляющем большинстве слу- чаев не превышал 10 – 20%, что свидетельствует о слабой и средней степени изменчивости показателей каждого размера и их однородности. у Критерии включения: – предполагаемая сосудистая патология головного мозга; – отсутствие патологии костей черепа; у – отсутствие аномалий развития костей черепа; В мужской и женской группах на СКТ по величи- не интервалов сигм этих размеров определили частоту встречаемости асимметрии различной степени выражен- ности. В мужской серии СКТ (рис.1) в верхнем, нижнем и боковом «веерах» различия в показателях с двух сторон не превышают 1 σ в 67,6 %, 74,9 % и 64,3 % случаев со- ответственно, различия в показателях в пределах 2 σ (в 25,7%, в 15,6 % и 30,3 % случаев соответственно), и в пре- делах 3 σ (в 6,8 %, в 8,6 % и 5,4 % случаев соответственно). У женщин на СКТ (рис. 2) в верхнем, нижнем и боковом «веерах» различия в показателях с двух сторон не превы- шают 1 σ в 68,8 %, 63,8 % и 65,0 % случаев, различия в по- казателях в пределах 2 σ (в 26,3 %, 32,5 % и 32,8 % случаев соответственно), и в пределах 3 σ (в 4,9 %, 3,7 % и 2,2 % случаев соответственно). – отсутствие аномалий развития черепа в целом – отсутствие аномалий развития черепа в целом; Критерии исключения: отсутствие аномалий развития черепа в целом; Критерии исключения: – детский и юношеский возраст (до окончания фор- мирования лицевого черепа); – макроскопические признаки патологических изме- нений костей черепа; р – аномалии развития черепа. р – аномалии развития черепа. Для анализа полученных изображений применяли ак- сиальные, MPR (multiplanar reconstruction) и SSD (surface shadow density) реконструкции в различных проекциях. Измерения производили с использованием стандартной цифровой линейки рабочей станции компьютерного то- мографа. р ф Форму мозгового и лицевого черепа определяли по 79 Медицинский вестник Юга России Medical Herald of the South of Russia 2021;12(3):78-85 Медицинский вестник Юга России Е.В. Чаплыгина, О.А. Каплунова, М.Г. Шепетюк, О.П. Суханова, И.М. Материалы и методы Чаплыгина, О.А. Каплунова, М.Г. Шепетюк, О.П. Суханова, И.М. Блинов ПОЛОВЫЕ ОСОБЕННОСТИ АСИММЕТРИИ ЛИЦЕВОГО ЧЕРЕПА ПО ДАННЫМ СПИРАЛЬНОЙ КОМПЬЮТЕРНОЙ ТОМОГРАФИИ ОРИГИНАЛЬНЫЕ СТАТЬИ Ss-ap. lat S 14,8±2,1 67-20-13 14,1 14,8±2,9 67-31-2 19,6 D 15,0±1,9 62-27-11 12,7 15,3±3,0 65-33-2 19,3 Ss-ap. Inf. S 6,5±1,7 84-9-7 25,8 6,7±1,6 56-38-6 24,7 D 6,8±1,6 60-38-2 23,8 7,3±1,8 58-40-2 24,1 Боковой веер / Side fan Zm-infr S 22,5±3,4 64-27-9 15,1 22,4±4,2 65-33-2 18,8 D 22,2±3,5 64-24-11 15,6 23,4±5,1 67-31-2 21,7 Zm-ap.lat S 20,9±3,6 62-31-7 17,3 20,2±3,6 62-37-2 17,7 D 21,2±4,1 64-27-9 19,3 20,8±4,1 52-44-4 19,6 Zm-ap.inf S 18,0±3,0 71-22-7 16,9 17,7±3,5 75-23-2 19,7 D 18,1±2,9 64-29-7 16,1 18,2±3,3 73-21-6 18,4 Zm-max S 35,7±8,2 64-31-4 23,1 32,7±6,8 56-44-0 20,7 D 35,8±8,0 58-36-7 22,4 33,5±7,1 63-37-0 21,3 Zm-Da S 29,3±4,3 69-24-7 14,6 29,6±4,8 67-29-4 16,3 D 30,0±4,2 60-33-7 13,9 31,0±5,3 65-35-0 17,1 Zm-min S 40,4±6,2 69-22-9 15,4 38,1±5,7 69-27-4 14,8 D 40,5±6,2 67-22-11 15,2 38,4±5,8 63-37-0 15,2 Zm-Fn* S 46,0±5,6 60-33-7 12,1 43,1±4,7 63-37-0 11,0 D 46,3±5,5 62-24-13 11,9 44,3±5,3 67-27-6 11,9 Примечания: * – достоверно значимые отличия между линейными размерами у черепов различного пола; kv – коэффициент вариации; N – Ft (назион – фронтотемпорале), N – Fmt (назион – фронтомаляре – темпорале), N – Da (назион – дакрион), N – Infr (назион – инфроорбитале), N – Zm (назион – зигомаксиляре); N – Fn (назион – фронтоназале); N – max (назион – точка наибольшего удаления носовых костей), N – ap.lat (назион – латеральный край грушевидного отверстия), N – ap inf (назион – нижний край грушевидного отверстия), N – min (назион – точка минимальной ширины носовых костей), Ss – Ft (супраспинале – фронтоназале), Ss – Fmt ( супраспинале –фронтомаляре – темпорале), Ss – Fn (супраспинале – фронтоназале), Ss – Da (супраспинале – дакрион), Ss – min (супраспинале – точка минимальной ширины носовых костей), Ss – infr (супраспинале – инфраорбитале), Ss – max (супраспинале – точка наибольшего удаления носовых костей), Ss – ap. Lat (супраспинале – латеральный край грушевидного отверстия), Ss – ap. Inf.(супраспинале – нижний край грушевидного отверстия), Zm – infr (зигомаксиля- ре – инфраорбитале), Zm – ap.lat (зигомаксиляре – латеральный край грушевидного отверстия), Zm – ap.inf (зигомаксиляре – нижний край грушевидного отверстия), Zm – max (зигомаксиляре – точка наибольшей ширины носовых костей), Zm – Da (зигомаксиляре – дакрион), Zm – min (зигомаксиляре – точка минимальной ширины носовых костей), Zm – Fn (зигомаксиляре – фронтоназале), s – слева; d – справа. Материалы и методы Notes: * – reliably signifi cant diﬀ erences between the linear dimensions of the skulls of diﬀ erent sex; kv – the coeﬃ cient of variation; N – Ft (nasion – frontotemporale), N – Fmt (nasion – frontomalare-temporal), N – Da (nasion – dacrion), N – Infr (nasion – infraorbital), N – Zm (nasion – zygomaxilar); N – Fn (nazion – frontonazale); N – max (nasion – the point of the greatest removal of the nasal bones), N – ap.lat (nasion – the lateral edge of the pear- shaped opening), N – ap inf (nasion – the lower edge of the pear-shaped opening), N – min (nasion – the point of the minimum width of the nasal bones), Ss – Ft (supraspinal – frontonazale), Ss – Fmt (supraspinal – frontonazale-temporal), Ss – Fn (supraspinal – frontonazale), Ss – Da (supraspinal – dacrion), Ss – min (supraspinal – point of minimum width of the nasal bones), Ss – infr (supraspinal – infraorbital), Ss – max (supraspinal – the point of the greatest removal of the nasal bones), Ss – ap. Lat (supraspinal – lateral edge of the pear-shaped foramen), Ss – ap. Inf. (Supraspinal – lower edge of the pear-shaped opening), Zm – infr (zygomaxilar – infraorbital), Zm – ap.lat (zygomaxilar – lateral edge of the pear-shaped opening), Zm – ap.inf (zygomaxilar – lower edge of the pear-shaped opening), Zm – max (zygomaxilar – the point of the greatest width of the nasal bones), Zm – Da (zygomaxilar – dacrion), Zm – min (zygomaxilar – the point of the minimum width of the nasal bones), Zm – Fn (zygomaxilar – frontonasale), s – left ; d – right. Рисунок 1. Асимметрия линейных размеров лицевого черепа у мужчин различной степени выраженности в % в пределах 1 сигмы (синий), 2 сигм (красный) и 3 сигм (зеленый). 1 – верхний «веер», 2 – нижний «веер», 3 – боковой «веер». Picture 1. Asymmetry of linear dimensions of the facial skull in men of varying severity in% within 1 sigma (blue), 2 sigma (red) and 3 sigma (green). 1 – upper “fan”, 2 – lower “fan”, 3 – lateral “fan”. 1 2 3 9% 24% 67% 11% 14% 75% 8% 28% 64% 1 9% 24% 67% 2 11% 14% 75% 3 8% 28% 64% 8% 1 1 2 3 Рисунок 1. Материалы и методы Блинов ПОЛОВЫЕ ОСОБЕННОСТИ АСИММЕТРИИ ЛИЦЕВОГО ЧЕРЕПА ПО ДАННЫМ СПИРАЛЬНОЙ КОМПЬЮТЕРНОЙ ТОМОГРАФИИ Таблица / Table 1 Линейные размеры лицевого черепа с различной степенью выраженности асимметрии у лиц обоего пола; p < 0,05, M ± m (мм), kv (%), σ (%) Th e linear dimensions of the facial skull with the highest degree of asymmetry in persons of both sexes, p < 0,05, M ± m (mm), kv (%), σ (%) Линейный размер / Linear dimension Мужчины / Men Женщины / Women Значения показателя справа (D) или слева (S) в мм / Indicator values to the right (D) or left (S) in mm Частота встречаемос- ти асимметрии размера в пределах 1σ-2σ- 3 σ (%) / Th e frequency of occurrence of size asymmetry within 1σ-2 σ-3σ (%) Kv размера (%) / Kv size (%) Значения показателя справа (D) или слева (S) в мм / Indicator values to the right (D) or left (S) in mm Частота встречаемости асимметрии размера в пределах 1σ-2 σ-3σ (%)/ Th e frequency of occurrence of size asymmetry within 1σ-2 σ-3σ (%) Kv размера (%) / Kv size (%) Верхний веер / Upper fan N-Ft S 46,8±4,6 64-29-7 9,9 47,9±6,4 75-21-4 13,4 D 47,4±4,9 71-22-7 10,3 48,3±6,6 75-19-6 13,7 N-Fmt S 48,3±5,5 67-27-7 11,3 48,7±6,6 69-25-6 13,6 D 48,9±5,7 71-24-4 11,6 49,2±6,8 75-19-6 13,9 N-Da S 15,0±3,1 62-33-4 20,3 15,8±4,1 63-35-2 26,2 D 14,9±2,8 67-27-7 18,9 15,8±3,9 62-37-2 24,7 N-Infr S 30,5±4,3 58-38-4 14,2 30,9±4,6 69-27-4 14,9 D 31,5±4,0 71-20-9 12,6 32,3±5,2 69-25-6 16,1 N-Zm* S 58,8±5,6 67-16-18 9,6 56,0±7,4 69-27-4 13,2 D 59,0±5,6 64-18-18 9,5 57,2±7,5 69-27-4 13,1 N-Fn* S 5,1±1,0 69-18-13 20,3 6,1±1,8 65-29-6 30,0 D 5,8±1,2 56-40-4 20,9 6,8±1,9 73-27-0 28,3 N-max S 26,5±4,1 67-22-11 15,5 25,0±3,9 56-37-8 15,7 D 26,2±4,0 69-24-7 15,2 25,0±3,7 63-35-2 15,0 N-ap.lat S 36,3±4,5 76-16-9 12,4 37,5±5,8 69-25-6 15,5 D 37,0±4,2 71-18-11 11,3 37,3±5,9 75-17-8 15,7 N-ap inf S 43,2±4,8 69-20-11 11,2 43,7±6,7 75-15-10 15,2 D 43,5±4,7 69-18-13 10,8 43,8±6,7 75-19-6 15,4 N-min S 19,0±3,4 71-24-4 17,7 18,9±2,9 62-33-6 15,2 D 18,8±3,1 73-22-4 16,7 19,2±2,9 65-29-6 15,0 Нижний веер / Lower fan Ss-Ft S 72,5±11,3 84-9-7 15,5 73,6±8,6 67-27-6 11,6 D 73,7±7,7 73-18-9 10,4 73,1±8,1 71-23-6 11,1 Ss-Fmt S 68,3±7,4 78-11-11 10,8 69,7±9,2 65-29-6 13,1 D 68,2±7,1 79-9-13 10,4 69,2±9,2 69-25-6 13,3 Ss-zm* S 46,0±5,4 67-16-18 11,8 42,8±4,5 71-25-4 10,6 D 45,3±5,2 67-22-11 11,4 42,7±4,9 75-23-2 11,4 Ss-Fn S 44,1±4,9 78-4-18 11,0 43,8±6,9 63-35-2 15,7 D 44,2±5,1 78-9-13 11,4 44,2±6,7 69-29-2 15,1 Ss-Da S 42,4±4,6 71-16-13 10,9 42,2±6,2 69-27-4 14,8 D 42,6±4,6 73-13-13 10,9 42,2±5,9 63-33-4 14,1 Ss-min S 28,3±4,4 80-13-7 15,4 28,9±5,9 62-35-4 20,5 D 28,1±4,3 80-13-7 15,3 28,6±6,2 71-25-4 21,6 Ss-infr S 33,2±4,5 80-9-11 13,5 33,0±3,8 65-33-2 11,5 D 33,3±4,0 80-9-11 11,9 32,9±4,1 71-25-4 12,3 Ss-max S 22,8±4,3 18-13-9 18,7 23,3±4,6 65-31-4 19,5 D 22,3±3,9 80-13-7 17,4 23,0±4,7 67-29-4 20,5 Медицинский вестник Юга России ц / йные размеры лицевого черепа с различной степенью выраженности асимметрии у лиц обоего пола p < 0,05, M ± m (мм), kv (%), σ (%)h 80 Е.В. Медицинский вестник Юга России Medical Herald of the South of Russia 2021;12(3):78-85 Материалы и методы Асимметрия линейных размеров лицевого черепа у мужчин различной степени выраженности в % в пределах 1 сигмы (синий), 2 сигм (красный) и 3 сигм (зеленый). 1 – верхний «веер», 2 – нижний «веер», 3 – боковой «веер». ( ) ( ) р Picture 1. Asymmetry of linear dimensions of the facial skull in men of varying severity in% within 1 sigma (blue), 2 sigma (red) and 3 sigma (green). 1 – upper “fan”, 2 – lower “fan”, 3 – lateral “fan”. Picture 1. Asymmetry of linear dimensions of the facial skull in men of varying severity in% within 1 sigma (blue), 2 sigma (red) and 3 sigma (green). 1 – upper “fan”, 2 – lower “fan”, 3 – lateral “fan”. 81 ОРИГИНАЛЬНЫЕ СТАТЬИ Е.В. Чаплыгина, О.А. Каплунова, М.Г. Шепетюк, О.П. Суханова, И.М. Блинов ПОЛОВЫЕ ОСОБЕННОСТИ АСИММЕТРИИ ЛИЦЕВОГО ЧЕРЕПА ПО ДАННЫМ Й Й ОРИГИНАЛЬНЫЕ СТАТЬИ Е.В. Чаплыгина, О.А. Каплунова, М.Г. Шепетюк, О.П. Суханова, И.М. Блинов ПОЛОВЫЕ ОСОБЕННОСТИ АСИММЕТРИИ ЛИЦЕВОГО ЧЕРЕПА ПО ДАННЫМ Е.В. Чаплыгина, О.А. Каплунова, М.Г. Шепетюк, О.П. Суханова, И.М. Блинов О.А. Каплунова, М.Г. Шепетюк, О.П. Суханова, И.М. Блинов ОРИГИНАЛЬНЫЕ СТАТЬИ , у , , у , ПОЛОВЫЕ ОСОБЕННОСТИ АСИММЕТРИИ ЛИЦЕВОГО ЧЕРЕПА ПО ДАННЫМ СПИРАЛЬНОЙ КОМПЬЮТЕРНОЙ ТОМОГРАФИИ Рисунок 2. Асимметрия линейных размеров лицевого черепа у женщин различной степени выраженности в % в пределах 1 сигмы (синий), 2 сигм (красный) и 3 сигм (зеленый). 1 – верхний «веер», 2 – нижний «веер», 3 – боковой «веер». Picture 2. Asymmetry of linear dimensions of the facial skull in women of varying severity in% within 1 sigma (blue), 2 sigma (red) and 3 sigma (green). 1 – upper “fan”, 2 – lower “fan”, 3 – lateral “fan”. 1 2 3 5% 26% 69% 4% 32% 64% 2% 33% 65% 3 2% 33% 65% 2 4% 32% 64% 1 5% 26% 69% 1 2 3 Рисунок 2. Асимметрия линейных размеров лицевого черепа у женщин различной степени выраженности в % в пределах 1 сигмы (синий), 2 сигм (красный) и 3 сигм (зеленый). 1 – верхний «веер», 2 – нижний «веер», 3 – боковой «веер». Picture 2. Asymmetry of linear dimensions of the facial skull in women of varying severity in% within 1 sigma (blue), 2 sigma (red) and 3 sigma (green). 1 – upper “fan”, 2 – lower “fan”, 3 – lateral “fan”. Выявлены линейные размеры лицевого черепа со ста- тистически значимыми различиями степени выраженно- сти асимметрии у лиц обоего пола. Материалы и методы В верхнем «веере» – это расстояния от назион до зигомаксиляре (N – Zm), от назион до фронтоназале (N – Fn), преобладающие справа у обоих полов. В нижнем «веере» – расстояние от супра- спинале до зигомаксиляре (Ss – Zm), с преобладанием и у женщин, и у мужчин с левой стороны. В боковом «веере» – расстояние от зигомаксиляре до фронтоназале (Zm – Fn), преобладающее справа у обоих полов. Заключение На СКТ выявлены половые отличия линейных разме- ров лицевого черепа, большинство из которых достовер- но преобладают у мужчин. Выполненная на СКТ краниометрия в верхнем, ниж- нем и боковом «веерах» выявила асимметрию линейных размеров лицевого черепа у мужчин и женщин в верхней, нижней и боковой частях черепа. У мужчин преобладает правосторонняя асимметрия линейных размеров лицевого черепа во всех «веерах», в женской серии – правосторонняя в верхнем и боковом «веерах», но левосторонняя в нижнем «веере». У мужчин преобладает правосторонняя асимметрия линейных размеров лицевого черепа во всех «веерах», в женской серии – правосторонняя в верхнем и боковом «веерах», но левосторонняя в нижнем «веере». Медицинский вестник Юга России Medical Herald of the South of Russia 2021;12(3):78-85 Обсуждение Выявлены линейные размеры лицевого черепа с наи- большей степенью выраженности асимметрии у лиц обо- его пола. В верхнем «веере» – это расстояния от назион до зигомаксиляре (N – Zm), от назион до фронтоназале (N – Fn ), преобладающие справа у обоих полов. В нижнем «веере» – расстояние от супраспинале до зигомаксиляре (Ss – Zm), а в боковом «веере» – расстояние от зигомакси- ляре до фронтоназале (Zm – Fn), преобладающее справа у обоих полов. Выявленные на СКТ половые отличия линейных раз- меров лицевого черепа проявляются достоверным пре- обладанием величины этих размеров у мужчин по срав- нению с женщинами. Эти данные соответствует данным С.А. Алиевой с соавт. [11]. У мужчин на СКТ выявлено преобладание правосто- ронней асимметрии линейных размеров лицевого черепа во всех «веерах», а у женщин – правосторонней в верхнем и боковом «веерах», но левосторонней в нижнем «веере». Эти данные не соответствуют выводам С.А. Алиевой с соавт. [11], но подтверждают данные А.И. Гайворонского с соавт. [13]. Выявленная асимметрия размеров лицевого черепа у обоих полов преимущественно незначительной и сред- ней степени выраженности определяет индивидуальную форму лица. Полученные данные как показатели кранио- метрии в норме могут быть использованы в клинической практике рентгенологами, пластическими хирургами, челюстно-лицевыми хирургами, а также в антропологии и судебной медицине. В мужской и женской группах по величине интерва- лов сигм линейных размеров лицевого черепа опреде- лили частоту встречаемости асимметрии различной степени выраженности. В мужской и женской группах в верхнем, нижнем и боковом «веерах» выявлены различия в показателях с двух сторон, преимущественно не пре- вышающие 1 или 2 ϭ. По мнению ряда авторов [10, 13], если различие показателей правой и левой сторон черепа преимущественно находятся в пределах 1 ϭ, то выявлен- ная асимметрия является незначительной или физиоло- гичной, в пределах 2 ϭ – средней степени выраженности асимметрии, что не требует коррекции. Финансирование. Исследование не имело спонсор- ской поддержки.h Finansing. Th e study did not have sponsorship. б Конфликт интересов. Авторы заявляют об отсут- ствии конфликта интересов.ll Confl ict of interest. Authors declares no confl ict of interest. 82 Медицинский вестник Юга России Medical Herald of the South of Russia 2021;12(3):78-85 Медицинский вестник Юга России Medical Herald of the South of Russia 2021;12(3):78-85 Е.В. Чаплыгина, О.А. Каплунова, М.Г. Шепетюк, О.П. Суханова, И.М. Блинов ПОЛОВЫЕ ОСОБЕННОСТИ АСИММЕТРИИ ЛИЦЕВОГО ЧЕРЕПА ПО ДАННЫМ СПИРАЛЬНОЙ КОМПЬЮТЕРНОЙ ТОМОГРАФИИ Е.В. Чаплыгина, О.А. Каплунова, М.Г. Шепетюк, О.П. Суханова, И.М. Блинов ПОЛОВЫЕ ОСОБЕННОСТИ АСИММЕТРИИ ЛИЦЕВОГО ЧЕРЕПА ПО ДАННЫМ СПИРАЛЬНОЙ КОМПЬЮТЕРНОЙ ТОМОГРАФИИ ОРИГИНАЛЬНЫЕ СТАТЬИ Рисунок 3. Медицинский вестник Юга России Medical Herald of the South of Russia 2021;12(3):78-85 ЛИТЕРАТУРА 1. Панина Н.Г., Перепелкин А.И., Краюшкин А.И. Совре- менные представления об асимметрии лица // Ураль- ский медицинский журнал. – 2014. - № 7. – С. 126-129. eLIBRARY ID: 22753142 1. Panina N.G., Perepelkin A.I., Krayushkin A.I. Modern views of facial asymmetry. Ural'skij medicinskij zhurnal. 2014;(7):126-129. (In Russ.) eLIBRARY ID: 22753142 2. Ko E.W.-C., Lin C.-H., Chen Y.-A, Chen Yu-R. Enhanced Surgical Outcomes in Patients with Skeletal Class III Facial Asymmetry by 3-Dimensional Surgical Simulation. Journal of Oral and Maxillofacial Surgery. 2018;76(5):1073-1083. DOI: 10.1016/j.joms.2017.09.009 2. Ko E.W.-C., Lin C.-H., Chen Y.-A, Chen Yu-R. Enhanced Surgical Outcomes in Patients with Skeletal Class III Facial Asymmetry by 3-Dimensional Surgical Simulation. // Journal of Oral and Maxillofacial Surgery. – 2018. – V.76 (5). -P. 1073- 1083. DOI: 10.1016/j.joms.2017.09.009 3. Mareev O.V., Nikolenko V.N., Aleshkina O.U., Mareev G.O., Markeeva M.V., et al. Computer craniometry with the help of modern technology in medical craniology. Morphological newsletter. 2015;(1):49-54. (In Russ.) eLIBRARY ID: 25456984 3. Мареев О.В., Николенко В.Н., Мареев Г.О., Алешкина О.Ю., Маркеева М.В., и др. Компьютерная краниометрия с помощью современных технологий в медицинской краниологии. // Морфологические ведомости. – 2015. – № 1(25). – С. 49-54. eLIBRARY ID: 25456984 4. Zhang D., Wang S., Li J., Zhou Y. Novel method of constructing a stable reference frame for 3-dimensional cephalometric analysis // American Jornal of Orthodontics and Dentofacial Orthopedics. – 2018. – V.154 (3). – P. 397- 404. DOI: 10.1016/j.ajodo.2017.11.038 4. Zhang D, Wang S, Li J, Zhou Y. Novel method of constructing a stable reference frame for 3-dimensional cephalometric analysis. Am J Orthod Dentofacial Orthop. 2018;154(3):397- 404. DOI: 10.1016/j.ajodo.2017.11.038 5. Jiang X, Zhang Y, Bai S, Chang X, Wu L, Ding Y. Th ree- dimensional analysis of craniofacial asymmetry and integrated, modular organization of human head. International Journal of Clinical and Experimental Medicine. 2017;10(8):11424-11431. 5. Jiang X, Zhang Y, Bai S, Chang X, Wu L, Ding Y. Th ree- dimensional analysis of craniofacial asymmetry and integrated, modular organization of human head. // International Journal of Clinical and Experimental Medicine. – 2017. – V.10 (8). – P.11424-11431. 6. Park H., Lee J., Cho J., Hwang H., Lee K. Accuracy of three- dimensional cephalograms generated using a biplanar imaging system // Korean Journal of Ortthodontics. – 2018. – V.48. (5). – P. 292-303. DOI: 10.4041/kjod.2018.48.5.292. 6. Park H., Lee J., Cho J., Hwang H., Lee K. Accuracy of three-dimensional cephalograms generated using a biplanar imaging system. ЛИТЕРАТУРА Korean Journal of Ortthodontics. 2018;48(5):292-303. DOI: 10.4041/kjod.2018.48.5.292. 7. Dos Santos R.M.G., De Martino J.M., Haiter Neto F., Passeri A.L. Cone-beam computed tomography-based three- dimensional McNamara cephalometric analyisis. Jornal of Craniofacial Surgery. 2018;29(4):895-899. DOI: 10.1097/ SCS.0000000000004248. 7. Dos Santos R.M.G., De Martino J.M., Haiter Neto F., Passeri A.L. Cone-beam computed tomography-based three- dimensional McNamara cephalometric analysis. // Jornal of Craniofacial Surgery. – 2018. – V.29 (4). – P.895-899. DOI: 10.1097/SCS.0000000000004248 8. Katsumuta A., Fujishita M., Maeda M., Ariji Y., Ariji E., Langlais R.R. 3D-CT evaluation of facial asymmetry. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2005;99(2):212–220. DOI: 10.1016/j.tripleo.2004.06.072. 8. Katsumuta A., Fujishita M., Maeda M., Ariji Y., Ariji E., Langlais R.R. 3D-CT evaluation of facial asymmetry. // Oral Surg Oral Med Oral Pathol Oral Radiol Endod. – 2005. – V.99 (2). – P.212–220. DOI: 10.1016/j.tripleo.2004.06.072. 9. Kreutz M., Fitze B., Blecher C., Marcello A., Simon R., et al. Facial asymmetry correction with moulded helmet therapy in infants with deformational skull base plagiocephaly. // Journal of Cranio-Maxillofacial Surgery. – 2018. – V.46 (1).- P.28-34. DOI: 10.1016/j.jcms.2017.10.013. 9. Kreutz M., Fitze B., Blecher C., Marcello A., Simon R., et al. Facial asymmetry correction with moulded helmet therapy in infants with deformational skull base plagiocephaly. Journal of Cranio-Maxillofacial Surgery. 2018;46(1):28-34. DOI: 10.1016/j.jcms.2017.10.013. 10. Гайворонский И. В., Дубовик Е. И., Крайник И. В Мор- фометрические показатели асимметрии лицевого чере- па у взрослого человека. // Морфология. – 2009. – Т.135, № 2. – С.74-79. DOI: 10.1016/j.jcms.2014.01.028. 10. Gajvoronskij I. V., Dubovik E. I., Krajnik I.V. Morphometric parameters of facial cranium asymmetry in adult man. Morfologiya. 2009; 135(2);74-79 (in Russ.). DOI: 10.1016/j.jcms.2014.01.028. 11. Алиева С.А., Шадлинский В.Б., Мовсумов Н.Т. Половые особенности асимметрии краниометрических показате- лей при различных формах лицевого черепа. // Морфо- логические ведомости. – 2019.- Т. 27.- № 4. – С. 9-15. DOI: 10.20340/mv-mn.19(27).04.9-15 11. Alieva SA, Shadlinsky VB, Movsumov NT. Sex-related features of the asymmetry of craniometrics parameters in various forms of the facial skull. Morfologicheskie Vedomosti – Morphological Newsletter. 2019;27(4):9-15. (In Russ.) DOI :10.20340/mvmn.19(27).04.9-15 12. Алексеев В.П., Дебец Г.Ф. Краниометрия: Методика ан- тропометрических исследований. Москва: Наука.1964. 128 с. 12. Alekseev V.P., Debec G.F. Kraniometrija: Metodika antropometricheskih issledovanij. Moskva: Nauka.1964. 128 s. 13. Гайворонский И. В., Дубовик Е. И., Крайник И. В., Дер- гачева Е.А. Асимметрия лицевого черепа у взрослого человека и возможности ее оценки. // Вестник Россий- ской Военно-медицинской академии. 2009. – № 1(25). – С. – 140-144. eLIBRARY ID: 12773593 13. Обсуждение СКТ черепа, вид спереди (SSD-изображение оттенённых поверхностей). Наиболее асимметричные статистически значимые линейные размеры лицевого черепа у обследованных мужчин (1) и женщин (2) показаны черной линией в верхнем «веере», белой линией – в нижнем и пунктирной линией – в боковом «веере» слева и справа. Picture 3. SKT of the skull, front view (SSD-image of shaded surfaces). Th e most asymmetric statistically signifi cant linear dimensions of the facial skull in the examined men (1) and women (2) are shown by a black line in the upper “fan”, a white line – in the lower and dashed lines – in the lateral “fan” on the left and right. Медицинский вестник Юга России Medical Herald of the South of Russia 021;12(3):78-85 Рисунок 3. СКТ черепа, вид спереди (SSD-изображение оттенённых поверхностей). Наиболее асимметричные статистически значимые линейные размеры лицевого черепа у обследованных мужчин (1) и женщин (2) показаны черной линией в верхнем «веере», белой линией – в нижнем и пунктирной линией – в боковом «веере» слева и справа. Picture 3. SKT of the skull, front view (SSD-image of shaded surfaces). Th e most asymmetric statistically signifi cant linear dimensions of the facial skull in the examined men (1) and women (2) are shown by a black line in the upper “fan”, a white line – in the lower and dashed lines – in the lateral “fan” on the left and right. 83 Е.В. Чаплыгина, О.А. Каплунова, М.Г. Шепетюк, О.П. Суханова, И.М. Блинов ПОЛОВЫЕ ОСОБЕННОСТИ АСИММЕТРИИ ЛИЦЕВОГО ЧЕРЕПА ПО ДАННЫМ СПИРАЛЬНОЙ КОМПЬЮТЕРНОЙ ТОМОГРАФИИ ОРИГИНАЛЬНЫЕ СТАТЬИ Е.В. Чаплыгина, О.А. Каплунова, М.Г. Шепетюк, О.П. Суханова, И.М. Блинов ПОЛОВЫЕ ОСОБЕННОСТИ АСИММЕТРИИ ЛИЦЕВОГО ЧЕРЕПА ПО ДАННЫМ СПИРАЛЬНОЙ КОМПЬЮТЕРНОЙ ТОМОГРАФИИ ЛИТЕРАТУРА Gajvoronskij I. V., Dubovik E. I., Krajnik I. V., Dergacheva E.A. Adult visceral cranium asymmetry and its assessment possibility. Vestnik Rossijskoj Voenno-medicinskoj akademii. 2009;1(25):140-144 (in Russ.). eLIBRARY ID: 12773593 14. Bahareva N.S. Features of the asymmetry of the linear dimensions of the facial skulls of residents of the South of Russia. Fundamental'nye issledovaniya. 2012;8-2:279-284. (In Russ.) eLIBRARY ID: 18304362 14. Бахарева Н.С. Особенности асимметрии линейных раз- меров лицевых черепов жителей Юга России. // Фунда- ментальные исследования. – 2012. – №8-2. – С.279-284. eLIBRARY ID: 18304362 84 Медицинский вестник Юга России Medical Herald of the South of Russia 2021;12(3):78-85 Медицинский вестник Юга России Medical Herald of the South of Russia 2021;12(3):78-85 Е.В. Чаплыгина, О.А. Каплунова, М.Г. Шепетюк, О.П. Суханова, И.М. Блинов ПОЛОВЫЕ ОСОБЕННОСТИ АСИММЕТРИИ ЛИЦЕВОГО ЧЕРЕПА ПО ДАННЫМ СПИРАЛЬНОЙ КОМПЬЮТЕРНОЙ ТОМОГРАФИИ ОРИГИНАЛЬНЫЕ СТАТЬИ 15. Ефимова Е.Ю., Краюшкин А.И., Ефимов Ю.В., Буянов Е.А. Линейные параметры черепа мезокранного типа. // Волгоградский научно-медицинский журнал. – 2018, – №4 (60). – С. 15-18. eLIBRARY ID: 36784831 15. Efi mova У. Yu., Krayushkin A. I., Efi mov Yu. V., Bujanov E. A.. Th e linear parameters of the skull of mesocranial type. Volgograd Medical Scientifi c Journal, 2018;4(60):15-18. (In Russ.) eLIBRARY ID: 36784831 Information about the authors Elena V. Chaplygina, Dr. Sci. (Med.), Professor; Rostov State Medical University, Rostov-on-Don, Russia. ORCID: 0000-0002-2855-42103; e-mail: ev.chaplygina@yandex.ru. Information about the authors Elena V. Chaplygina, Dr. Sci. (Med.), Professor; Rostov State Medical University, Rostov-on-Don, Russia. ORCID: 0000-0002-2855-42103; e-mail: ev.chaplygina@yandex.ru. Чаплыгина Елена Викторовна, д.м.н., проф., Ро- стовский государственный медицинский университет, Ростов-на-Дону, Россия. ORCID: 0000-0002-2855-42103; e-mail: ev.chaplygina@yandex.ru. Olga A. Kaplunova, Dr. Sci. (Med.), Professor, Ro stov State Medical University, Rostov-on-Don, Russia. ORCID: 0000-0002-5860-112X; e-mail: kaplunova @bk.ru. Olga A. Kaplunova, Dr. Sci. (Med.), Professor, Ro stov State Medical University, Rostov-on-Don, Russia. ORCID: 0000-0002-5860-112X; e-mail: kaplunova @bk.ru. Каплунова Ольга Антониновна, д.м.н., проф., Ро- стовский государственный медицинский университет, Ростов-на-Дону, Россия. ORCID: 0000-0002-5860-112X; e-mail: kaplunova @bk.ru. Каплунова Ольга Антониновна, д.м.н., проф., Ро- стовский государственный медицинский университет, Ростов-на-Дону, Россия. ORCID: 0000-0002-5860-112X; e-mail: kaplunova @bk.ru. Maxim G. Shepetyuk, assistant, Rostov State Medical University, Rostov-on-Don, Russia. ORCID: 0000-0003- 3950-0590; e-mail: mykshorosh@mail.ru. Maxim G. Shepetyuk, assistant, Rostov State Medical University, Rostov-on-Don, Russia. ORCID: 0000-0003- 3950-0590; e-mail: mykshorosh@mail.ru. Шепетюк Максим Геннадьевич, ассистент, Ро- стовский государственный медицинский университет, Ростов-на-Дону, Россия. ORCID: 0000-0003-3950-0590; e-mail: mykshorosh@mail.ru. Olga P. Suhanova, assistant, Rostov State Medical University, Rostov-on-Don, Russia. ORCID: 0000-0002- 8190-791X; e-mail: Suhanova1949@mail.ru. Olga P. Suhanova, assistant, Rostov State Medical University, Rostov-on-Don, Russia. ORCID: 0000-0002- 8190-791X; e-mail: Suhanova1949@mail.ru. y Суханова Ольга Петровна, ассистент, Ростовский государственный медицинский университет, Ростов- на-Дону, Россия. ORCID: 0000-0002-8190-791X; e-mail: Suhanova1949@mail.ru. Igor M. Blinov, Rostov State Medical University, Rostov- on-Don, Russia. ORCID: ORCID: 0000-0003-3116-0560; e-mail: bim-bim@mail.ru. Блинов Игорь Михайлович, Ростовский государ- ственный медицинский университет, Ростов-на-Дону, Россия. ORCID: 0000-0003-3116-0560; e-mail: bim-bim@ mail.ru. Медицинский вестник Юга России Medical Herald of the South of Russia 2021;12(3):78-85 Получено/ Received: 28.05.2021 Принято к печати/ Accepted: 29.06.2021 Вклад авторов: Authors’ contribution: E.V. Chaplygin – editing the article; O.A. Kaplunova, M.G. Shepetyuk – obtaining and analysis of the data; O.P. Suchanov I.M. Blinov – obtaining research materials. р Е.В. Чаплыгина – редактирование статьи; р р О.А. Каплунова, М.Г. Шепетюк – получение и анализ данных; О.П. Суханова, И.М. Блинов – получение материалов исследования. Получено/ Received: 28.05.2021 Принято к печати/ Accepted: 29.06.2021 Принято к печати/ Accepted: 29.06.2021 Медицинский вестник Юга России Medical Herald of the South of Russia 2021;12(3):78-85 85
https://openalex.org/W4392817716	https://www.qeios.com/read/29U36Y/pdf	English	null	Review of: "Patterns of Social Dynamics Surrounding Suicide Notes on Facebook Platforms: A Netnographic Approach"	null	2,024	cc-by	426	Qeios, CC-BY 4.0 · Review, March 14, 2024 Jussara Carvalho dos Santos1 1 Universidade de São Paulo Potential competing interests: No potential competing interests to declare. Review of: "Patterns of Social Dynamics Surrounding Suicide Notes on Facebook Platforms: A Netnographic Approach" Jussara Carvalho dos Santos1 Potential competing interests: No potential competing interests to declare. The present article addresses a very important topic; however, it still needs to be further refined. Some points that need to be improved for the article to be published include: 1. More detailed methodology: Although the article mentions the use of netnography as a research method, it is important to provide more details on how netnography was conducted, including the selection of Facebook groups, data collection, analysis, and ethical considerations. Additionally, it is important to highlight how sampling was done and how participants were recruited. 1. More detailed methodology: Although the article mentions the use of netnography as a research method, it is important to provide more details on how netnography was conducted, including the selection of Facebook groups, data collection, analysis, and ethical considerations. Additionally, it is important to highlight how sampling was done and how participants were recruited. 2. Methodological rigor: It is essential to ensure that the methodology used is robust and that the results are reliable. This may include a more detailed description of content analysis techniques, network observation, and netnographic observation, as well as validation of results through triangulation methods. 3. In-depth discussion of results: The article could benefit from a more in-depth discussion of the results, highlighting the practical and theoretical implications of the findings. Additionally, it would be helpful to relate the results to existing literature on the topic and highlight how the results contribute to advancing knowledge in the field of mental health. 4. Ethical considerations: Although the article mentions the importance of ethical considerations, it is fundamental to provide details on how informed consent was obtained from participants and how privacy and anonymity were protected. Moreover, it is important to highlight how researchers prioritized the well-being of participants and adhered to ethical guidelines when conducting the study. 5. Original contribution: The article should clearly highlight its original contribution to the field, whether through new discoveries, innovative methodological approaches, or theoretical insights. This may include a more in-depth discussion of how the study results fill gaps in existing literature and how they can inform mental health interventions and policies. By addressing these points, the article will be better prepared for consideration for publication in a mental health journal. Qeios ID: 29U36Y · https://doi.org/10.32388/29U36Y 1/1
W1989047251.txt	https://journals.plos.org/plospathogens/article/file?id=10.1371/journal.ppat.1003115&type=printable	en		Complement Mediated Signaling on Pulmonary CD103+ Dendritic Cells Is Critical for Their Migratory Function in Response to Influenza Infection	PLOS pathogens	2,013	cc-by	11,005	Complement Mediated Signaling on Pulmonary CD103+ Dendritic Cells Is Critical for Their Migratory Function in Response to Influenza Infection Matheswaran Kandasamy1, Poon C. Ying1, Adrian W. S. Ho2, Hermi R. Sumatoh1, Andreas Schlitzer2, Timothy R. Hughes3, David M. Kemeny4, B. Paul Morgan3, Florent Ginhoux2, Baalasubramanian Sivasankar1* 1 Infection and Immunity Programme, Singapore Institute for Clinical Sciences, Agency for Science, Technology and Research (ASTAR), Singapore, 2 Singapore Immunology Network (SIgN), Agency for Science, Technology and Research (ASTAR), Singapore, 3 Institute of Infection and Immunity, School of Medicine, Cardiff University, Cardiff, Wales, United Kingdom, 4 Immunology Programme and Department of Microbiology, National University of Singapore, Singapore Abstract Trafficking of lung dendritic cells (DCs) to the draining lymph node (dLN) is a crucial step for the initiation of T cell responses upon pathogen challenge. However, little is known about the factors that regulate lung DC migration to the dLN. In this study, using a model of influenza infection, we demonstrate that complement component C3 is critically required for efficient emigration of DCs from the lung to the dLN. C3 deficiency affect lung DC-mediated viral antigen transport to the dLN, resulting in severely compromised priming of virus-specific T cell responses. Consequently, C3-deficient mice lack effector T cell response in the lungs that affected viral clearance and survival. We further show that direct signaling by C3a and C5a through C3aR and C5aR respectively expressed on lung DCs is required for their efficient trafficking. However, among lung DCs, only CD103+ DCs make a significant contribution to lung C5a levels and exclusively produce high levels of C3 and C5 during influenza infection. Collectively, our findings show that complement has a profound impact on immune regulation by controlling tissue DC trafficking and highlights a potential utility for complement as an adjuvant in novel vaccine strategies. Citation: Kandasamy M, Ying PC, Ho AWS, Sumatoh HR, Schlitzer A, et al. (2013) Complement Mediated Signaling on Pulmonary CD103+ Dendritic Cells Is Critical for Their Migratory Function in Response to Influenza Infection. PLoS Pathog 9(1): e1003115. doi:10.1371/journal.ppat.1003115 Editor: Andrew Pekosz, Johns Hopkins University - Bloomberg School of Public Health, United States of America Received June 13, 2012; Accepted November 20, 2012; Published January 10, 2013 Copyright: ß 2013 Kandasamy et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded by the Agency for Science, Technology and Research (ASTAR; http://www.a-star.edu.sg/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. E-mail: shiv@sics.a-star.edu.sg both types of lung resident DCs migrate to the dLN to prime T cells [10] henceforth referred to as migratory DC subsets (mDCs). The complement system is an essential component of the innate immune network and has evolved as an important bridge between innate and adaptive immune systems, similarly to DCs, but at the molecular level [11,12,13,14]. In particular, complement component C3 has been shown to impact antiviral T cell immunity and allograft rejection in a mechanism independent of intrinsic C3 expression in T cells [15,16,17,18]. These observations suggested the involvement of an additional cell type expressing C3 that control T cell activation. Recent studies using bone marrow derived DCs have revealed that C3 is essential for DCs to efficiently stimulate T cells [19,20], suggesting that DCs mediate these aforementioned complement effects [15,16,17] on T cells by acting both as complement producing and sensing cells. However, the in vivo functional relevance of C3 in peripheral tissue DCs, which play a central role in the induction of immunity by virtue of their location [21], remains largely unexplored, necessitating further studies. Influenza infection is known to activate complement both locally in the lung and systemically [22,23], but the biological significance of complement activation during influenza infection remains poorly understood. The functional importance of C3 in T Introduction Influenza is a global health problem and current vaccination strategies are still inadequate at providing protection against seasonal and epidemic outbreaks [1]. Vaccination strategies aiming to induce a protective CD8+ T cell response hold tremendous potential, since CD8+ T cells are able to recognize core epitopes conserved across a wide range of influenza strains [2,3]. Hence, there is a pressing need to improve our understanding on the mechanisms that contribute to the orchestration of CD8+ T cell responses during influenza infection. Influenza-specific T cell responses are initiated and maintained by lung dendritic cells (DCs) which are strategically localized within the respiratory tract to mediate this process effectively [4,5]. DCs comprise a heterogeneous population of antigen sensing and presenting cells that control the initiation of T cell responses thus bridging innate and adaptive immune responses [6]. Different subsets of DCs with unique homeostasis and immune functions had been described in both lymphoid and non-lymphoid tissues [7]. In this regard, lung resident DCs can be divided into CD103+CD11b2 (CD103+ DCs) and CD1032CD11b+ (CD11b+ DCs) based on the expression of the integrins aEb7 (CD103) and ITGAM (CD11b) respectively [8,9]. During influenza infection, PLOS Pathogens \| www.plospathogens.org 1 January 2013 \| Volume 9 \| Issue 1 \| e1003115 Complement Mediates Lung DC Migration resulted in 100% mortality by day 12 (Fig. 1A and B). Subsequent evaluation of effector T cell responses and viral load in the lungs recapitulated previous observations [16], showing decreased effector T cell responses and viral clearance in the C32/2 mice (Fig. S1). Absence of C3 in T cells does not directly alter their function [16], hence we reasoned that these defects in T cell responses were mediated by the lack of C3 in DCs, as suggested in previous reports [19,20]. In order to investigate whether priming of antigen-specific T cells in the dLN in response to influenza infection was altered in C32/2 mice, we adoptively transferred CFSE labeled OT-I CD8+ transgenic T cells into mice prior to infection with either wild type PR8 influenza virus or recombinant PR8 influenza virus containing the OVA epitope SIINFEKL (PR8-OT-I). We then harvested lung dLNs at indicated time points post-infection to assess proliferation by CFSE dilution on SIINFEKL-Kb tetramer positive CD8+ T cells. As expected, proliferation was observed only when mice were inoculated with PR8-OT-I, and no proliferation was observed with PR8 influenza virus alone, reflecting the specificity of the response (Fig. 1C). Strikingly, the level of proliferation was significantly reduced in the C32/2 mice in comparison with WT mice (Fig. 1C). CD4+ T cell priming in the dLN was also evaluated using OT-II (CD4) transgenic T cells and PR8 influenza virus containing the OVA epitope ISQAVHAAHAEINEAGR (PR8-OT-II). Similarly, we found that the priming of CD4+ T cells was significantly reduced in the C32/2 mice when compared to WT controls (Fig. 1D). Since C3 has been reported to influence the expression of costimulatory molecules on DCs [19], we tested whether diminished levels of costimulation could explain the decreased T cell responses by measuring the expression of costimulatory molecules on mDCs in the dLN of both WT and C32/2 mice on day 2 post-infection. Resident and mDC subsets in the dLN were characterized as shown in Fig. S2. We found comparable levels of CD86 and CD40 expression, while CD80 expression was even increased in the C32/2 mDCs (Fig. 1E and F), excluding an intrinsic maturation defect in C32/2 DCs. Finally, to understand whether the T cell priming defect observed in vivo was due to a decreased priming ability by C32/2 mDCs (Fig. 1C and D), we sorted CD103+ DCs and CD11b+ DCs on day 2 post infection with PR8-OT-I and evaluated their priming ability ex vivo. A strong proliferation of OT-I CD8+ T cells was observed only when CD8+ T cells were cocultured with CD103+ DCs, but not with CD11b+ DCs, an observation supporting the predominant role of CD103+ DCs in early CD8+ T cell priming [10]. However, ex vivo priming ability was comparable between WT and C3-deficient CD103+ DCs (Fig. 1G), demonstrating that there was no intrinsic priming defect in C3-deficient CD103+ DCs. Author Summary Influenza is a global health problem frequented by epidemics and pandemics. Current vaccines against influenza offer limited protection hence the need for reformulation and repeated vaccination. There is a pressing need to develop newer vaccines that are able to generate T cell response. In order to develop such vaccines, there is a need to understand how T cell responses are generated during influenza infection. Influenza specific T cell responses are generated by the dendritic cells (DCs) in the lung. Upon influenza infection, DCs in the lung carry viral peptides to the draining lymph node (dLN) to initiate an immune response. Thus, migration of DCs from the lung to the dLN is an important step in the initiation of influenza specific T cell response. We now show that activation products of the complement system interact with their receptors on the DCs, which signals for the DCs to migrate from the lung to the dLN. Thus, our results reveal a previously unknown function for complement in mediating lung DC migration during influenza infection and highlight its potential as an adjuvant in novel vaccine strategies. cell immunity during influenza infection was initially demonstrated by Kopf et al., who showed that C32/2 mice exhibited reduced T cell response to influenza infection and attributed this to a possible priming defect mediated by DCs [16]. Thus, we hypothesized that C3 may be critically involved in the control of the T cell priming function of lung mDCs during influenza infection. In this study, we used C3-deficient (C32/2) mice to show that C3 was critical for survival during influenza infection, and C3 deficiency was associated with attenuated T cell priming in the dLN and reduced development of effector T cell responses in the lung as previously shown [16]. However, we found that this defective priming was not due to altered priming ability of C3deficient DCs but rather due to a defect of mDC migration from the lung to the dLN in C32/2 mice. We further demonstrated that the direct interaction of complement activation products C3a and C5a with their receptors expressed on the surface of mDCs was critical for their migration. Finally, we identified CD103+ DCs as the sole mDC subset capable of secreting C3 and C5 and one of the major source of lung C5a during influenza infection. Altogether, our results establish a previously unidentified role for complement activation products C3a and C5a in mediating migratory function of mDCs and highlight the crucial role of CD103+ DC subset as an unique complement sensing and producing DC population controlling its own migration and the migration of CD11b+ DCs. C32/2 mice show defective trafficking of mDC subsets from the lung to the dLN during influenza infection Results Next, we investigated the composition of mDC subsets in the lung and lung dLN at steady state and during the course of influenza infection to test whether the priming defect could be explained by a defect of the mDC network in the lung and dLN of C32/2 mice. The gating strategy employed for characterizing lung mDCs is shown in Fig. S3 [25,26]. Under steady state conditions, the relative numbers of CD103+ DCs and CD11b+ DCs were comparable between C32/2 and WT mice, whereas enumeration of absolute numbers showed a slight decrease for CD103+ DCs only in the C32/2 mice (Fig. 2A–C). Upon influenza infection, both the relative and absolute numbers of lung CD103+ DCs and CD11b+ DCs became comparable between C32/2 and WT mice during influenza infection (Fig. 2A–C). Complement component C3 is critical for survival during influenza infection C3 has been shown to be important for eliciting T cell responses and viral clearance during influenza infection in mice [16], although the impact of C3 deficiency on survival after influenza infection was not well reported. In order to evaluate this, we infected WT and C32/2 mice with a sub-lethal dose (optimized in WT mice, 15 PFU for female and 25 PFU for male) of influenza virus and monitored their weight loss and survival. WT mice showed ,20% weight loss at the peak of infection and recovered with 20% mortality as previously reported [24]. In stark contrast, C32/2 mice showed greater weight loss on days 5 and 7, and PLOS Pathogens \| www.plospathogens.org 2 January 2013 \| Volume 9 \| Issue 1 \| e1003115 Complement Mediates Lung DC Migration Figure 1. C32/2 mice show greater weight loss and mortality during influenza infection. (A) Percentage of body weight loss after influenza infection. A weight loss of ,20% and recovery represents a sub-lethal infection. (B) Survival curve comparing WT and C32/2 mice during influenza infection (n = 6–7 in each group). (C) Plots represent ex vivo analysis of CFSE-labeled OT-I CD8+ T cell proliferation in the dLN 3 days after infection with PR8 and PR8-OT-I (bottom). The graph on the right shows the respective division index for the proliferating OT-I CD8+ T cells for each group of mice. (D) Plots represent ex vivo analysis of CFSE-labeled OT-II CD4+ T cell proliferation in the dLN 3 days after infection with PR8 and PR8OT-II (bottom). The graph on the right shows the respective division index for the proliferating OT-II CD4+ T cells for each group of mice. (E) Histogram showing expression of costimulatory molecules CD86, CD80 and CD40 on CD103+ DCs and CD11b+ DCs in the dLN on day 2 post infection. Filled histogram: Isotype control, dashed histogram: Naı̈ve, open histogram: infected. (F) Bar graphs show the mean fluorescent intensity (MFI) of CD86, CD80 and CD40 expression on CD103+DC and CD11b+ DC subsets in the dLN of naı̈ve and influenza infected (Day 2) WT or C32/2 mice. (G) Plots represent CFSE-labeled OT-I CD8+ T cell proliferation 3 days after co-culture with sorted CD103+ DCs and CD11b+ DCs obtained from pooled dLN of PR8-OT-I influenza infected mice at a ratio of 1:10 (DC:T cells). Results shown are representative of four experiments with similar results. The values are expressed as mean 6 SEM. doi:10.1371/journal.ppat.1003115.g001 PLOS Pathogens \| www.plospathogens.org 3 January 2013 \| Volume 9 \| Issue 1 \| e1003115 Complement Mediates Lung DC Migration Figure 2. Migratory DC subsets are reduced in the dLN in C32/2 mice after influenza infection. (A) Kinetics of CD103+ DCs and CD11b+ DCs in the lungs during influenza infection in WT and C32/2 mice. Populations were gated as show in Fig. S2. Numbers within the dot plot represent average percentage of cells for the group. (B and C) Absolute numbers of CD103+ DC (B) and CD11b+ DC (C) subsets in lungs during steady state and during influenza infection. (D) Kinetics of CD103+ DCs and CD11b+ DCs in the dLN during influenza infection in WT and C32/2 mice. Populations were gated as shown in Fig. S1. Numbers within the dot plot represent average percentage of cells for the group. (E and F) Absolute numbers of CD103+ DC (E) and CD11b+ DC (F) subsets in dLN during steady state and during influenza infection. The values are expressed as mean 6 SEM. The data are representative of three different experiments with similar results. , P,0.05, and P,0.01. doi:10.1371/journal.ppat.1003115.g002 by day 3 in the WT mice. However, CFSE+ mDC accumulation (independent of subsets) was significantly reduced in the C32/2 dLN (Fig. 3A left panel and B). Upon enumeration of CD103+ DCs and CD11b+ DCs within the CFSE+ mDC population, we found that migration of both CD103+ DCs and CD11b+ DCs was severely compromised in the C32/2 mice (Fig. 3A, C and D). In order to understand whether a strong inflammatory signal is able to overcome the defective signaling on mDCs due to the lack of C3, we administered LPS intratracheally shortly after influenza infection and then evaluated mDC migration in C32/2 mice. Our observations indicated that LPS administration was not able to overcome the defective trafficking of both subset of lung mDCs in the C32/2 mice, although a mild effect was observed for CD11b+ DCs (Fig. 3E). These observations indicate that the critical importance of complement mediated signaling in lung mDC trafficking is independent of inflammatory signals. Altogether, our observations demonstrate that migration of C32/2 mDCs to the dLN is significantly reduced compared to WT during inflammation. These results show that the T cell priming deficit observed in C32/2 mice is not due to a defect in the differentiation of lung mDCs. Next, we assessed whether the migration ability of lung mDCs was compromised. Under steady state, lung mDCs constitutively migrate at a slow rate to the dLN, a process strongly increased under inflammatory conditions [8]. As expected, in WT mice during the course of infection, the absolute numbers of both CD103+ DCs and CD11b+ DCs increased in the dLN by day 1 post infection and further by day 2 post infection (Fig. 2D–F). However, absolute numbers of both CD103+ DCs and CD11b+ DCs were significantly decreased in C32/2 mice, suggesting a decreased migration capacity of CD103+ DCs and CD11b+ DCs at both steady state and at all time points tested after influenza infection (Fig. 2D–F). To confirm these data, the migration of mDCs from the lung to the dLN was tracked using CFSE delivered intranasally into the lungs at different time points after influenza infection [27]. As expected, the relative numbers of CFSE+ mDCs in the dLN increased after influenza infection, reaching a maximum PLOS Pathogens \| www.plospathogens.org 4 January 2013 \| Volume 9 \| Issue 1 \| e1003115 Complement Mediates Lung DC Migration Figure 3. Tracking of mDC migration from the lung to the dLN demonstrates defective mDC migration in C32/2 mice during influenza infection and intratracheal administration of LPS does not recover the defective mDC migration during influenza infection. WT and C32/2 mice were infected with influenza virus; 16 hours before sacrificing, cells in the lungs were labeled with 8 mM CFSE by intranasal instillation. dLN was harvested and analysed for CFSE+ mDCs using gating strategy shown in Fig. S1. (A) Dot plots show CFSE+ mDCs (first panel) in the dLN and subsequent panels show CD103+ DCs and CD11b+ DCs after gating on CFSE+ mDCs at indicated times after influenza infection. Numbers within the dot plot represent cell number. (B) Kinetics of frequency of CFSE+ mDCs in the dLN of influenza infected mice. Absolute number of CFSE+ CD103+ DCs (C) and CFSE+ CD11b+ DCs (D) in the dLN at indicated time points after influenza infection. LPS (2 mg in PBS) was administered intratracheally after 6 hours to either uninfected or flu infected C32/2 mice and the migration of mDCs from the lung to the dLN were followed as described earlier using CFSE on day 2 post infection. (E) Number of CFSE+ CD103+ (left) and CD11b+ (right) DCs in LPS administered C32/2 mice in comparison with WT mice without LPS administration. The values are expressed as mean 6 SEM. The data are representative of three different experiments with similar results. , P,0.05, P,0.01 and * P,0.001. doi:10.1371/journal.ppat.1003115.g003 defective mDCs migration in C32/2 mice, we examined their antigen uptake capacity using DiD labeled influenza virus as previously described [25]. We first infected mice with unlabelled PR8 influenza virus to establish an infection and its associated inflammation, and subsequently inoculated the same mice with DiD labeled PR8 influenza virus 16 hours Viral uptake and maturation is not affected in mDCs from C32/2 mice Complement component C3 mediated opsonization has been shown to affect viral uptake by DCs [28]. To test whether reduced viral uptake by C32/2 DCs could explain the PLOS Pathogens \| www.plospathogens.org 5 January 2013 \| Volume 9 \| Issue 1 \| e1003115 Complement Mediates Lung DC Migration Figure 4. C32/2 mDCs display similar capacity for virus uptake and maturation. (A) Dot plots show the percentage of DiD flu+ uptake by CD103+ DC (left panel) and CD11b+ DC (right panel) in WT and C32/2 mice 16 hrs after DiD flu administration on day 1 post infection with PR8. (B) Bar graph shows the average numbers of DiD flu+ CD103+ DCs and DiD flu+ CD11b+ DCs in the lungs of WT and C32/2 mice. (C)Single cell preparations from the lungs were enriched for DCs by a density gradient method. Enriched DCs were infected with influenza virus under ex vivo culture conditions and six hours later the CD103+ and CD11b+ DCs were flow sorted. Q-RT-PCR for the indicated cytokines were performed on the RNA obtained from the CD103+ and CD11b+ DC populations. Bar graph shows % relative expression in comparison to un-infected control. (D) WT and C32/2 mice were infected with flu and 24 hours later the CD103+ and CD11b+ DCs from the lungs were flow sorted. Q-RT-PCR for the indicated cytokines were performed on the RNA obtained from the CD103+ and CD11b+ DC populations. Bar graph shows % relative expression in comparison to un-infected control. (E) Bar graphs shows the mean fluorescent intensity of CD86, CD40 and CD80 expression on CD103+DC and CD11b+ DC subsets in the lungs of naı̈ve and PR8 infected WT or C32/2 mice. The data are representative of three different experiments with similar results. The values are expressed as mean 6 SEM. doi:10.1371/journal.ppat.1003115.g004 PLOS Pathogens \| www.plospathogens.org 6 January 2013 \| Volume 9 \| Issue 1 \| e1003115 Complement Mediates Lung DC Migration before harvesting the lungs. DiD+ mDC subsets were comparable between C32/2 and WT mice suggesting that the lack of C3 did not affect antigen uptake (Fig. 4A and B). Migratory DCs upon exposure to antigen under inflammatory conditions undergo maturation before migrating to the dLN and C3 is known to influence the expression of costimulatory molecules [29]. In order to understand whether the mDCs in the C32/2 mice produced sufficient inflammatory mediators comparable to the WT mice, we examined the expression levels of inflammatory cytokines in the mDCs after ex vivo and in vivo influenza infection. Expression levels of IL-1b and IL-6 were comparable in the mDCs under both ex vivo and in vivo conditions. However IL-12p40 expression were higher in the C32/2 mice when mDCs were infected under ex vivo conditions whereas comparable under in vivo conditions. (Fig. 4C and D). Subsequently, we evaluated the maturation of mDC subsets by analyzing the expression of costimulatory molecules after influenza infection (Fig. 4E). Expression levels of CD86, CD80 and CD40 on mDCs were comparable between C32/2 and WT mice during the course of infection. CCR7 expression on mDCs has been shown to be important for their migration to dLN [29,30,31], hence we evaluated the expression of CCR7+ at the surface of lung mDCs. Under steady state, the frequency of CCR7 expressing mDCs was lower in C32/2 mice (Fig. 5A–C). After influenza infection, the relative numbers of CCR7 expressing mDCs increased in both groups to a similar extent. However due to the reduced initial frequency of CCR7+ mDCs in C32/2 mice at steady state, the proportion of CCR7+ mDCs upon flu infection remained lower than WT (Fig. 5A–C). These observations suggest that lack of C3 did not affect the up- regulation of CCR7 expression on mDCs under inflammatory conditions. Altogether, these results show that C3-deficient mDCs are not defective in viral uptake and are fully competent in the expression of inflammatory mediators and co-stimulatory molecules for T-cell priming, but are less migratory. Complement components C3 and C5 are produced by CD103+ DCs upon influenza infection The absence of C3 causes a complete block in complement activation and hence C32/2 mice lack the ability to generate both C3a and C5a [22]. To ascertain whether these activation products are directly involved in mediating mDC migration, we first quantified the levels of C3a and C5a in the bronchioalveolar lavage fluid before and after influenza infection in WT mice by ELISA. At steady state, C3a levels were high, further increasing by approximately 4-fold on day 2 after infection and returning to background level by day 4 (Fig. 6A). In the case of C5a, steady state levels were very low, increasing by approximately 7-fold and 17-fold on days 2 and 4 respectively post infection, before returning to background levels by day 7 post infection (Fig. 6A). Next, we investigated the cellular source of C3a and C5a and assessed the ability of lung mDCs to synthesize and secrete C3 and C5 during infection. Lung DCs before and after influenza infection were sorted and C3 and C5 mRNA expression levels were determined by qRT-PCR. Both C3 and C5 mRNA levels were significantly upregulated after influenza infection in CD103+ DCs; C3 levels were upregulated by approximately 3-fold on day 1 post infection and 50-fold on day 3 after infection (Fig. 6B), while C5 levels were upregulated 2-fold on day 1 post infection and 5fold on day 3 (Fig. 6C). No modulation of C3 and C5 mRNA expression was observed in CD11b+ DCs after influenza infection (Fig. 6B and C). Since only CD103+ DCs showed increased C3 and C5 mRNA expression upon influenza infection, we evaluated the contribution of CD103+ DCs to the observed increase in C3a and C5a levels in the lung during influenza infection. For this purpose, we used langerin-DTR mice to specifically deplete CD103+ DCs [10]. Langerin-DTR mice specifically expressed DTR on CD103+ DCs in the lung and DT administration efficiently depleted lung CD103+ DCs (Fig. S4), and subsequent influenza infection in DTtreated langerin-DTR mice did not cause increased C3a and C5a Figure 5. C32/2 mDCs show similar fold CCR7 up regulation upon influenza infection. (A) Representative FACS plots for CCR7 expression in lung mDC subsets of naı̈ve and PR8 infected WT or C32/2 mice. Percentage CCR7 expression on CD103+ DCs (B) and CD11b+ DCs (C) during the course of influenza infection. The values are expressed as mean 6 SEM. doi:10.1371/journal.ppat.1003115.g005 PLOS Pathogens \| www.plospathogens.org 7 January 2013 \| Volume 9 \| Issue 1 \| e1003115 Complement Mediates Lung DC Migration Figure 6. Lung C3a and C5a levels are up regulated during influenza infection by exclusive contribution from CD103+ DCs. (A) Bar graph shows the amounts of C3a and C5a per 100 mg protein in BAL fluid from naı̈ve and PR8 infected WT mice on day 2, 4 and 7 post infection. (B and C) Bar graphs showing the relative mRNA expression levels of C3 and C5 in FACS sorted lung CD103+ DC and CD11b+ DC subsets in naı̈ve or PR8 infected WT mice on day 1 and 3 post infection. Highest expression level in the naı̈ve mice is taken as 100%. (D and E) Bar graphs show the amount of C3a and C5a per 100 mg protein in BAL fluid from naı̈ve and PR8 infected langerin DTR mice with or without the depletion of CD103+ DCs. The data are representative of three different experiments with similar results. The values are expressed as mean 6 SEM. doi:10.1371/journal.ppat.1003115.g006 levels in the lungs on days 2 and 4 when compared with CD103+ DCs sufficient mice (Fig. 6D and E). Importantly, when depletion of lung CD103+ DCs in langerinDTR mice was followed by infection with PR8-OT-I, priming of OT-I CD8+ T cells in the dLN was severely reduced when compared with PR8-OT-I infected WT (Fig. 7A), similar to our observations in the C32/2 mice (Fig. 1C). CD103+ DCs depleted langerin-DTR mice infected with influenza displayed greater weight loss (Fig. 7B), higher mortality (Fig. 7C), reduced lung effector T cell response (Fig. 7D–G) and increased viral load (Fig. 7H) as compared to WT, paralleling our observations in C32/2mice (Fig. 1A and B). Since DT administered langerinDTR mice showed rapid mortality, we evaluated whether DT administration induced any toxicity during influenza infection in WT mice. Our observation did not indicate any toxicity for DT in WT mice during influenza infection (Fig. S5). Among the mDC subsets, only the CD103+ DCs were found to produce the complement components C3 and C5 upon influenza infection (Fig. 6B and C) and depletion of CD103+ DCs significantly reduced the availability of C3a and C5a in the lungs (Fig. 6D and E). We hypothesized that CD11b+ DCs rely on complement produced by CD103+ DCs for their migration to the dLN. Therefore, we followed the migration of CD11b+ DCs in the langerin-DTR mice after depleting the CD103+ DCs. Our results suggested that depletion of CD103+ DCs in langerin-DTR mice PLOS Pathogens \| www.plospathogens.org significantly affected the migration of CD11b+ DCs, supporting our hypothesis (Fig. 7I and J). Altogether, these observations suggest that C3a and C5a produced by CD103+ DCs are crucial for their migration to the dLN in order to initiate protective T cells responses and also control the migration of CD11b+ DCs. Locally produced C3a and C5a provide migratory signals by interacting with their receptors on mDCs Our data suggest that sensing of complement activation products C3a and C5a by mDCs is crucial for their migration to the dLNs, which could be mediated by expression of the receptors C3aR and C5aR on mDCs during infection. Thus, we examined the expression of C3aR and C5aR mRNA in mDC subsets by qRT-PCR. In CD103+ DCs, there was a slight increase in the expression of C3aR mRNA on day1 post infection, and a 4-fold increase by day 2 post infection. C5aR also increased by 4-fold on day 1 and by 90-fold on day 2 post infection (Fig. 8A and B). In CD11b+ DCs, the expression of both C3aR and C5aR was higher even under steady state as compared to CD103+ DCs and showed a modest increase on day 2 post infection (Fig. 8A and B). To determine whether mDC migration to the dLNs was directly controlled through interaction of C3a and C5a with their receptors, we blocked C3aR and C5aR either alone or together in vivo using specific, high affinity competitive antagonists [32,33]. Compstatin was used as the control peptide since it is known to 8 January 2013 \| Volume 9 \| Issue 1 \| e1003115 Complement Mediates Lung DC Migration Figure 7. Langerin-DTR mice show effector T cells response and survival characteristics similar to those of C32/2 mice upon influenza infection and has defective CD11b+ DC migration. (A) Plots represent ex vivo analysis of CFSE-labeled OT-I CD8+ T cells proliferation in the dLN 3 days after infection with PR8 and PR8-OT-I (bottom) in WT and CD103+ DCs depleted langerin-DTR mice. (B) Percentage of body weight loss after influenza infection. (C) Survival curve comparing WT, C32/2 and Langerin –DTR mice during influenza infection (n = 6 per group). (D) Graph shows the relative mRNA expression level of influenza M protein in lung tissues of WT and CD103+ DCs depleted langerin-DTR mice infected with 4 PFU of PR8. (E) Graph shows the frequency of IFNc secreting CD4+ T cells in lungs by ex vivo overnight stimulation with MHC-II flu peptide on day 7 post infection. (F) Bar graph shows the absolute numbers of IFNc secreting CD4+ T cells in lungs on day 7 post infection (G) Graph shows the frequency of Flu specific CTL response in lung as measured by Flu peptide (ASNENMETM (NP 366–374)/H-2Db tetramer staining on day 7 post infection. (H) Bar graph shows the absolute numbers of flu specific CD8+ T cells in lungs by tetramer staining on day 7 post infection. DT treated WT and Langerin-DTR mice were flu infected and the migration of CD103+ and CD11b+ DCs were evaluated as described before on day 3 post infection. (I) Dot plots show CD103+ and CD11b+ DCs mDCs in the dLN of DT treated and untreated WT and Langerin-DTR mice. Numbers within the dot plot represent cell number. (J) Total number of CD103+(top) and CD11b+(bottom) DCs in the dLN of flu infected mice. Results shown are representative of at least three different experiments with similar results. The values are expressed as mean 6 SEM. doi:10.1371/journal.ppat.1003115.g007 PLOS Pathogens \| www.plospathogens.org 9 January 2013 \| Volume 9 \| Issue 1 \| e1003115 Complement Mediates Lung DC Migration Figure 8. C3aR and C5aR expression on mDCs is upregulated upon influenza infection and blockade of the receptors inhibits mDC migration to the dLN. C3aR (A) and C5aR (B) expression in FACS sorted lung CD103+ DC and CD11b+ DC subsets in naı̈ve or PR8 infected WT mice on day 1 and 3 post infection. Highest expression level in a naı̈ve mouse is taken as 100%. (C and D) Tracking of mDC from the lung to the dLN (as described in Fig. 3) in mice treated with PBS or compstatin or C3aR antagonist (C3aRA) or C5aR antagonist (C5aRA) or both C3aRA and C5aRA together. Bar graphs show the absolute numbers of CFSE+ CD103+ DC (C) and CD11b+ DC (D) subsets in the draining lymph nodes on day 2 post infection. The data are representative of three different experiments with similar results. The values are expressed as mean 6 SEM. (E–G) Total CD11c+ cells were purified from the lungs WT and C3aR2/2C5aR2/2 mice that has been flu infected 18 hours before. Purified cells were CFSE labeled and then transferred (56105 cells obtained by pooling CD11c+ cells from 5–6 mice) into a WT mice intratracheally that has been previously (36 hours before) infected with flu. CFSE+ mDCs were analysed for the number of CD103+ and CD11b+ DCs in the dLN of recipient mice 18 hours after adoptive transfer of cells. (E) Right panel shows the number of CD103+ and CD11b+ DCs within the CFSE+ mDC population in the dLN. Numbers indicate the number of cells within the gate. (F) Percentage of CFSE+ mDC in the dLN gated from CD11chi MHC IIhi cells as shown in Fig. S2. (G) Bar graph showing the number of CD103+ and CD11b+ DCs within the CFSE+ mDCs in the dLN of adoptively transferred recipient mice. The data are representative of three different experiments with similar results. doi:10.1371/journal.ppat.1003115.g008 PLOS Pathogens \| www.plospathogens.org 10 January 2013 \| Volume 9 \| Issue 1 \| e1003115 Complement Mediates Lung DC Migration The specific production of C3 and C5 by CD103+ DCs underlines their central role in transporting antigen from the lung to the dLN, allowing the priming of T-cells during the early phase of influenza infection [10,25]. It is also interesting to note that CD103+ DCs control the migration of CD11b+ DCs based on the fact that CD11b+ DCs do not produce C3and C5 upon influenza infection, and that their migratory function is facilitated through the production and generation of complement activation products from CD103+ DCs. Thus, to the best of our knowledge, our data demonstrates for the first time how one subset of DC is able to control the functioning of another in a complement dependent manner. Therefore, the defective priming observed in the C32/2 mice may be primarily due to a profound defect in DC-dependent transport of viral antigen to the dLN. In addition the frequency of resident DCs (CD8a+ DCs) in dLN were comparable between WT and C32/2 mice (Fig. S2), ruling out their contribution to this defect. This is further supported by our observations in CD103+ DC-depleted langerin-DTR mice which showed deleterious effects on survival and effector T cell responses similar to those observed in C32/2 mice upon influenza infection. Our results also indicated that the percentage of mDCs that express CCR7 was low under steady state in the C32/2 mice and remained low during influenza infection despite CCR7 upregulation. These results suggest that lack of complement affects CCR7 expression only under steady state, but that CCR7 upregulation during maturation was unaffected in the absence of complement mediated signaling (Fig. 5A–C). Although CCR7 has been shown to be critical for mDC migration to the dLN, its upregulation alone was insufficient to compensate for normal mDC migration, suggesting that multiple signals control DC migration to the LNs [35,36,37]. Supporting this view, NLRP10, a nucleotide-binding domain leucine-rich-repeat-containing receptors (NLR) has been recently implicated in DC migration, independently of CCR7 mediated signaling [38]. Of note, expression of factor-H and CD59a, genes that control complement activation was found to be altered in DCs from NLRP102/2 mice, suggesting a possible coordination between complement and NLRP10 in facilitating DC migration [38]. Previous studies have shown that DC-derived and -activated C3 and C5 could signal via C3aR and C5aR in an autocrine manner to promote T-cell activation during cognate interaction [19]. In addition, C5aR2/2 DCs exhibited attenuated proinflammatory cytokine production, lower expression of MHC-II and costimulatory molecules in response to LPS challenge, as well as reduced capacity for allospecific T-cell stimulation [39]. Similarly, C3deficient macrophages exhibited lower MHC-II expression and poor ability to expand alloreactive T-cells [40]. These studies, however, were limited by the fact that experiments were performed under in vitro conditions and were confined to bone marrow-derived DCs or macrophages. Furthermore, it is not clearly well established how much these in vitro derived cells are related to resident DCs within non-lymphoid organs. Tissue mDCs are derived from committed circulating DC precursors which migrate from the bone marrow to the periphery and differentiate into distinct DC subsets [7], whereas bone-marrow DCs were proposed to be rather of monocytic origin [41,42]. Furthermore, monocyte-derived DCs or inflammatory DCs are absent at steady state and only appear during inflammatory conditions, which permits their differentiation from Ly6Chi blood monocytes [31,41]. Apart from their late appearance, these monocyte-derived DCs possess little ability to acquire viral particles, although their soluble protein uptake capacity is comparable with mDCs [43]. Furthermore, we observed that C3 does not play a costimulatory role on lung mDCs as the expression possess no complement inhibiting property in mice [33]. Treatment with antagonists started 2 days before infection and was continued daily. CFSE instillation was done 16 hours before sacrificing the mice. At day 2 post infection, the number of CD103+ DCs and CD11b+ DCs were quantified within the CFSE+ mDCs in the dLN. CD103+ DC numbers were marginally reduced when either C3aR or C5aR was blocked but were significantly reduced when both were blocked together (Fig. 8C). A significant reduction in CD11b+ DC migration was observed when C3aR was blocked alone or together with C5aR (Fig. 8D). These results indicate that direct signaling through both complement receptors C3aR and C5aR is critical for the migration of mDCs from the lung to the dLNs. To confirm that the C3aR and C5aR receptors on mDCs directly mediate their migration rather than by the receptors on other cells in the lungs, we purified total CD11c+ DCs from the lungs of WT and C3aR2/2C5aR2/2 mice that had been flu infected 18 hours before, CFSE labeled and transferred them intratracheally to flu infected WT recipient mice. We then determined the number of donor CD103+ DCs and CD11b+ DCs in the dLN 18 hours after adoptive transfer. We observed a significantly reduced CD103+ DCs in the mice that received CD11c+ DCs from C3aR2/2C5aR2/2 mice in comparison with mice that received CD11c+ DCs from WT mice. Although the CD11b+ DCs showed a similar trend, it is important to note that the CD11b+ DCs have delayed migration kinetics as compared to CD103+ DCs. These results suggest a direct role for these receptors on these mDCs in their migration to the dLN during influenza infection (Fig. 8E–G). Discussion Influenza infection is known to activate complement in the lung with an increase in complement and complement activation products, although the pathway through which it is activated remains unknown [22,23]. Our study has uncovered the role of lung resident DCs in this process and highlighted their importance as complement producing and sensing cells. Our observations showed that under steady state conditions, lung C3a levels are high and are likely contributed by cells other than CD103+ DCs. However, it is interesting to note that upon influenza infection, CD103+ DCs exclusively produced high levels of C3 and C5, yielding activation fragments C3a and C5a. These anaphylotoxins, in turn, interacted with their receptors on both CD103+ DCs and CD11b+ DCs to promote DC migration. Furthermore, our studies using C3aRA and C5aRA demonstrate that C3a and C5a exhibit overlapping but not fully redundant functions because blockade of both their receptors has a significantly more profound effect on mDC migration than the blockade of either alone. Also adoptive transfer experiments using both receptor (C3aR2/2C5aR2/2) deficient lung DCs indicated that signaling mediated by both C3a and C5a on mDCs is required for their effective migration. Our results also highlighted the requirement of C3 in the maintenance of CD103+ DCs under steady state. Complement is known to regulate DC induced inhalation tolerance through C3’s opsonisation property on innocuous antigen [34]. Hence in the absence of C3, innocuous antigens are less efficiently taken up by CD103+ DCs, decreasing their ability to migrate (our data shows decreased number mDC in the dLN under steadystate) and thus proliferating less under steady state. However, the requirement of C3 in the maintenance of CD103+ DCs was limited to steady state, since under strong inflammatory conditions, deficiency of C3 seems not to affect CD103+ DCs number in the lung. PLOS Pathogens \| www.plospathogens.org 11 January 2013 \| Volume 9 \| Issue 1 \| e1003115 Complement Mediates Lung DC Migration with the Law and Guidelines for Animal Experiments of the Biological Resource Center (BRC) of Agency for Science, Technology and Research (A*STAR), Singapore. These guidelines were established by the national advisory committee for laboratory animal research as per the Animals and Birds Act 2002. of maturation markers (Fig. 1E,F, Fig. 4E and Fig. 5A–C) and the priming ability of mDCs from the dLN were comparable between WT and C32/2 mice during influenza infection (Fig. 1G). Supporting this similarly, the expression of costimulatory molecules on cDCs was not affected after infection with Listeria monocytogenes in C32/2 mice [44], suggesting that complement mediated signaling is dispensable for DC maturation during infection. These results are in sharp contrast with previous observations using bone marrow-derived DCs, stimulated with soluble ovalbumin or LPS [19,20]. It is thus important to draw a distinction between these two DC subtypes as it is apparent that complement signaling may mediate different functions on these cells. We also observed that acute administration of LPS was unable to overcome the defective mDC migration in C32/2 mice suggesting that complement mediated signaling operate independently of the inflammatory signal in mediating lung mDC trafficking during influenza infection. CD8+ T cell responses are critical in the protection against influenza infection [45], and it is noteworthy that C3 deficiency affects T cell immunity, viral clearance and survival [16,22]. Early lethality in CD103+ DC depleted langerin DTR mice paralleling C32/2 mice though surprising, highlights the critical importance of C3 and its contribution by CD103+ DCs in the control of early viral replication. C32/2 mice showed increased early viral replication kinetics as compared to WT mice (data not shown) suggesting a role for C3 in mediating innate antiviral immunity. Thus, our observations reiterate the critical importance of C3 during influenza infection, and we surmise increased viral load leading to pneumonia as a possible cause of mortality in the C32/2 mice. This is further strengthened by the report in the recent 2009 H1N1 pandemic patients wherein individuals who had severe disease had lower C3 levels in the serum, while the C3 concentrations were higher in moderately ill subjects [46]. These observations clearly suggest that complement has a key role in determining the outcome of influenza infection in both mice and humans. Because of its crucial role in protection against influenza, defects in many of the complement proteins, although rare, may be associated with increased susceptibility to influenza infection. Strikingly, deficiency or a defect in factor H and factor I are known to increase the susceptibility to bacterial infections, due to lack of C3 regulation [47]. Similarly, deficiency of mannose-binding lectin (MBL), an activator of complement via the lectin pathway, is more prevalent as compared to other complement component deficiency and is known to be associated with increased susceptibility to upper respiratory tract infections in human and to influenza infection in mice [48,49]. Although complement deficiency is rare in humans, genetic polymorphisms in complement proteins such as factor I, factor B, C3 and factor H are known to affect their availability, activity, and susceptibility to chronic disease conditions [50,51,52,53,54,55,56]. Thus, it is possible that complement deficiency/polymorphisms which affect the level and functioning of C3 in an individual may be associated with higher susceptibility to influenza infection as a consequence of compromised DC migration, T-cell priming and decreased viral clearance resulting in severe disease outcome. By demonstrating a novel role for C3 in regulating tissue DC trafficking, our data may also provide a rationale for complement to be exploited as a target for controlling tissue immunity by modulating mDC emigration and as an adjuvant in novel vaccine strategies for enhancing mDC migration to the lymph node to initiate stronger T-cell responses. Mice C57BL/6 mice were obtained from the BRC, C32/2 C3aR2/2 and C5aR2/2 mice were purchased from The Jackson Laboratory. C3aR2/2 C5aR2/2 double receptor mice were generated by crossing C3aR2/2 mice and C5aR2/2 mice. Langerin DTR, OT-I Rag12/2 and OT-II Rag22/2 mice (Taconic) were obtained from Mutant Mouse Collection Core Service, Singapore Immunology Network (SIgN), Singapore. Homozygous OT-II Rag22/2 mice were crossed once with homozygous CD45.1 mice (Jax) and the offspring from the F1 generation, referred to hereafter as CD45.1+OT-II, were used for adoptive transfer studies. Experiments were generally performed with sex matched mice at 6–10 week of age. Animals were bred under specific pathogen-free conditions at the BRC. Influenza virus infection Influenza virus strain A/PR/8/34 (H1N1) was obtained from National Institute for Medical Research (NIMR, London, UK). Recombinant influenza A/PR/8/34 strains containing the chicken OVA epitope SIINFEKL (PR8 OT-I) and chicken OVA peptide ISQAVHAAHAEINEAGR (323–339; PR8 OT-II) were a gift of P. Thomas (St. Jude Children’s Research Hospital, Memphis, TN). Mice were infected intranasally with one of the influenza strains at 15 or 25 plaque forming units (PFU) in 25 ml for female or male mice respectively. The influenza dose was optimized to give ,20% weight loss in WT mice during the peak of infection and recovery without incidence of mortality. In some experiments, diphtheria toxin was administered intraperitoneally (at 7 ng/g of mouse weight) before and/or during the course of influenza infection for depleting langerin-expressing CD103+ DCs. In some experiments LPS was administered intratracheally shortly followed by influenza infection. Flow cytometry and cell sorting Anti-mouse Abs used for FACS analysis were CD3-APC from BD Pharmingen; CD11c- PerCPCy5.5, MHCII-PB, CD103-PE, CD11b-PE-Cy7, B220-APCCy7, CD4-PE, CD86- FITC, CD80FITC, CD40-APC and CD8-PE-Cy7 from eBioscience; Ly6G/ Ly6C-APC, CCR7-APC and IFNc-APC from Biolegend. For live cell gating, live/dead fixable dye (Molecular Probes, Invitrogen) was used. Lung DCs were identified as low auto fluorescent CD11chigh, MHC-II high, B220 negative and Ly6G negative cells. Detection was performed using secondary Ab, goat anti-rat-FITC or Chicken-anti-goat-Alexa Flour 647 from Jackson Immuno Research Laboratories, Molecular Probes and Invitrogen respectively. Intracellular staining of IFNc in CD4+ T cells was performed by re-stimulating lung lymphocytes with 1 mM of Influenza virus nucleoprotein MHC-II restricted peptide (311– 325, QVYSLIRPNENPAHK) overnight in the presence of brefeldin A (Sigma). Cells were stained for surface markers and fixed with fixation/permeabilization buffer (BD biosciences) before staining for intracellular IFNc. For enumerating virus specific CD8+ T cells, enriched lymphocytes from the lungs were stained with PE labelled H-2Db tetramer with the NP 366–374 epitope ASNENMETM (Immudex) for 15 min at room temperature followed by staining with the following antibodies: CD3e-APC and CD8 PE-Cy7. For C3aR or C5aR staining, cells were incubated with primary Ab for 30 min, followed by secondary Ab for Materials and Methods Ethics statement Experiments were performed under the approval of the Institutional Animal Care and Use Committee in compliance PLOS Pathogens \| www.plospathogens.org 12 January 2013 \| Volume 9 \| Issue 1 \| e1003115 Complement Mediates Lung DC Migration 30 min. All Ab staining was performed at 4uC. Fc blocking Ab (Anti-Mouse CD16/CD32) was used during all FACS staining. Flow cytometric analysis was performed using BD FACS Canto or BD LSR II and analyzed using FlowJo software (Tree Star, San Carlos, CA). Cell sorting of lung and dLN DCs was performed using MoFlo (Beckman Coulter) and BD FACS Aria. influenza infected mice, CFSE labeled and then administered into the lungs of influenza infected mice. The migration of recipient mDCs in the donor mice were analysed in the dLN 18 hours after adoptive transfer. Reverse transcription of mRNA, RT-PCR, and primers Total RNA was obtained from lung tissue/sorted DC population using the RNeasy kit (Qiagen), following which cDNA was then obtained using QuantiTect Reverse Transcription kit (Qiagen). Both kits were used as per manufacturer’s protocol. Real-time PCR was performed on an ABI7500 real time PCR system using SYBR Green (Applied Biosystems) Primers used for qRT-PCR are as follows: Influenza M-protein-forward: 59-GGA CTG CAG CGT TAG ACG CTT-39 and reverse: 59-CAT CCT GTT GTA TAT GAG GCC CAT-39; C3-forward: 59-AAG CAT CAA CAC ACC CAA CA-39 and reverse: 59- CTT GAG CTC CAT TCG TGA CA-39; C5-forward: 59-GCA TTT CTG ACA CCA GGC TTC-39 and reverse: 59- AGC GCA CAG TCA GCT TCC A-39; C3aR-forward: 59-TGA AAG CAG GGA GTG TTG AG-39 and reverse: 59-TGC TCA CTT GCT CAC ATG AA-39; C5aR-forward: 59- CCA TGG ACG ACT CCT AAG GT-39 and reverse: 59-CTC CTC TAC ACC GCC TGA CT-39 Measurement of C3a and C5a in BAL fluid by ELISA C3a and C5a levels in bronchioalveolar lavage fluid were quantified by a sandwich ELISA as described below. MaxiSorp immuno modules (NUNC) were coated with 100 ml rat anti-mouse C3a or C5a capture antibody (BD pharmingen) at 1 or 2 mg/ml in carbonate buffer pH 9.6 or phosphate buffer pH6.5 (as recommended by the manufacturer) respectively and incubated overnight at 4uC. The plates were then washed three times with wash buffer (PBS with 0.05% v/v Tween 20) and blocked with 200 ml blocking buffer (PBS with 10% FBS), following which the plates were washed 3 times before the addition of 100 ml standards or 1/ 50–250 diluted BAL fluid and incubated for 2 hours at room temperature (RT). The plates were washed again, and then 100 ml of biotinylated rat-anti mouse C3a or C5a detecting Ab (BD pharmingen) was added at 2 mg/ml concentration to each well (RT, 1 hour). After washing, 100 ml of horseradish peroxidase (HRP) conjugated streptavidin at 1 mg/ml (Jackson Immuno research laboratories) was added and incubated for 30 minutes at RT. Subsequently, the plates were washed and 100 ml TMB substrate solution (Pierce Biotechnology Inc) was added to each well, and incubated in the dark (RT, 30 min). The reaction was stopped with the addition of 50 ml stop solution (2N H2SO4) to each well. The absorbance was read at 450 nm; reference wavelength 570 nm (Tecan GENios/Magellan). C3a or C5a levels were expressed as ng/100 mg of BAL fluid protein. Total protein in the BAL fluid was quantified by Bradford method (BioRad) following manufacturer’s instructions. Statistical analysis Data were analyzed using Prism GraphPad software. Statistical significance was determined by one-way ANOVA or the unpaired Student t test. Supporting Information Figure S1 C32/2 mice show decreased effector T cell response and viral clearance upon infection with influenza. WT and C32/2 mice were infected with flu and on day 7 post infection the flu specific CD4+ and CD8+ T cell response were evaluated. (A) Graph shows the frequency of IFNc secreting CD4+ T cells in lungs by ex vivo overnight stimulation with MHC-II flu peptide on day 7 post infection. (B) Bar graph shows the absolute numbers of IFNc secreting CD4+ T cells in lungs on day 7 post infection (C) Graph shows the frequency of Flu specific CTL response in lung as measured by Flu peptide (ASNENMETM (NP 366–374)/H-2Db tetramer staining on day 7 post infection. (D) Bar graph shows the absolute numbers of flu specific CD8+ T cells in lungs by tetramer staining on day 7 post infection.(E) Graph shows the relative mRNA expression level of influenza M protein in lung tissues of WT and C32/2 mice at the indicated days after influenza infection. (PDF) Analysis of T cell proliferation in vivo and ex vivo CD4+ T cells (from OT-II mice) and CD8+ T cells (from OT-I mice) were enriched using MACS beads (Miltenyi Biotec) from spleen and lymph node single cell suspension. Enriched cells were labelled with 5 mM of CFSE violet (Molecular Probes, Invitrogen) following manufacturer’s instructions. Approximately 26106 CD8+ T cells or 56106 CD4+ T cells in 200 ml volume were injected retro-orbitally followed by intranasal flu infection. dLN were harvested on day 3 and T cell proliferation was determined by CFSE dilution. For ex vivo analysis mDC subsets were sorted from the dLN of OT-I PR8 infected mice and co-cultured in U bottomed plates at a 1:10 ratio with CFSE labelled OT-I CD8+ T cells for 3 days. T cell divisions were measured by flow cytometry. The division index was calculated using Flow Jo software. Figure S2 Characterization of dLN mDC subsets. Dot plots show the flow cytometric analysis of dLN mDC subsets and resident CD8a DCs in naı̈ve WT and C32/2 mice. Gate III (resident DCs) and IV (mDCs) were selected on the basis of CD11chi MHC IIhi expression. Gate IV were subsequently divided on the basis of CD103 (gate V) and CD11b (gate VI) expression. Gate III were subsequently gated for CD8a DCs (gateVII) on the basis CD8a and CD11b expression (PDF) DC migration studies Influenza infected mice were intranasally instilled with 25 ml 8 mM CFSE (Molecular Probes, Invitrogen) for labelling lung DC in vivo at indicated time points. Mice were sacrificed 16 hours later and the dLN were removed and analysed for DCs. In some experiments, mice were treated intraperitoneally with either C3aR antagonist N2-[(2,2-Diphenylethoxy) acetyl]-L-arginine (SB290157, Calbiochem) at 500 mg/mouse or C5aR antagonistcyclic hexapeptide AcF[OPdChaWR] at 1 mg of peptide/g of mouse weight or both together. Compstatin at a concentration of 1 mg of peptide/g of mouse weight was used as the control. Treatment was started 2 days before influenza infection and continued daily until the mice were sacrificed. In some experiments CD11c+ DCs were purified from the pooled lungs of PLOS Pathogens \| www.plospathogens.org Figure S3 Characterization of lung mDC subsets. Dot plots show the flow cytometric analysis of lung DC subsets in naı̈ve WT and C32/2 mice. Autofluorescent cells were excluded from the analysis (gate III) and subsequently plasmacytoid DCs (pDCs) and Gr-1+ cells were gated out on the basis of B220 and Ly6C expression (gate IV) respectively. The remaining DCs were defined as CD11c+ MHC-II+ (gate 13 January 2013 \| Volume 9 \| Issue 1 \| e1003115 Complement Mediates Lung DC Migration Figure S5 Diphtheria toxin (DT) does not show any toxicity during influenza infection. (A) Percentage of body weight loss after influenza infection. A weight loss of ,20% and recovery represents a sub-lethal infection. (B) Survival curve comparing influenza infected +/2 DT. (n = 6–7 in each group). (PDF) V) which were further divided on the basis of the expression of CD103 (gate VI) and CD11b (gate VII). (PDF) Figure S4 A. Flow cytometric analysis for the expres- sion of langerin-EGFP on lung DCs and other lineage cells in langerin-DTR mouse. Single cell preparations from the lungs of WT and langerin-DTR were gated for live cells (DAPI-ve) and CD45+ve cells and then analyzed for indicated lineage markers. Expression of langerin was analyzed through the expression of GFP (langerin-DTR mice expresses GFP under the control of langerin) on the indicated cell types. Histograms shows the expression of langerin-GFP in the depicted populations. Grey : WT, Open: langerin-DTR mice) B. Flow cytometry data to show specific depletion of CD103+ DCs in the lungs of langerin-DTR mice. WT and langerin-DTR mice were either treated with DT or not and the number of CD103+ and CD11b+ DCs in the lungs were evaluated by flow cytometry after 48 hours after DT administration.. Numbers indicate the number of cells within each gate. (PDF) Acknowledgments We thank the staffs at BRC, Biopolis for the excellent support in animal husbandry, Paul Edward Hutchinson and Komathi Paramasivam for their excellent support with FACS sorting and Dr. Christiane Ruedl for discussion and critical review. Author Contributions Conceived and designed the experiments: BS MK FG. Performed the experiments: MK PCY AWSH HRS AS. Analyzed the data: MK BS. Contributed reagents/materials/analysis tools: DMK TRH BPM FG. Wrote the paper: BS AWSH FG BPM. References 20. Li K, Fazekasova H, Wang N, Peng Q, Sacks SH, et al. (2012) Functional modulation of human monocytes derived DCs by anaphylatoxins C3a and C5a. Immunobiology 217: 65–73. 21. Allenspach EJ, Lemos MP, Porrett PM, Turka LA, Laufer TM (2008) Migratory and lymphoid-resident dendritic cells cooperate to efficiently prime naive CD4 T cells. Immunity 29: 795–806. 22. O’Brien KB, Morrison TE, Dundore DY, Heise MT, Schultz-Cherry S (2011) A protective role for complement C3 protein during pandemic 2009 H1N1 and H5N1 influenza A virus infection. PLoS One 6: e17377. 23. Ohta R, Torii Y, Imai M, Kimura H, Okada N, et al. (2011) Serum concentrations of complement anaphylatoxins and proinflammatory mediators in patients with 2009 H1N1 influenza. Microbiol Immunol 55: 191–198. 24. McKinstry KK, Strutt TM, Buck A, Curtis JD, Dibble JP, et al. (2009) IL-10 deficiency unleashes an influenza-specific Th17 response and enhances survival against high-dose challenge. J Immunol 182: 7353–7363. 25. Ho AW, Prabhu N, Betts RJ, Ge MQ, Dai X, et al. (2011) Lung CD103+ dendritic cells efficiently transport influenza virus to the lymph node and load viral antigen onto MHC class I for presentation to CD8 T cells. J Immunol 187: 6011–6021. 26. Moltedo B, Li W, Yount JS, Moran TM (2011) Unique type I interferon responses determine the functional fate of migratory lung dendritic cells during influenza virus infection. PLoS Pathog 7: e1002345. 27. Ballesteros-Tato A, Leon B, Lund FE, Randall TD (2010) Temporal changes in dendritic cell subsets, cross-priming and costimulation via CD70 control CD8(+) T cell responses to influenza. Nat Immunol 11: 216–224. 28. Banki Z, Posch W, Ejaz A, Oberhauser V, Willey S, et al. (2010) Complement as an endogenous adjuvant for dendritic cell-mediated induction of retrovirusspecific CTLs. PLoS Pathog 6: e1000891. 29. Grayson MH, Ramos MS, Rohlfing MM, Kitchens R, Wang HD, et al. (2007) Controls for lung dendritic cell maturation and migration during respiratory viral infection. J Immunol 179: 1438–1448. 30. Heer AK, Harris NL, Kopf M, Marsland BJ (2008) CD4+ and CD8+ T cells exhibit differential requirements for CCR7-mediated antigen transport during influenza infection. J Immunol 181: 6984–6994. 31. Shortman K, Naik SH (2007) Steady-state and inflammatory dendritic-cell development. Nat Rev Immunol 7: 19–30. 32. Zhang X, Kimura Y, Fang C, Zhou L, Sfyroera G, et al. (2007) Regulation of Toll-like receptor-mediated inflammatory response by complement in vivo. Blood 110: 228–236. 33. Kim AH, Dimitriou ID, Holland MC, Mastellos D, Mueller YM, et al. (2004) Complement C5a receptor is essential for the optimal generation of antiviral CD8+ T cell responses. J Immunol 173: 2524–2529. 34. Kohl J, Wills-Karp M (2007) Complement regulates inhalation tolerance at the dendritic cell/T cell interface. Mol Immunol 44: 44–56. 35. Bouchon A, Hernandez-Munain C, Cella M, Colonna M (2001) A DAP12mediated pathway regulates expression of CC chemokine receptor 7 and maturation of human dendritic cells. J Exp Med 194: 1111–1122. 36. Randolph GJ, Ochando J, Partida-Sanchez S (2008) Migration of dendritic cell subsets and their precursors. Annu Rev Immunol 26: 293–316. 37. Sanchez-Sanchez N, Riol-Blanco L, Rodriguez-Fernandez JL (2006) The multiple personalities of the chemokine receptor CCR7 in dendritic cells. J Immunol 176: 5153–5159. 1. Brown LE, Kelso A (2009) Prospects for an influenza vaccine that induces crossprotective cytotoxic T lymphocytes. Immunol Cell Biol 87: 300–308. 2. Lee LY, Ha do LA, Simmons C, de Jong MD, Chau NV, et al. (2008) Memory T cells established by seasonal human influenza A infection cross-react with avian influenza A (H5N1) in healthy individuals. J Clin Invest 118: 3478–3490. 3. Teijaro JR, Verhoeven D, Page CA, Turner D, Farber DL (2010) Memory CD4 T cells direct protective responses to influenza virus in the lungs through helperindependent mechanisms. J Virol 84: 9217–9226. 4. Legge KL, Braciale TJ (2003) Accelerated migration of respiratory dendritic cells to the regional lymph nodes is limited to the early phase of pulmonary infection. Immunity 18: 265–277. 5. Belz GT, Smith CM, Kleinert L, Reading P, Brooks A, et al. (2004) Distinct migrating and nonmigrating dendritic cell populations are involved in MHC class I-restricted antigen presentation after lung infection with virus. Proc Natl Acad Sci U S A 101: 8670–8675. 6. Steinman RM (2007) Dendritic cells: understanding immunogenicity. Eur J Immunol 37 Suppl 1: S53–60. 7. Ginhoux F, Liu K, Helft J, Bogunovic M, Greter M, et al. (2009) The origin and development of nonlymphoid tissue CD103+ DCs. J Exp Med 206: 3115–3130. 8. del Rio ML, Rodriguez-Barbosa JI, Kremmer E, Forster R (2007) CD103- and CD103+ bronchial lymph node dendritic cells are specialized in presenting and crosspresenting innocuous antigen to CD4+ and CD8+ T cells. J Immunol 178: 6861–6866. 9. Sung SS, Fu SM, Rose CE, Jr., Gaskin F, Ju ST, et al. (2006) A major lung CD103 (alphaE)-beta7 integrin-positive epithelial dendritic cell population expressing Langerin and tight junction proteins. J Immunol 176: 2161–2172. 10. GeurtsvanKessel CH, Willart MA, van Rijt LS, Muskens F, Kool M, et al. (2008) Clearance of influenza virus from the lung depends on migratory langerin+CD11b- but not plasmacytoid dendritic cells. J Exp Med 205: 1621–1634. 11. Pepys MB (1974) Role of complement in induction of antibody production in vivo. Effect of cobra factor and other C3-reactive agents on thymus-dependent and thymus-independent antibody responses. J Exp Med 140: 126–145. 12. Carroll MC (2004) The complement system in regulation of adaptive immunity. Nat Immunol 5: 981–986. 13. Kimberley FC, Sivasankar B, Paul Morgan B (2007) Alternative roles for CD59. Mol Immunol 44: 73–81. 14. Ricklin D, Hajishengallis G, Yang K, Lambris JD (2010) Complement: a key system for immune surveillance and homeostasis. Nat Immunol 11: 785–797. 15. Suresh M, Molina H, Salvato MS, Mastellos D, Lambris JD, et al. (2003) Complement component 3 is required for optimal expansion of CD8 T cells during a systemic viral infection. J Immunol 170: 788–794. 16. Kopf M, Abel B, Gallimore A, Carroll M, Bachmann MF (2002) Complement component C3 promotes T-cell priming and lung migration to control acute influenza virus infection. Nat Med 8: 373–378. 17. Mehlhop E, Diamond MS (2006) Protective immune responses against West Nile virus are primed by distinct complement activation pathways. J Exp Med 203: 1371–1381. 18. Vieyra M, Leisman S, Raedler H, Kwan WH, Yang M, et al. (2011) Complement regulates CD4 T-cell help to CD8 T cells required for murine allograft rejection. Am J Pathol 179: 766–774. 19. Strainic MG, Liu J, Huang D, An F, Lalli PN, et al. (2008) Locally produced complement fragments C5a and C3a provide both costimulatory and survival signals to naive CD4+ T cells. Immunity 28: 425–435. PLOS Pathogens \| www.plospathogens.org 14 January 2013 \| Volume 9 \| Issue 1 \| e1003115 Complement Mediates Lung DC Migration 48. Garred P, Larsen F, Madsen HO, Koch C (2003) Mannose-binding lectin deficiency–revisited. Mol Immunol 40: 73–84. 49. Eisen DP, Minchinton RM (2003) Impact of mannose-binding lectin on susceptibility to infectious diseases. Clin Infect Dis 37: 1496–1505. 50. Hageman GS, Anderson DH, Johnson LV, Hancox LS, Taiber AJ, et al. (2005) A common haplotype in the complement regulatory gene factor H (HF1/CFH) predisposes individuals to age-related macular degeneration. Proc Natl Acad Sci U S A 102: 7227–7232. 51. Pickering MC, de Jorge EG, Martinez-Barricarte R, Recalde S, Garcia-Layana A, et al. (2007) Spontaneous hemolytic uremic syndrome triggered by complement factor H lacking surface recognition domains. J Exp Med 204: 1249–1256. 52. Gold B, Merriam JE, Zernant J, Hancox LS, Taiber AJ, et al. (2006) Variation in factor B (BF) and complement component 2 (C2) genes is associated with agerelated macular degeneration. Nat Genet 38: 458–462. 53. Yates JR, Sepp T, Matharu BK, Khan JC, Thurlby DA, et al. (2007) Complement C3 variant and the risk of age-related macular degeneration. N Engl J Med 357: 553–561. 54. Prosser BE, Johnson S, Roversi P, Herbert AP, Blaum BS, et al. (2007) Structural basis for complement factor H linked age-related macular degeneration. J Exp Med 204: 2277–2283. 55. Heurich M, Martinez-Barricarte R, Francis NJ, Roberts DL, Rodriguez de Cordoba S, et al. (2011) Common polymorphisms in C3, factor B, and factor H collaborate to determine systemic complement activity and disease risk. Proc Natl Acad Sci U S A 108: 8761–8766. 56. Fagerness JA, Maller JB, Neale BM, Reynolds RC, Daly MJ, et al. (2009) Variation near complement factor I is associated with risk of advanced AMD. Eur J Hum Genet 17: 100–104. 38. Eisenbarth SC, Williams A, Colegio OR, Meng H, Strowig T, et al. (2012) NLRP10 is a NOD-like receptor essential to initiate adaptive immunity by dendritic cells. Nature 484: 510–513. 39. Peng Q, Li K, Wang N, Li Q, Asgari E, et al. (2009) Dendritic cell function in allostimulation is modulated by C5aR signaling. J Immunol 183: 6058–6068. 40. Zhou W, Patel H, Li K, Peng Q, Villiers MB, et al. (2006) Macrophages from C3-deficient mice have impaired potency to stimulate alloreactive T cells. Blood 107: 2461–2469. 41. Geissmann F, Manz MG, Jung S, Sieweke MH, Merad M, et al. (2010) Development of monocytes, macrophages, and dendritic cells. Science 327: 656– 661. 42. Naik SH, Metcalf D, van Nieuwenhuijze A, Wicks I, Wu L, et al. (2006) Intrasplenic steady-state dendritic cell precursors that are distinct from monocytes. Nat Immunol 7: 663–671. 43. Kim TS, Braciale TJ (2009) Respiratory dendritic cell subsets differ in their capacity to support the induction of virus-specific cytotoxic CD8+ T cell responses. PLoS One 4: e4204. 44. Nakayama Y, Kim SI, Kim EH, Lambris JD, Sandor M, et al. (2009) C3 promotes expansion of CD8+ and CD4+ T cells in a Listeria monocytogenes infection. J Immunol 183: 2921–2931. 45. Topham DJ, Tripp RA, Doherty PC (1997) CD8+ T cells clear influenza virus by perforin or Fas-dependent processes. J Immunol 159: 5197–5200. 46. Monsalvo AC, Batalle JP, Lopez MF, Krause JC, Klemenc J, et al. (2011) Severe pandemic 2009 H1N1 influenza disease due to pathogenic immune complexes. Nat Med 17: 195–199. 47. Reis ES, Falcao DA, Isaac L (2006) Clinical aspects and molecular basis of primary deficiencies of complement component C3 and its regulatory proteins factor I and factor H. Scand J Immunol 63: 155–168. PLOS Pathogens \| www.plospathogens.org 15 January 2013 \| Volume 9 \| Issue 1 \| e1003115
https://openalex.org/W2193454486	https://europepmc.org/articles/pmc3007966?pdf=render	English	null	Redetermination of Fe<sub>2</sub>[BP<sub>3</sub>O<sub>12</sub>]	Acta crystallographica. Section E	2,010	cc-by	2,916	Table 1 Explorations of phases in the quaternary FeIII–BIII–PV–O system prepared by the high temperature solution growth (HTSG) method led to single-crystal growth of anhydrous diiron(III) borotriphosphate, Fe2[BP3O12]. This phase has been synthesized previously as a microcrystalline material and its structure reﬁned in space group P3 from powder X-ray diffraction data using the Rietveld method [Chen et al. (2004). J. Inorg. Mater. 19, 429-432]. In the current single-crystal study, it was shown that the correct space group is P63/m. The three-dimensional structure of the title compound is built up from FeO6 octahedra (3.. symmetry), trigonal–planar BO3 groups (6 symmetry) and PO4 tetrahedra (m.. symmetry). Two FeO6 octahedra form Fe2O9 dimers via face-sharing, while the anionic BO3 and PO4 groups are connected via corner-sharing to build up the [BP3O12]6 anion. Both units are inter- connected via corner-sharing. Data collection: CrystalClear (Rigaku, 2004); cell reﬁnement: CrystalClear; data reduction: CrystalClear; program(s) used to solve structure: SHELXS97 (Sheldrick, 2008); program(s) used to reﬁne structure: SHELXL97 (Sheldrick, 2008); molecular graphics: DIAMOND (Brandenburg, 2004); software used to prepare material for publication: SHELXTL (Sheldrick, 2008) and PLATON (Spek, 2009). The authors acknowledge the Doctoral Foundation of Henan Polytechnic University (B648174). Supplementary data and ﬁgures for this paper are available from the IUCr electronic archives (Reference: WM2377). Supplementary data and ﬁgures for this paper are available from the IUCr electronic archives (Reference: WM2377). Acta Cryst. (2010). E66, i63 inorganic compounds Acta Crystallographica Section E Structure Reports Online ISSN 1600-5368 Z = 2 Mo K radiation = 4.15 mm1 T = 293 K 0.15 0.05 0.05 mm Data collection Rigaku Mercury70 CCD diffractometer Absorption correction: multi-scan (ABSCOR; Higashi, 1995) Tmin = 0.575, Tmax = 0.819 3247 measured reﬂections 345 independent reﬂections 338 reﬂections with I > 2(I) Rint = 0.042 Reﬁnement R[F 2 > 2(F 2)] = 0.035 wR(F 2) = 0.072 S = 1.07 345 reﬂections 33 parameters max = 0.58 e A˚ 3 min = 0.78 e A˚ 3 Acta Crystallographica Section E Structure Reports Online ISSN 1600-5368 T = 293 K 0.15 0.05 0.05 mm T = 293 K 0.15 0.05 0.05 mm a = 8.0347 (8) A˚ c = 7.4163 (13) A˚ V = 414.63 (9) A˚ 3 Fei Fei Li,* Hui Ju Zhang and Li Na Zhang Department of Physics and Chemistry, Henan Polytechnic University, Jiaozuo, Henan 454000, People’s Republic of China Correspondence e-mail: lifeifei@hpu.edu.cn Received 8 July 2010; accepted 27 July 2010 Key indicators: single-crystal X-ray study; T = 293 K; mean (P–O) = 0.004 A˚; R factor = 0.035; wR factor = 0.072; data-to-parameter ratio = 10.5. Table 1 Selected bond lengths (A˚ ). Fe1—O3i 1.929 (2) Fe1—O2 2.103 (2) P1—O3 1.507 (3) P1—O2 1.538 (3) P1—O1 1.586 (3) O1—B1 1.357 (3) Symmetry code: (i) x þ 2; y þ 1; z þ 1. References Brandenburg, K. (2004). DIAMOND. Crystal Impact GbR, Bonn, Germany. Chen, H. H., Ge, M. H., Yang, X. X., Mi, J. X. & Zhao, J. T. (2004). J. Inorg. Mater. 19, 429–432. Ewald, B., Huang, Y. X. & Kniep, R. (2007). Z. Anorg. Allg. Chem. 633, 1517– 1540. Higashi, T. (1995). ABSCOR. Rigaku Corporation, Tokyo, Japan. Kniep, R., Engelhardt, H. & Hauf, C. (1998). Chem. Mater. 10, 2930–2934. Meisel, M., Pa¨ch, M., Wilde, L. & Wulff-Molder, D. (2004). Z. Anorg. Allg. Chem. 630, 983–985. Mi, J. X., Zhao, J. T., Mao, S. Y., Huang, Y. X., Engelhardt, H. & Kniep, R. (2000). Z. Kristallogr. New Cryst. Struct. 215, 201–202. Rigaku (2004). CrystalClear. Rigaku Corporation, Tokyo, Japan. Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122. Spek, A. L. (2009). Acta Cryst. D65, 148–155. Zhao, D., Cheng, W. D., Zhang, H., Huang, S. P., Xie, Z., Zhang, W. L. & Yang, S. L. (2009). Inorg. Chem. 48, 6623–6629. Brandenburg, K. (2004). DIAMOND. Crystal Impact GbR, Bonn, Germany. Chen, H. H., Ge, M. H., Yang, X. X., Mi, J. X. & Zhao, J. T. (2004). J. Inorg. Mater. 19, 429–432. Reviews on the crystal chemistry of borophosphates were given by Kniep et al. (1998) and Ewald et al. (2007). For the previous powder study of Fe2[BP3O12], see: Chen et al. (2004). For the structure of a related borophosphate, see: Zhao et al. (2009). Meisel et al. (2004) have reported the structure of V2[BP3O12] and Mi et al. (2000) that of Cr2[BP3O12]. Ewald, B., Huang, Y. X. & Kniep, R. (2007). Z. Anorg. Allg. Chem. 633, 1517– 1540. igashi, T. (1995). ABSCOR. Rigaku Corporation, Tokyo, Japa Higashi, T. (1995). ABSCOR. Rigaku Corporation, Tokyo, Japan. Kniep, R., Engelhardt, H. & Hauf, C. (1998). Chem. Mater. 10, 2930–2934. ( ) ( ) Kniep, R., Engelhardt, H. & Hauf, C. (1998). Chem. Mater. 10, 2930–2934. ( ) Meisel, M., Pa¨ch, M., Wilde, L. & Wulff-Molder, D. (2004). Z. Anorg. Allg. Chem. 630, 983–985. Mi, J. X., Zhao, J. T., Mao, S. Y., Huang, Y. X., Engelhardt, H. & Kniep, R. Experimental Crystal data Fe2[BP3O12] Mr = 407.42 Hexagonal, P63=m a = 8.0347 (8) A˚ c = 7.4163 (13) A˚ V = 414.63 (9) A˚ 3 (2000). Z. Kristallogr. New Cryst. Struct. 215, 201–202. Experimental Experimental Crystal data Fe2[BP3O12] Mr = 407.42 Hexagonal, P63=m Rigaku (2004). CrystalClear. Rigaku Corporation, Tokyo, Experimental Crystal data Fe2[BP3O12] Mr = 407.42 Hexagonal, P63=m Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122. Spek, A. L. (2009). Acta Cryst. D65, 148–155. a = 8.0347 (8) A˚ c = 7.4163 (13) A˚ V = 414.63 (9) A˚ 3 a = 8.0347 (8) A˚ c = 7.4163 (13) A˚ V = 414.63 (9) A˚ 3 Zhao, D., Cheng, W. D., Zhang, H., Huang, S. P., Xie, Z., Zhang, W. L. & Yang, S. L. (2009). Inorg. Chem. 48, 6623–6629. Li et al. i63 doi:10.1107/S1600536810029818 Comment The systematic development of borophosphates has led to a broad spectrum of new borophosphate compounds with quite different anionic partial structures, such as oligomeric units, chains, ribbons, layers, and three-dimensional frameworks. (Kniep et al., 1998; Ewald et al., 2007; Zhao et al., 2009). Most of the borophosphate compounds were synthesized under hydrothermal conditions; hence, their structures usually incorporate water molecules, hydroxy groups or organic templates. There are considerably less anhydrous borophosphate compounds known, which might have better chemical and thermal stability than the hydrous or templated phases to ensure the feasibility of industrial applications. Herein, we report the redetermined structure of the anhydrous diiron(III) borotri- phosphate, Fe2[BP3O12]. The basic building units of the three-dimensional structure of the title compound are FeO6 octahedra (3.. symmetry), trigonal-planar BO3 groups (6 symmetry) and PO4 tetrahedra (m.. symmetry) (Fig. 1). Two neighboring FeO6 octahedra are connected via their faces to form Fe2O9 dimers. Trigonal-planar BO3 units and PO4 tetrahedra are isolated. Each BO3 triangle connects three PO4 tetrahedra via corner-sharing O atoms and each PO4 connects three Fe2O9 groups and one BO3 group also via corner sharing. As shown in Fig. 2, the aforementioned groups are interconnected to form the three-dimen- sional framework of the title compound. Chen et al. (2004) have previously refined the structure of Fe2[BP3O12] in space group P3 using the Rietveld method. The analogous chromium compound Cr2[BP3O12] (Mi et al., 2000) is isotypic to this structure model. The differences between the previous and the current model are discussed in the Refinement Section. The three-dimensional frameworks of Cr2[BP3O12] (space group P3) and our model of Fe2[BP3O12] (space group P63/m) are very similar, and the differences mainly lie in the distortion of the MO6 octahedra (M = Cr, Fe). The asymmetric unit of Cr2[BP3O12] consists of four Cr atoms, and the Cr–O bond distances range from 1.88 (2) to 2.07 (2) Å, while there is only one Fe site in the asymmetric unit of Fe2[BP3O12] with Fe–O bond distances ranging from 1.929 (2) to 2.103 (2) Å. Based on the current findings, a space group change from P3 to P63/m seems to be most likely for the Cr compound but has to be evidenced experimentally. Meisel et al. (2004) have reported the analogous vanadium(III) compound V2[BP3O12] in space group P63/m, but with a tripled unit cell (a of the V compound ≈ 31/2 × a of the Fe and Cr compounds). Comment However, a comparison of the three structures shows very similar frameworks. F. F. Li, H. J. Zhang and L. N. Zhang F. F. Li, H. J. Zhang and L. N. Zhang supplementary materials Acta Cryst. (2010). E66, i63 [ doi:10.1107/S1600536810029818 ] supplementary materials supplementary materials Redetermination of Fe2[BP3O12] F. F. Li, H. J. Zhang and L. N. Zhang supplementary materials and then quenched to room temperature. The obtained crystals were light-red and of prismatic shape. The dimensions of the used sample were typical for the grown crystals in this batch. and then quenched to room temperature. The obtained crystals were light-red and of prismatic shape. The dimensions of the used sample were typical for the grown crystals in this batch. Experimental Single crystals of Fe2[BP3O12] have been prepared by the high temperature solution growth (HTSG) method in air. A powder mixture of Fe2O3, B2O3 and NaPO3 at the molar ratio of Fe: B: Na: P = 1:5:10:10 was first ground in an agate mortar and then transferred to a platinum crucible. The sample was gradually heated in air at 1173 K for 24 h. In this stage, the reagents were completely melted. After that, the intermediate product was slowly cooled to 673 K at the rate of 2 K h-1 sup-1 supplementary materials Refinement Chen et al. (2004) have refined the structure of Fe2[BP3O12] using the Rietved method from powder X-ray data and de- termined the space group to be P3, in analogy with the chromium compound Cr2[BP3012] (Mi et al., 2000). However, in our study we determined the structure from single-crystal X-ray diffraction data in the centrosymmetric space group P63/m. In the progress of the space group determination using XPREP (Sheldrick, 2008), the mean \|EE-1\| statistics gave a value of 0.948 revealing that the structure is centrosymmetric; the CFOM (combined figure-of-merit) value for each space group determination were P3 (16.06), P3 (7.16), P63 (7.56), P63/m (1.75). So we selected the latter space group to solve the structure. The final refinement converged with satisfactory results (R1(gt) = 0.0348). Furthermore, the final refined model was checked with the ADDSYM algorithm using the program PLATON (Spek, 2009), and no higher symmetry was found. Hence, our final structure model is considered to be reasonable and corrects the previous model by Chen et al. (2004). The highest peak in the difference electron density map is located at a distance of 1.41 Å from the Fe1 site while the deepest hole is at a distance of 0.83 Å from the same site. Figures Fig. 1. Section of the structure of Fe2[BP3012] with the atom labelling scheme. The displace- ment ellipsoids are drawn at the 50% probability level. [Symmetry codes: (i) 2 - x, 1 - y, 1 - z; (ii) x-y, -1 + x, 1 - z; (iii) y, 1 - x + y, 1 - z; (iv) 1 - x + y, 1 - x, 0.5 - z; (v) 1 - y, x-y, z; (vi) x, y, 0.5 - z; (vii) 2 - y, 1 + x-y, z; (viii) 1 - x + y, 2 - x, 0.5 - z.] Fig. 2. View of the crystal structure of Fe2[BP3012] in a projection along [001]. diiron(III) borotriphosphate Crystal data Fe2[BP3O12] Dx = 3.263 Mg m−3 Mr = 407.42 Mo Kα radiation, λ = 0.71073 Å Hexagonal, P63/m Cell parameters from 1049 reflections Hall symbol: -P 6c θ = 4.0–27.4° a = 8.0347 (8) Å µ = 4.15 mm−1 c = 7.4163 (13) Å T = 293 K V = 414.63 (9) Å3 Prism, light-red Z = 2 0.15 × 0.05 × 0.05 mm F(000) = 396 Figures Fig. 1. Refinement diiron(III) borotriphosphate Dx = 3.263 Mg m−3 Mo Kα radiation, λ = 0.71073 Å Cell parameters from 1049 reflections θ = 4.0–27.4° µ = 4.15 mm−1 T = 293 K Prism, light-red 0.15 × 0.05 × 0.05 mm sup-2 supplementary materials Data collection Rigaku Mercury70 CCD diffractometer 345 independent reflections Radiation source: fine-focus sealed tube 338 reflections with I > 2σ(I) Graphite Monochromator Rint = 0.042 Detector resolution: 14.6306 pixels mm-1 θmax = 27.4°, θmin = 2.9° ω scans h = −10→10 Absorption correction: multi-scan (ABSCOR; Higashi, 1995) k = −10→10 Tmin = 0.575, Tmax = 0.819 l = −9→6 3247 measured reflections Data collection Rigaku Mercury70 CCD diffractometer 345 independent reflections Radiation source: fine-focus sealed tube 338 reflections with I > 2σ(I) Graphite Monochromator Rint = 0.042 Detector resolution: 14.6306 pixels mm-1 θmax = 27.4°, θmin = 2.9° ω scans h = −10→10 Absorption correction: multi-scan (ABSCOR; Higashi, 1995) k = −10→10 Tmin = 0.575, Tmax = 0.819 l = −9→6 3247 measured reflections 345 independent reflections 338 reflections with I > 2σ(I) Rint = 0.042 θmax = 27.4°, θmin = 2.9° h = −10→10 k = −10→10 l = −9→6 345 independent reflections Refinement Refinement on F2 Least-squares matrix: full R[F2 > 2σ(F2)] = 0.035 wR(F2) = 0.072 S = 1.07 345 reflections 33 parameters 0 restraints 0 restraints Primary atom site location: structure-invariant direct methods Secondary atom site location: difference Fourier map Secondary atom site location: difference Fourier map w = 1/[σ2(Fo 2) + (0.0168P)2 + 3.P] where P = (Fo 2 + 2Fc 2)/3 (Δ/σ)max < 0.001 Δρmax = 0.58 e Å−3 Δρmin = −0.78 e Å−3 w = 1/[σ2(Fo 2) + (0.0168P)2 + 3.P] w = 1/[σ2(Fo 2) + (0.0168P)2 + 3.P] where P = (Fo 2 + 2Fc 2)/3 (Δ/σ)max < 0.001 Δρmax = 0.58 e Å−3 Δρmin = −0.78 e Å−3 33 parameters Refinement Section of the structure of Fe2[BP3012] with the atom labelling scheme. The displace- ment ellipsoids are drawn at the 50% probability level. [Symmetry codes: (i) 2 - x, 1 - y, 1 - z; (ii) x-y, -1 + x, 1 - z; (iii) y, 1 - x + y, 1 - z; (iv) 1 - x + y, 1 - x, 0.5 - z; (v) 1 - y, x-y, z; (vi) x, y, 0.5 - z; (vii) 2 - y, 1 + x-y, z; (viii) 1 - x + y, 2 - x, 0.5 - z.] Fig. 2. View of the crystal structure of Fe2[BP3012] in a projection along [001]. 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 mat- rix. 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, convention- al 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) Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) x y z Uiso/Ueq Fe1 0.6667 0.3333 0.45190 (12) 0.0072 (3) P1 1.04513 (17) 0.68473 (17) 0.2500 0.0064 (3) O2 0.8735 (5) 0.4782 (5) 0.2500 0.0080 (7) O1 0.9428 (5) 0.8099 (5) 0.2500 0.0097 (8) B1 1.0000 1.0000 0.2500 0.0101 (19) O3 1.1626 (3) 0.7289 (3) 0.4200 (4) 0.0115 (6) sup-3 supplementary materials Symmetry codes: (i) −x+2, −y+1, −z+1; (ii) x−y, x−1, −z+1; (iii) y, −x+y+1, −z+1; (iv) −x+y+1, −x+1, −z+1/2; (v) −y+1, x−y, z; (vi) x, y, −z+1/2; (vii) −y+2, x−y+1, z; (viii) −x+y+1, −x+2, −z+1/2. supplementary materials Atomic displacement parameters (Å2) U11 U22 U33 U12 U13 U23 Fe1 0.0076 (3) 0.0076 (3) 0.0063 (5) 0.00381 (15) 0.000 0.000 P1 0.0055 (6) 0.0057 (6) 0.0077 (7) 0.0025 (5) 0.000 0.000 O2 0.0067 (15) 0.0059 (16) 0.0099 (19) 0.0021 (13) 0.000 0.000 O1 0.0060 (16) 0.0056 (16) 0.017 (2) 0.0022 (13) 0.000 0.000 B1 0.008 (2) 0.008 (2) 0.014 (5) 0.0040 (12) 0.000 0.000 O3 0.0116 (12) 0.0115 (12) 0.0127 (15) 0.0066 (10) −0.0045 (11) −0.0019 (10) Geometric parameters (Å, °) Fe1—O3i 1.929 (2) P1—O2 1.538 (3) Fe1—O3ii 1.929 (2) P1—O1 1.586 (3) Fe1—O3iii 1.929 (2) O2—Fe1iv 2.103 (2) Fe1—O2iv 2.103 (2) O1—B1 1.357 (3) Fe1—O2 2.103 (2) B1—O1vii 1.357 (3) Fe1—O2v 2.103 (2) B1—O1viii 1.357 (3) P1—O3 1.507 (3) O3—Fe1i 1.929 (2) P1—O3vi 1.507 (3) O3i—Fe1—O3ii 97.83 (11) O3—P1—O3vi 113.6 (2) O3i—Fe1—O3iii 97.83 (11) O3—P1—O2 111.85 (12) O3ii—Fe1—O3iii 97.83 (11) O3vi—P1—O2 111.85 (12) O3i—Fe1—O2iv 93.65 (11) O3—P1—O1 108.24 (12) O3ii—Fe1—O2iv 91.53 (11) O3vi—P1—O1 108.24 (12) O3iii—Fe1—O2iv 164.04 (11) O2—P1—O1 102.37 (18) O3i—Fe1—O2 91.53 (11) P1—O2—Fe1 129.13 (10) O3ii—Fe1—O2 164.04 (11) P1—O2—Fe1iv 129.14 (10) O3iii—Fe1—O2 93.65 (11) Fe1—O2—Fe1iv 90.78 (13) O2iv—Fe1—O2 74.92 (10) B1—O1—P1 136.3 (3) O3i—Fe1—O2v 164.04 (11) O1vii—B1—O1viii 120.000 (1) O3ii—Fe1—O2v 93.65 (11) O1vii—B1—O1 120.000 (1) O3iii—Fe1—O2v 91.53 (11) O1viii—B1—O1 120.000 (1) O2iv—Fe1—O2v 74.92 (10) P1—O3—Fe1i 142.54 (17) O2—Fe1—O2v 74.92 (10) Symmetry codes: (i) −x+2, −y+1, −z+1; (ii) x−y, x−1, −z+1; (iii) y, −x+y+1, −z+1; (iv) −x+y+1, −x+1, −z+1/2; (v) −y+1, x−y, z; (vi) x, y, −z+1/2; (vii) −y+2, x−y+1, z; (viii) −x+y+1, −x+2, −z+1/2. Atomic displacement parameters (Å2) sup-4 supplementary materials Fig. 1 Fig. 1 Fig. 1 sup-5 supplementary materials Fig. 2 Fig. 2 sup-6
https://openalex.org/W3195380190	https://pure.rug.nl/ws/files/191479514/Oral_contraceptives_depressive_and_insomnia_symptoms_in_adult_women_with_and_without_depression.pdf	English	null	Oral contraceptives, depressive and insomnia symptoms in adult women with and without depression	Psychoneuroendocrinology	2,021	cc-by	9,965	University of Groningen Oral contraceptives, depressive and insomnia symptoms in adult women with and without depression Morssinkhof, Margot W L; Lamers, Femke; Hoogendoorn, Adriaan W; de Wit, Anouk E; Riese, Harriëtte; Giltay, Erik J; van den Heuvel, Odile A; Penninx, Brenda W; Broekman, Birit F P Published in: University of Groningen Oral contraceptives, depressive and insomnia symptoms in adult women with and without depression Morssinkhof, Margot W L; Lamers, Femke; Hoogendoorn, Adriaan W; de Wit, Anouk E; Riese, Harriëtte; Giltay, Erik J; van den Heuvel, Odile A; Penninx, Brenda W; Broekman, Birit F P Published in: University of Groningen Published in: Psychoneuroendocrinology DOI: 10.1016/j.psyneuen.2021.105390 IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Publication date: 2021 Publication date: 2021 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Morssinkhof, M. W. L., Lamers, F., Hoogendoorn, A. W., de Wit, A. E., Riese, H., Giltay, E. J., van den Heuvel, O. A., Penninx, B. W., & Broekman, B. F. P. (2021). Oral contraceptives, depressive and insomnia symptoms in adult women with and without depression. Psychoneuroendocrinology, 133, 1-9. https://doi.org/10.1016/j.psyneuen.2021.105390 Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license. More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne- amendment. Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Citation for published version (APA): Morssinkhof, M. W. L., Lamers, F., Hoogendoorn, A. W., de Wit, A. E., Riese, H., Giltay, E. J., van den Heuvel, O. A., Penninx, B. W., & Broekman, B. F. P. (2021). Oral contraceptives, depressive and insomnia symptoms in adult women with and without depression. Psychoneuroendocrinology, 133, 1-9. https://doi.org/10.1016/j.psyneuen.2021.105390 Download date: 24-10-2024 Oral contraceptives, depressive and insomnia symptoms in adult women with and without depression Margot W.L. Morssinkhof a,b,c,, Femke Lamers b, Adriaan W. Hoogendoorn b,d, Anouk E. de Wit e, Harri¨ette Riese e, Erik J. Giltay f, Odile A. van den Heuvel g, Brenda W. Penninx b,d, Birit F.P. Broekman a,b,h a OLVG Hospital, Department of Psychiatry and Medical Psychology, Amsterdam, The Netherlands a OLVG Hospital, Department of Psychiatry and Medical Psychology, Amsterdam, The Netherlands b Amsterdam UMC, Vrije Universiteit Amsterdam, Department of Psychiatry, Amsterdam Public Health Research Institute, Amsterdam, The Netherlands c Amsterdam UMC, Vrije Universiteit Amsterdam, Department of Endocrinology, Department of Internal Medicine, Amsterdam, The Netherlands d GGZ inGeest, Department of Research and Innovation, Amsterdam, The Netherlands e University of Groningen, University Medical Center Groningen, Department of Psychiatry, Interdisciplinary Center Psychopathology and Emotional regulation G i Th N h l d a OLVG Hospital, Department of Psychiatry and Medical Psychology, Amsterdam, The Netherlands b Amsterdam UMC, Vrije Universiteit Amsterdam, Department of Psychiatry, Amsterdam Public Health Research Institute, Amsterdam, The Netherlands c Amsterdam UMC, Vrije Universiteit Amsterdam, Department of Endocrinology, Department of Internal Medicine, Amsterdam, The Netherlands d GGZ inGeest, Department of Research and Innovation, Amsterdam, The Netherlands f e University of Groningen, University Medical Center Groningen, Department of Psychiatry, Interdisciplinary Center Psychopathology and Emotional regulation, Groningen, The Netherlands f Leiden University Medical Center, Department of Psychiatry, Leiden, The Netherlands Leiden University Medical Center, Department of Psychiatry, Leiden, The Netherlands g Amsterdam UMC, Vrije Universiteit Amsterdam, Department of Psychiatry, Department of Anatomy and Neuroscience, Amsterdam Neuroscience, Amsterdam, Netherlands g Amsterdam UMC, Vrije Universiteit Amsterdam, Department of Psychiatry, Department of Anatomy and Neuroscience, Amsterdam Neuroscien Netherlands h Singapore Institute for Clinical Sciences, Agency for Science, Technology and Research (ASTAR), Singapore * Correspondence to: OLVG Hospital, Department of Psychiatry and Medical Psychology, P.O. Box 95500, 1090 HM Amsterdam, The Netherlands. E-mail address: m.morssinkhof@amsterdamumc.nl (M.W.L. Morssinkhof). Available online 12 August 2021 0306-4530/© 2021 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). https://doi.org/10.1016/j.psyneuen.2021.105390 Received 21 April 2021; Received in revised form 11 August 2021; Accepted 11 August 2021 Take-down policy Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Psychoneuroendocrinology 133 (2021) 105390 1. Introduction complaints and more daytime sleepiness compared to no use of OC (Bezerra et al., 2020). On the other hand, OC use has also been associ ated with longer sleep durations compared to no use in sleep EEG as sessments (Guida et al., 2020). The association between OC use and insomnia has not been examined at large. Use of oral contraceptives (OC), commonly called “the pill”, is a method of birth control used by many women. It is currently the most popular form of contraception in Europe, North America and Australia with user rates in reproductive aged women between 35% and 63% (UN. Population Division, 2019). OC works through administration of exog enous sex steroids, most commonly a combination of synthetic estradiol and progesterone (e.g. combined oral contraceptives), which suppress ovulation, thus preventing pregnancy and regulating the menstrual cycle. The potential deleterious effects of OC use have gained much attention. Among reported adverse mental health effects are depressive symptoms and, to a lesser extent, sleep problems (Bezerra et al., 2020; Hall et al., 2012). Although some women may experience negative mood symptoms while using OC (Skovlund et al., 2016), most women seem unaffected (Schaffir et al., 2016). Previous studies have also not assessed the association of OC use with specific subtypes of depression. Atypical depression, which is charac terized by mood reactivity and can include increased appetite and/or weight gain, hypersomnia, leaden paralysis and interpersonal sensi tivity, is more prevalent in women than in men (Lamers et al., 2010), suggesting a role for sex hormones. In addition, Halbreich and Kahn (2007) noted that symptoms of postpartum depression and premenstrual dysphoric disorder strongly overlap with symptoms of atypical depres sion. Following their conclusion, OC use might also be associated with symptoms of atypical depression, but this has not yet been studied. i Our study firstly aimed to investigate the association between self- reported OC use and depressive symptom severity (including atypical depressive symptoms) and insomnia symptom severity in adult women, as well as the association between OC use and concurrent diagnoses of major depressive disorder (MDD) and dysthymia. Secondly, we aimed to assess if a previous or current depression diagnosis moderates the as sociation between OC use and depressive and insomnia symptom severity. A B S T R A C T M.W.L. Morssinkhof et al. M.W.L. Morssinkhof et al. Psychoneuroendocrinology 133 (2021) 105390 Discussion: The study findings showed consistent associations between OC use and more severe insomnia symptoms, but no consistent associations between OC and depressive symptoms or diagnoses. Instead, post-hoc analyses showed that associations between OC and depression differed between within- and between person- estimates. This indicates that, although OC shows no associations on the overall level, some individuals might experience OC-associated mood symptoms. Our findings underscore the importance of accounting for individual differences in experiences during OC use. Furthermore, it raises new questions about mechanisms underlying associations between OC, depression and insomnia. 1. Introduction We hypothesized that measurements during OC use would show a higher depressive and insomnia symptom severity and a higher prevalence of concurrent MDD and dysthymia than women not using OC. Furthermore, we hypothesized that the association between OC use and severity of symptoms of depression and insomnia would be stronger in women with a previous or current diagnosis of a depressive disorder compared to women without a diagnosis. i Studies on the association between OC use and depression have shown mixed results. Skovlund et al. (2016) report that first use of OC is associated with a higher risk for a first clinical diagnosis of depression, as well as with a higher risk of first use of an antidepressant. However, most prospective studies have not shown a significant worsening in depressive symptoms in women using OC (as reviewed by Schaffir et al. (2016), Worly et al. (2018)). There are indications that variables such as young age (De Wit et al., 2020; Skovlund et al., 2016), composition of OC (Skovlund et al., 2016), but also previous experience of mood deterioration during OC use (Engman et al., 2018; Gingnell et al., 2013), may modify the risk of experiencing depressive symptoms during OC use. However, it is not yet clear whether a history of depression makes women more vulnerable for possible negative effects of current OC use such as depressive symptoms or insomnia. This information is important to enable well-informed choices on OC use. 2. Methods Up till now, few studies explored the relationship between OC use and adverse mood states in women with a previous or current depressive disorder. In a randomized controlled trial, women with previous or current psychiatric disorders (anxiety, depressive and eating disorders, n = 59) were more likely to report adverse mood during use of combined OCs compared to placebo, although only six women in this sample had a current depressive disorder (Bengtsdotter et al., 2018). Further studies showed that women with current OC-induced mood complaints were more likely to have comorbid mood disorders than women without OC-induced mood complaints in a cross-sectional comparison (Sege bladh et al., 2009). Joffe et al. (2003) retrospectively found that pres ence of a previous depressive episode was a significant predictor of OC-associated mood deterioration and Hall et al. (2012) found that in young women (aged 13–24) depressed moods at start of OC use increased the likelihood of reporting OC-related moodiness. However, there are only a few prospective studies on OC that specifically assessed women with diagnoses of depression. This means that clinicians and OC users currently have very limited guidance on whether OC use could affect women with a history of depression diagnoses differently than they affect non-depressed women. A B S T R A C T Keywords: Oral Contraceptives Depression Sleep Problems Insomnia Sex Hormones Background: Worldwide, oral contraceptive (OC) use is a very common form of birth control, although it has been associated with symptoms of depression and insomnia. Insomnia is a risk factor for major depressive disorder (MDD) but may also be a symptom of the disorder. Despite the large number of women who use OC, it is yet unknown whether women with previous or current diagnosis of depression are more likely to experience more severe depressive and insomnia symptoms during concurrent OC use than women without diagnosis of depression. Aim: This study examined associations between OC use and concurrent symptoms of depression (including atypical depression) and insomnia as well as between OC and prevalences of concurrent dysthymia and MDD. Participants were adult women with and without a history of MDD or dysthymia. We hypothesized that OC use is associated with concurrent increased severity of depressive symptoms and insomnia symptoms, as well as with an increased prevalence of concurrent diagnoses of dysthymia and MDD. We also hypothesized that a history of MDD or dysthymia moderates the relationship between OC use and depressive and insomnia symptoms. Methods: Measurements from premenopausal adult women from the Netherlands Study of Depression and Anxiety (NESDA) were grouped, based on whether participants were using OC or naturally cycling (NC). OC use, timing and regularity of the menstrual cycle were assessed with a structured interview, self-reported symptoms of depression (including atypical depression), insomnia with validated questionnaires, and MDD and dysthymia with structured diagnostic interviews. Results: We included a total of 1301 measurements in women who reported OC use and 1913 measurements in NC women (mean age 35.6, 49.8% and 28.9% of measurements in women with a previous depression or current depression, respectively). Linear mixed models showed that overall, OC use was neither associated with more severe depressive symptoms (including atypical depressive symptoms), nor with higher prevalence of diagnoses of MDD or dysthymia. However, by disentangling the amalgamated overall effect, within-person estimates indicated increased depressive symptoms and depressive disorder prevalence during OC use, whereas between- person estimated indicated lower depressive symptoms and prevalence of depressive disorders. OC use was consistently associated with more severe concurrent insomnia symptoms, in the overall estimates as well as in the within-person and between-person estimates. Presence of current or previous MDD or dysthymia did not mod erate the associations between OC use and depressive or insomnia symptoms. 2.1. Participants For this study we used data from the Netherlands Study on Depres sion and Anxiety (NESDA). NESDA is an ongoing longitudinal cohort study assessing the long-term course and consequences of MDD and anxiety disorders in the Netherlands. From 2003 until 2007, NESDA recruited 2981 adult participants from the community, via primary healthcare and outpatient mental healthcare providers. At recruitment, 78% of participants had (past or present) depressive and/or anxiety disorders, and 22% of participants were included as non-depressed controls. Exclusion criteria were having a primary clinical diagnosis of bipolar disorder, obsessive compulsive disorder, severe substance use disorder, psychotic disorder, or organic psychiatric disorder (all confirmed by Composite International Diagnostic Interview or CIDI) or being not sufficiently proficient in Dutch language. For more details on the NESDA study and details on recruitment and methods, see Penninx et al. (2021). All participants provided informed consent and the study was approved by the Medical Ethical Committee of the VUmc (reference number 2003/183) and other participating centers. The NESDA study followed participants 2, 4, 6 and 9 years after the baseline measurement, meaning every participant could contribute up to 5 measurements to the full dataset. At every assessment, participants underwent a structured diagnostic interview for assessment of mental disorders and questionnaires on depressive symptoms, insomnia Insomnia is an important component of depression, both as a symptom as well as a risk factor (Baglioni et al., 2011; Fava, 2004; Paunio et al., 2015). Previous studies showed that OC use was associated with less deep sleep as revealed with EEG measurements, and a longer time to fall asleep (Burdick et al., 2002), more frequent insomnia 2 M.W.L. Morssinkhof et al. Psychoneuroendocrinology 133 (2021) 105390 symptoms, lifestyle and socioeconomic factors. This included self-report questions on the presence and duration of a menstrual cycle, possible pregnancy, menopause status and contraceptive use status. placement and subsequent unknown quantity of hormone release by the IUD. Observations in women who were pregnant at the time of the assessment (n = 195), who gave birth in the last year (n = 159) or were breastfeeding (n = 7) were also excluded from the analysis. 2.1. Participants Women who continued OC for more than 90 days, meaning they skipped multiple placebo weeks or “stop weeks”, were also excluded (n = 37), as well as naturally cycling women who reported abnormal menstrual cycle durations, defined as current or regular cycles were shorter than 21 days or longer than 35 days (Nederlandse Vereniging voor Obstetrie en Gynaecologie, 2004; n = 357). To assess possible se lection biases that could stem from excluding women with abnormal cycle durations, an additional sensitivity analysis was conducted on the full sample, including the women with abnormal cycle durations (re ported in Appendix, see Supplementary materials B). Additionally, ob servations were also excluded if participants were using other hormonal medications (World Health Organization Anatomical Therapeutic Chemical (ATC) code G03, n = 22) or using hormone antagonists (ATC code L02, n = 2) at the time. Fig. 1. Visualisation of how hypothetical participants can contribute multiple observations (panel A), and the amount of measurement observations per group (panel B). OC = oral contraceptive use, NC = naturally cycling. 2.5. Statistical analyses Statistical analyses were conducted using R (version 3.6.1; R Core Team, 2020). Demographic differences between the measurement groups were analyzed with Chi square tests for categorical variables and t-tests for continuous variables. p y y To assess the severity of depressive symptoms, all participants filled out the well-validated Self Rated Inventory of Depressive Symptom atology (IDS-SR) at every assessment wave. The IDS-SR has excellent internal consistency (Cronbach’s alpha = 0.94, Rush et al., 1996). The items in the IDS-SR are scaled on a Likert scale from 0 to 3 with 0 meaning the symptom is not present, and 3 meaning high symptom severity. The IDS-SR has 30 items, of which the scores of individual items can be summed to form a sum score that represents the depressive symptom severity. Sub-scores for atypical depression were calculated based on characteristics of atypical depression according to the DSM-IV by calculating the sum of item scores for hypersomnia (item 4), mood reactivity (item 8, reverse scored to represent absence of anhedonia), increased appetite (item 12), weight gain (item 14), leaden paralysis (item 29) and interpersonal sensitivity (item 30) from the IDS-SR, resulting in a sum score ranging from 0 to 18 (Novick et al., 2005). i To analyze associations between OC use and symptoms of depression or insomnia, all outcomes were compared using (generalized) linear mixed models. Models were created using the ‘lme4′ package (Barr, 2013), and analyzed with ANOVA tests (type 3) from the ‘car’ package (Fox and Weisberg, 2020). Model estimates were estimated using the ‘summary’ function from the R package lmerTest (Kuznetsova et al., 2017) using an unstructured variance-covariance structure. In all models, a random intercept per participant was added to account for multiple measures within the same participants. This random intercept also enabled within-participant comparisons between OC and NC mea surements in a subset of the measurements. Generalized linear (logistic) mixed models were used for analysis of categorical outcomes (e.g., di agnoses of MDD and dysthymia) and linear mixed models were used for the continuous variables (e.g. IDS scores, WHI-IRS scores, IDS atypical depression subscores). To assess insomnia symptom severity, participants filled-out the Women’s Health Insomnia Rating Scale (WHI-IRS) by Levine et al. (2003) at every assessment wave. The WHI-IRS consists of five items on a scale of 0–4, with a sum score ranging from 0 to 20. 2.3. Depressive disorders, and symptoms of depression and insomnia 2.3. Depressive disorders, and symptoms of depression and insomnia To assess the presence of MDD or dysthymia (as defined by DSM-IV, American Psychiatric Association, 1994) the Composite International Diagnostic Interview (CIDI, version 2.1; Nelson, 1999) was used. This means that the knowledge on all current and previous diagnoses in this study was acquired using clinically validated methods. The CIDI was conducted at every assessment by trained research staff. Diagnoses of MDD and dysthymia within the last 6 months were used as an outcome measure for current depressive or dysthymic disorder. i 2.5.1. Univariable model and multivariable model First, outcomes were assessed using a univariable model that included only the contraceptive status (e.g., OC status or NC status at time of measurement) as fixed or independent variable and MDD or dysthymia diagnoses, depressive symptom scores (including atypical depressive symptoms) or insomnia symptom score as outcomes. Second, we used a multivariable model with adjustment for previously mentioned confounders to estimate differences between OC and NC measurements in all outcomes (e.g., MDD and dysthymia diagnoses, depressive symptoms, atypical depressive symptoms and insomnia symptoms). 2.4. Covariates Sociodemographic variables in this study included age, highest ed ucation, country of birth, partner status and body mass index (BMI). Participants’ level of education was defined as the highest number of years of education that the participant reported over the whole study duration. This was used instead of the number of years of education at each assessment because the number of years of education and partici pants’ age were highly codependent, violating statistical assumptions. Since contraceptive choices in the Netherlands significantly differ be tween groups of different ethnicities (Marra et al., 2020), we adjusted for participants’ birth country in the analyses. Birth country was grouped based on whether the participant was born in the Netherlands or outside the Netherlands. Partner status was reported at every assessment wave by asking about marriage and partner status (e.g., “Are 2.5.2. Moderation analysis of depression status M.W.L. Morssinkhof et al. The remaining observations were then grouped based on contra ceptive status, into either observations during OC use (e.g. OC mea surements) or observations during which participants were naturally cycling (NC measurements). Observations in women who did not use any hormonal contraceptives and reported a regular menstrual cycle were clustered in the NC observations (this included women who re ported using a copper IUD or condoms for contraception), resulting in a total of 1913 observations. Observations in women who reported using OC at time of the measurement were clustered in the OC group, resulting in 1301 observations. This enabled a cross-sectional comparison of measurements based on contraceptive status (either during OC use or during non-OC use) within the longitudinal NESDA study setup. Fig. 1B shows the sample sizes of both contraceptive status groups and sample sizes over time, including the breakdown of group size. This process resulted in a total sample of 1205 women who contributed 3214 ob servations (mean number of observations per woman = 2.7). you married or do you have a person you consider your long-term partner?”). Age, education level and birth country were incorporated into the multivariate adjusted model, partner status and BMI were included only if these differed between the OC and NC measurements, meaning they could be confounding effect estimates. As an additional covariate, the course of participants’ depression diagnoses (obtained through CIDI interviews) before and throughout the study was determined to categorize observations into three groups. Observations were categorized in either the never depressed control group, the remitted group or the depressed group. Observations in participants who had no MDD or dysthymia diagnoses in their lifetime were labeled as never depressed, observations in participants with a previous but no current diagnosis of MDD or dysthymia were labeled as remitted, observations in participants with a current (within the last 6 months) MDD or dysthymia diagnosis were labeled as depressed. This method of labeling meant that a participant who was recruited without a history of depression could contribute her first observations to the never depressed group, but if she were subsequently diagnosed with a depressive disorder she would contribute her next measurement to the depressed group-observations, and if she next would recover from the depressive disorder her next measurement would be grouped into the remitted group. 2.2. Grouping measurements based on contraceptive status The present study included premenopausal women who had partic ipated at least once but up to five times in assessments between baseline and follow-up, as is visualized in Fig. 1A. To exclude postmenopausal women, observations in women aged 55 years or older (n = 2391) were excluded. Observations in women who had missing data on the question of contraceptive methods (n = 263), or who used hormonal contracep tives other than OC such as a hormonal intrauterine device (IUD; n = 373), a hormone implant (n = 23), vaginal ring (n = 57) or hormone injections (n = 34) or other hormonal methods (n = 18) were also excluded. Observations during use of hormone implants, vaginal rings and hormone injections were excluded due to low sample sizes, obser vations in hormonal IUDs were excluded due to unknown date of Visualisation of how hypothetical participants can contribute multiple observations (panel A), and the amount of measurement observations h h h i l i i ib l i l b i ( l ) d h f b Fig. 1. Visualisation of how hypothetical participants can contribute multiple observations (panel A), and the amount of measurement observations per group (panel B). OC = oral contraceptive use, NC = naturally cycling. 3 Psychoneuroendocrinology 133 (2021) 105390 M.W.L. Morssinkhof et al. 2.5. Statistical analyses Items inquire about difficulty falling asleep, waking up during the night, early awakening, getting back to sleep after wakefulness and feeling rested after sleep. The WHI-IRS has an acceptable Cronbach’s alpha of 0.78 (Levine et al., 2003). 3.5.1. Post-hoc within- and between-participant results The models with separate predictors for within- and between subject estimates for the IDS-SR scores, the atypical depression IDS subscores and the MDD prevalence had a better model fit (p < 0.001; ∆AIC = 19; 6 and 12, respectively) than the previous adjusted models of the main analysis. This analysis showed a disparity between the within- and be tween subject estimates. As displayed in Table 2, depressive symptoms (B = 1.43, 95%-CI = (0.37, 2.50), p = 0.0009), symptoms of atypical depression (B = 0.45, 95%-CI = (−0.03, 0.92), p = 0.07), as well as MDD diagnoses (OR = 1.70, 95%-CI = (1.16, 2.49), p = 0.0006) were estimated higher in OC measurements when assessing within-subject estimates. However, between-subject estimates indicated lower depression symptoms (B = −2.97, 95%-CI = (−4.57, −1.38), 3.1. Demographics To assess whether vulnerability for depressive disorder (MDD and/or dysthymia) moderates the relationship between OC status and severity of depressive symptoms or insomnia symptoms, an analysis was con ducted which incorporated depression group in interaction with OC status. There was no significant interaction effect of OC status X remitted depression on IDS scores (B = −0.005, 95%-CI = (−1.89, 1.78), p = 0.96), nor on atypical depression scores (B = −0.40, 95%-CI = (−1.20, 0.40), p = 0.32) or WHI-IRS scores (B = −0.34, 95%-CI = (−1.17, 0.48), p = 0.41). Similarly, no significant interaction effect of OC status X current depression was found on IDS scores (B = −0.59, 95%-CI = (−2.52, 1.34), p = 0.55), atypical depression scores (B = −0.22, 95%-CI = (−1.07, 0.63), p = 0.61) or WHI-IRS scores (B = −0.54, 95%-CI = (−1.42, 0.34), p = 0.23). We analyzed data from 1205 participants who contributed to 3214 observations. Of these observations, 1301 (40.5%) reported current OC use and 1913 (59.5%) reported they were naturally cycling at the time of measurement (see Fig. 1B). A total of 612 women contributed only to NC measurements (a total of 1467 measurements), 327 women contributed only to OC measurements (a total of 803 measurements), and 266 women contributed both to OC and NC measurements (a total of 944 measurements). Participants were on average 35.6 (SD 9.0) years old, had received 13.5 years of education (SD 3.2), and 91.4% were born in the Netherlands. OC use was significantly associated with younger age compared to NC measurements, and measurements in OC users were more likely to be in participants born in the Netherlands, but level of education, partner status, or BMI did not differ between OC measure ments and NC measurements. Means and percentages of sociodemo graphic variables are displayed in Table 1. 2.5.3. Post hoc analyses A post-hoc analysis was conducted to enable differentiation in within- and between-person estimates of OC use. Within- and between- person effects were assessed through the incorporation of two predictors for OC use instead of one: one predictor was OC use centered per participant (e.g. within cluster centering, with each participant repre senting one cluster of measurements), the other predictors was OC use but centered within the group mean, as recommended in Enders and Tofighi (2007) and Hamaker and Muth´en (2019). These between- and within-person predictors were assessed for all main outcomes (diagnoses of depression and dysthymia, severity of depressive symptoms (including atypical symptoms of depression), and insomnia symptoms in the multivariate models which included adjustments for age, highest education and birth country. M.W.L. Morssinkhof et al. M.W.L. Morssinkhof et al. Psychoneuroendocrinology 133 (2021) 105390 2.5.3. Post hoc analyses Furthermore, measurements during OC use did not show a signifi cantly different prevalence of MDD diagnoses in comparison to the NC measurements in the adjusted model (Odds Ratio (OR) = 0.99, 95%- CI = (0.77, 1.27), p = 0.93; Table 2). Prevalence of dysthymia di agnoses also did not differ significantly between OC and NC measure ments (adjusted model: OR = 0.65, 95%-CI = (0.32, 1.27), p = 0.21; Table 2). The full model results are displayed in Table 2, and scores and estimates per depression group are visualized in Fig. 2. 3. Results 3. Results 3.2. Current oral contraceptive status and depression We observed a statistically significant difference in the IDS-SR scores between OC and NC measurements of 0.90 points (95%-CI = (0.00, 1.80); p = 0.049; Table 2), although this difference was small in terms of effect size (Cohen’s d = 0.07) due to large differences within the groups, resulting in a high SD. This difference in IDS-SR scores was no longer significant after adjustment for the demographic covariates (B = 0.10, 95%-CI = (−0.80, 1.00), p = 0.83; Table 2). OC use was not associated with higher atypical depressive sub-scores in both the unadjusted and adjusted model. 3.5. Post-hoc analysis 3.5.1. Post-hoc within- and between-participant results 3.3. Current oral contraceptive status and insomnia We observed a higher WHI-IRS score during OC use in the unadjusted model, although this difference was not significant. The adjusted model, which included adjustments for age, country of birth and education resulted in a significant difference between OC and NC measurements of 0.54 points (B = 0.54, 95%-CI = (0.17, 0.91), p = 0.004; Table 2). The Cohen’s d effect size of the difference in WHI-IRS score was 0.12, meaning the effect size for the association between OC use and insomnia symptoms is small (Cohen, 1998). The results are summarized in Table 2, and the scores per depression group are displayed in Fig. 2. Furthermore, in post-hoc analysis, individual sleep-related items from the IDS-SR and the WHI-IRS were analyzed per item to explore whether specific sleep complaints (e.g., trouble falling asleep, waking up during the night) were associated with contraceptive status. Results for this analysis are provided in Supplementary materials A, see Appendix. Table 1 Table 1 Description of the sociodemographic variables across measurements. Contraceptive status during measurement Naturally cycling Oral contraceptive use p-value # of observations 1913 1301 – Age in years (mean, SD) 38.1 (8.0) 31.9 (9.1) <0.001 Highest education in years (mean, SD) 13.5 (3.2) 13.4 (3.2) 0.48 BMI in kg/m2 (mean, SD) 25.1 (5.2) 24.7 (5.2) 0.054 Born in the Netherlands (n, %) 1703 (89.0) 1234 (94.8) <0.001 Has a partner (n, %) 1376 (71.9) 942 (72.8) 0.62 Depression status (n, %) No MDD or dysthymia 433 (22.6) 268 (20.6) 0.22 Remitted from MDD or dysthymia 920 (48.1) 664 (51.0) Current MDD or dysthymia 560 (29.3) 369 (28.4) BMI = Body Mass Index, SD = standard deviation. p-values obtained from independent samples t-test or from chi-square test for cross-tabulation. Table 1 Description of the sociodemographic variables across measurements. 2.5.2. Moderation analysis of depression status To assess whether contraceptive status was associated with depres sive symptoms (including atypical depressive symptoms) and insomnia symptoms in never depressed controls differently than in (previously) depressed participants, the psychiatric history was incorporated in two interaction terms (one for remitted depression and one for current depression). Participants’ depression status (i.e., never, remitted or currently depressed) was then added through the addition of interaction terms with the OC use variable in the multivariable models. 4 Table 1 Description of the sociodemographic variables across measurements. The insomnia scores were still estimated somewhat higher, both in between-participant estimates (B = 0.51, 95%-CI = (−0.07, 1.10), p = 0.09) as well as within-participant estimates (B = 0.56, 95%-CI = (0.1, 1.02), p = 0.02). measurements (B = 0.28, 95%-CI = (0.16, 0.39), p < 0.001 and B = 0.19, 95%-CI = (0.08, 0.31), p = 0.001, respectively). See Supple mentary materials A in Appendix for the full model results for all sleep- specific IDS-SR items and WHI-IRS items. 4. Discussion This study examined associations between current OC use and depressive disorders, severity of depressive symptoms, and insomnia symptoms in a large cohort of adult women, of whom the diagnoses and symptoms of depression were well phenotyped over the course of 9 years. Our first findings showed no indications that self-reported OC status was associated with more severe depressive symptoms (including symptoms of atypical depression), nor with higher prevalence of MDD or dysthymia. Moreover, having a previous or current diagnosis of MDD or dysthymia did not moderate the associations between OC use and insomnia or depressive symptoms. Although our overall results on OC and depression did not confirm our hypotheses, which were that OC use Table 1 Description of the sociodemographic variables across measurements. Table 2 Association of oral contraceptive status with depression diagnoses and symptoms analyzed using linear mixed models. ceptive status with depression diagnoses and symptoms analyzed using linear mixed models. Association of oral contraceptive status with depression diagnoses and symptoms analyzed using linear mixed models. OC = oral contraceptives, NC = naturally cycling, MDD = major depressive disorder, IDS = Inventory of Depressive Symptoms, WHI-IRS = Women’s Health In ventory Insomnia Rating Scale. OR = odds ratio, B = unstandardized estimate, CI = confidence interval, SD = standard deviation. The adjusted model contains adjustment for age, education level and country of birth. i OC = oral contraceptives, NC = naturally cycling, MDD = major depressive disorder, IDS = Inventory of Depressive Symptoms, WHI-IRS = Women’s Health In ventory Insomnia Rating Scale. OR = odds ratio, B = unstandardized estimate, CI = confidence interval, SD = standard deviation. The adjusted model contains adjustment for age, education level and country of birth. a Model which includes between and within person estimates was significantly better than the model that estimated the general estimate as described in the text j g , y a =Model which includes between- and within-person estimates was significantly better than the model that estimated the general estimate as described in the text. j g , y a =Model which includes between- and within-person estimates was significantly better than the model that estimated the general estimate as described in the text. a =Model which includes between- and within-person estimates was significantly better than the model that estimated the ge Fig. 2. Visualization of mean scores and adjusted estimations and 95% confidence intervals of the IDS score, IDS atypical depression symptom score and WHI-IRS score from the adjusted model in Table 2. The estimate is adjusted for age, education level and birth country. OC = oral contraceptives, NC = naturally cycling, IDS- SR = Self-Reported Inventory of Depressive Symptoms, WHI-IRS = Women’s Health Initiative Insomnia Rating Scale, SE = Standard Error. Fig. 2. Visualization of mean scores and adjusted estimations and 95% confidence intervals of the IDS score, IDS atypical depression symptom score and WHI-IRS score from the adjusted model in Table 2. The estimate is adjusted for age, education level and birth country. OC = oral contraceptives, NC = naturally cycling, IDS- SR = Self-Reported Inventory of Depressive Symptoms, WHI-IRS = Women’s Health Initiative Insomnia Rating Scale, SE = Standard Error. Table 1 Description of the sociodemographic variables across measurements. Fig. 2. Visualization of mean scores and adjusted estimations and 95% confidence intervals of the IDS score, IDS atypical depression symptom score and WHI-IRS score from the adjusted model in Table 2. The estimate is adjusted for age, education level and birth country. OC = oral contraceptives, NC = naturally cycling, IDS- SR = Self-Reported Inventory of Depressive Symptoms, WHI-IRS = Women’s Health Initiative Insomnia Rating Scale, SE = Standard Error. Fig. 2. Visualization of mean scores and adjusted estimations and 95% confidence intervals of the IDS score, IDS atypical depression symptom score and WHI-IRS score from the adjusted model in Table 2. The estimate is adjusted for age, education level and birth country. OC = oral contraceptives, NC = naturally cycling, IDS- SR = Self-Reported Inventory of Depressive Symptoms, WHI-IRS = Women’s Health Initiative Insomnia Rating Scale, SE = Standard Error. p < 0.0001), lower symptom scores for atypical depression (B = −0.53, 95%-CI = (−1.04, −0.04), p = 0.03) and lower prevalence of MDD di agnoses (OR = 0.66, 95%-CI = (0.47, 0.92), p = 0.014) in OC mea surements compared to NC measurements. The models with separate predictors for within- and between-person estimates for dysthymia as well as for insomnia severity did not show a better model fit (∆AIC = +2 and ∆AIC = +2, respectively), and estimates for dysthymia still showed no indications of associations between OC use and dysthymia preva lence. The insomnia scores were still estimated somewhat higher, both in between-participant estimates (B = 0.51, 95%-CI = (−0.07, 1.10), p = 0.09) as well as within-participant estimates (B = 0.56, 95%-CI = (0.1, 1.02), p = 0.02). p < 0.0001), lower symptom scores for atypical depression (B = −0.53, 95%-CI = (−1.04, −0.04), p = 0.03) and lower prevalence of MDD di agnoses (OR = 0.66, 95%-CI = (0.47, 0.92), p = 0.014) in OC mea surements compared to NC measurements. The models with separate predictors for within- and between-person estimates for dysthymia as well as for insomnia severity did not show a better model fit (∆AIC = +2 and ∆AIC = +2, respectively), and estimates for dysthymia still showed no indications of associations between OC use and dysthymia preva lence. Table 1 Description of the sociodemographic variables across measurements. # of observations Age in years (mean, SD) Highest education in years (mean, SD) BMI in kg/m2 (mean, SD) Born in the Netherlands (n, %) Has a partner (n, %) Depression status (n, %) Psychoneuroendocrinology 133 (2021) 105390 Psychoneuroendocrinology 133 (2021) 105390 M.W.L. Morssinkhof et al. Table 2 Association of oral contraceptive status with depression diagnoses and symptoms analyzed using linear mixed models. Contraceptive status during measurement Estimated unadjusted difference, (OC vs. NC measurements) Estimated adjusted difference, (OC vs. NC measurements) Estimated adjusted difference (OC vs. NC measurements), within-and between-person estimates Naturally cycling (n = 1913) Oral contraceptive use (n = 1301) B or OR, 95%-CI p- value B or OR, 95%-CI p- value Estimate B or OR, 95%-CI p- value Depressive symptoms IDS score (mean, SD) 17.6 (12.9) 16.8 (12.4) B = 0.90 (0.00, 1.80) 0.049 B = 0.10 (−0.80, 1.00) 0.83 Between- persona B = −2.97 (−4.57, −1.38) 0.0003 Within-persona B = 1.43 (0.37, 2.50) 0.009 Atypical IDS subscore (mean, SD) 5.1 (4.3) 4.9 (4.2) B = 0.03 (−0.30, 0.37) 0.86 B = −0.05 (−0.40, 0.29) 0.75 Between- persona B = −0.53 (−1.04, −0.04) 0.03 Within-persona B = 0.45 (−0.03, 0.92) 0.07 Depression diagnoses MDD in the last 6 months (n, %) 523 (27.3) 355 (27.3) OR = 1.10 (0.87, 1.39) 0.40 OR = 0.99 (0.77, 1.27) 0.93 Between- persona OR = 0.66 (0.47, 0.92) 0.014 Within-persona OR = 1.70 (1.16, 2.49) 0.006 Dysthymia in the last 6 months (n, %) 80 (6.1) 158 (8.3) OR = 0.76 (0.39, 1.46) 0.42 OR = 0.65 (0.32, 1.27) 0.21 Between- person OR = 0.43 (0.13, 1.30) 0.15 Within-person OR = 0.81 (0.35, 1.94) 0.63 Insomnia WHI-IRS (mean, SD) 6.8 (4.7) 7.0 (4.7) B = 0.29 (−0.06, 0.65) 0.11 B = 0.54 (0.17, 0.91) 0.004 Between- person 0.51 (−0.07, 1.10) 0.09 Within-person 0.56 (0.1, 1.02) 0.02 OC = oral contraceptives, NC = naturally cycling, MDD = major depressive disorder, IDS = Inventory of Depressive Symptoms, WHI-IRS = Women’s Health In ventory Insomnia Rating Scale. OR = odds ratio, B = unstandardized estimate, CI = confidence interval, SD = standard deviation. The adjusted model contains adjustment for age, education level and country of birth. a =Model which includes between- and within-person estimates was significantly better than the model that estimated the general estimate as described in the text. 3.5.2. Insomnia per-item analysis Third, as we excluded women who breastfed, gave birth in the past year or had an abnormal cycle length, hormonal abnormalities related to pregnancy, breastfeeding or meno pause did most likely not affect the analyses. Fourth, the repeated measurements within the same participants enabled within-person comparisons between OC and NC measurements within the same per son, which have evidently provided novel insights into the association between OC and depression. Based on this idea of the “healthy survivor effect”, our findings do seem to follow a pattern that was previously found in the literature: studies which compared population-based groups of OC users with groups of non-users found OC to be associated lower depression preva lence (as seen in Toffol et al. (2011), Keyes et al. (2013)), whereas studies that have prospectively assessed OC use over time have found adverse effects of OC on depression diagnoses and symptoms (Skovlund et al., 2016)). Within the context of this previous work, our findings accentuate some important points. First, it could be that OC negatively affects depression symptoms in some women, who might then discon tinue their OC use, while other women remain unaffected. Previous work has already highlighted possible mechanisms involving proges terone and GABA receptors, in which there are strong individual dif ferences in sensitivity to sex hormone-associated mood symptoms (Schweizer-Schubert et al., 2021) Secondly, this highlights the impor tance of within-person comparisons in the field of sex hormones and mood. This point has been raised before in menstrual cycle research (Schmalenberger et al., 2020) and our findings highlight the relevance of accounting for within- and between-participant comparisons. i This study has limitations in other aspects. These limitations lie mostly in the domain of OC assessment, since the NESDA cohort origi nally was not aimed to assess OC use. We did not have information on the age of first use of OC, nor on the duration of current OC use. This limited the study in two ways. Firstly, recent studies found that OC use during adolescence could increase the likelihood of a depression diag nosis in adulthood, even if women did not use OC anymore (Zettermark et al., 2018; Anderl et al., 2020). The current study, however, could not take previous OC use during adolescence into account. Additionally, we only had data on concurrent OC use, meaning we did not have data on recency of OC use. 3.5.2. Insomnia per-item analysis Multivariable models, conducted for the sleep-specific items from the IDS-SR (items 1, 2, 3 and 4) and all WHI-IRS items showed that specif ically WHI-IRS items 2 (waking up during the night) and 3 (waking earlier than planned) were higher during OC use compared to NC 6 Psychoneuroendocrinology 133 (2021) 105390 M.W.L. Morssinkhof et al. significantly moderate the association between OC and insomnia scores, indicating that the OC-insomnia association is not dependent on history of a diagnosis of MDD or dysthymia. Our post hoc analyses indicate that OC use might be specifically associated with waking up multiple times during the night and earlier awakening. This suggests that differences in insomnia symptom severity during OC use (compared to non-use) could be more related to the area of maintaining sleep than initiating sleep. Our findings are in line with the study by Bezerra et al. (2020), who found that OC users report worse sleep quality than non-users. Also other studies found possible effects of OC on sleep, showing that OC users have less slow wave sleep (Burdick et al., 2002), have higher melatonin secretion and higher nocturnal body temperatures (Baker and Driver, 2007). However, the association between OC and sleep disrup tions could also be caused or mediated by lifestyle factors, such as working hours, caffeine or alcohol consumption, or presence of small children (Wilsmore et al., 2013). Although the effect size of the associ ation between OC use and insomnia was small, further studies in the domain of fundamental sleep research could offer more insight to what extent biological changes influence the association between OC use and sleep disruptions and how this may impact on health and functioning. would be associated with more severe depressive symptoms and higher prevalence of depressive disorders, the results from the post-hoc analysis indicated that our original lack of accounting for the within- and between-participant measurements might have obscured underlying associations between OC and depression outcomes, because the be tween- and within-person estimates showed opposite directions. Furthermore, insomnia symptoms were consistently found to be more severe during OC use, both in the overall analysis as well as in the be tween- and within person estimates, although the effect size was small. 3.5.2. Insomnia per-item analysis To understand our contradictory results from between- and within- subject estimates, it is important to realize that in our study setup, OC use was not randomized or assigned, which means OC use during the study purely relied on participant’s own contraceptive use choice. The choice to use or not use OC can depend on medical history or comor bidities, as seen in for example, medical advice to not use OC in women with history of thrombosis, or to use OC in women with polycystic ovary syndrome or endometriosis, as well as on personal experiences using OC: women who had adverse experiences using OC were found to be less likely to continue OC use (Westhoff et al., 2007). This implies the dis tribution of OC use across the participants was most likely not random. Following this reasoning, it would mean that measurements in women in our study who experience less adverse mood during OC use could be overrepresented in the OC measurements, whereas measurements in women who do experience more adverse mood effects could be over represented in either NC measurements or in the group of measurements in women who “switched” OC use during the study (e.g. the group of participants who contributed most to the within-subject estimates). This form of selection bias, specifically the idea that women who experience adverse effects during OC use are less likely to be present in long-term users of OC, has previously been deemed the “healthy survivor effect” (Zettermark et al., 2018). i Our study has some particular strengths. First, our study consisted of a large sample of participants, many of whom have a history or a current depressive disorder, meaning our results can be well-generalized to women with a vulnerability for depressive disorders. As depression is common in society, this helps to generalize results to the broader pop ulation. We were able to also assess interaction effects of OC with a history of depressive disorders, which has rarely been done before. Second, psychiatric diagnoses were assessed prospectively using vali dated clinical interviews as well as self-reported depressive symptoms. This is fairly unique, since many studies on OC and depression rely only on self-reported depressive symptoms and retrospective questions about previous diagnoses of depression. 3.5.2. Insomnia per-item analysis Another limitation is related to the exclu sion of women with abnormal cycle duration, which meant that our results could not be generalized to women who report abnormally long or short cycles. Since abnormal cycle lengths can be a reason to start with OC, there was a risk of sample bias, since this meant that we mostly excluded women with abnormal cycle during NC measurements. We addressed this by conducting a sensitivity analysis on the larger sample including women with abnormal cycle lengths. The results of this sensitivity analysis show similar results to analysis in the original sam ple, which only included women with regular cycle lengths (see Sup plementary materials B in the Appendix). Barr, D.J., 2013. Random effects structure for testing interactions in linear mixed-effects models. Front. Psychol. 4, 3–4. https://doi.org/10.3389/fpsyg.2013.00328. Bengtsdotter, H., Lundin, C., Gemzell Danielsson, K., Bixo, M., Baumgart, J., Marions, L., Sundstr¨om Poromaa, I., 2018. Ongoing or previous mental disorders predispose to adverse mood reporting during combined oral contraceptive use. Eur. J. Contracept. Reprod. Health Care 23 (1), 45–51. i Bezerra, A.G., Andersen, M.L., Pires, G.N., Banzoli, C.V., Polesel, D.N., Tufik, S., Hachul, H., 2020. Hormonal contraceptive use and subjective sleep reports in women: an online survey. J. Sleep. Res. 29 (6), 12983. https://doi.org/10.1111/ jsr.12983. Burdick, R.S., Hoffmann, R., Armitage, R., 2002. Short note: oral contraceptives and sleep in depressed and healthy women. Sleep 25, 2001–2003. Cohen, J., 1998. Statistical Power Analysis for the Behavioral Sciences - Jacob Cohen - Google Boeken. In summary, our study found consistent associations between OC use and insomnia symptoms, although effect sizes of these estimates were small. Furthermore, we found no overall associations between OC use and depression outcomes, both with regards to symptom severity and diagnosis prevalence. However, post-hoc analyses showed that the within- and between-person estimates indicated significant associations between depression and OC use. This indicates that, although OC overall was not associated with depression outcomes, some individuals might experience OC-associated depression symptoms. The effect sizes of the found associations were small, and future research should be conducted to assess whether effects of OC on depression risk are robust and clini cally relevant. Nonetheless, our findings on this topic show novel results on within-person associations between OC and depression diagnoses obtained through clinical interviews, which underscore the importance of accounting for individual differences in OC research. 3.5.2. Insomnia per-item analysis We used 6 month-latency of diagnoses of MDD and dysthymia, meaning a participant could hypothetically have been depressed before they started OC use. As a consequence of these limi tations we were unable to investigate exact causal relationships between OC use and depressive disorders. Furthermore, the composition or brand and dosage of the used OC and duration of use was unknown. Previous studies, such as Skovlund et al. (2016), found that progestin-only con traceptives were more strongly associated with depressive disorders than combined contraceptives. In the Netherlands, 95.8% of women use a form of combined oral contraceptives, with both estradiol and pro gesterone in its formulation. Moreover, ethinylestradiol/levonorgestrel 0.03/0.15 mg (or “Microgynon 30′′) is the most used form of OC in the Netherlands, with 90.6% of combined OC users using this formulation (data from 2018; GIP databank, 2021). Hence, it is expected that the majority of the OC-users in our study used this form. In the current study we could only rely on population-based estimates of what forms of OC are mostly used, but future studies should take OC formulation into The finding that severity of depressive symptoms were equally pre sent in OC measurements in women with or without previous or current diagnosis of MDD or dysthymia, is not line with previous studies on mood disorders and depressive symptoms during OC use, which showed that women with previous or current mood disorders were more likely to report adverse mood during OC use (Hall et al., 2012; Joffe et al., 2003; Bengtsdotter et al., 2018). However, one should keep in mind that we found different within- and between-person estimates for the association between OC use and severity of depressive symptoms, and it is yet un clear to what extent a role history of depression might have influenced these effects. Interestingly, we found an association between OC use and concur rent severity of insomnia symptoms, both in the overall estimates as well as in the within- and between-person estimates. Although severity of insomnia symptoms are generally higher in previously or currently depressed participants (Prather et al., 2015), psychiatric history did not 7 M.W.L. Morssinkhof et al. Psychoneuroendocrinology 133 (2021) 105390 Baker, F.C., Driver, H.S., 2007. Circadian rhythms, sleep, and the menstrual cycle. Sleep. Med. 8 (6), 613–622. account whenever possible. 3.5.2. Insomnia per-item analysis Although our study was limited in some aspects of OC use, such as duration of use, our findings raise new questions on which women might be susceptible to possible adverse mood symptoms during OC use, and whether insomnia symptoms might be affected by OC. Future research should focus on studying underlying mechanisms between OC, insomnia and depression. De Wit, A.E., Booij, S.H., Giltay, E.J., Joffe, H., Schoevers, R.A., Oldehinkel, A.J., 2020. Association of use of oral contraceptives with depressive symptoms among adolescents and young women. JAMA Psychiatry 77, 52–59. https://doi.org/ 10.1001/jamapsychiatry.2019.2838. i Enders, C.K., Tofighi, D., 2007. Centering predictor variables in cross-sectional multilevel models: a new look at an old issue. Psychol. Methods 12 (2), 121–138. Engman, J., Sundstr¨om Poromaa, I., Moby, L., Wikstr¨om, J., Fredrikson, M., Gingnell, M., 2018. Hormonal cycle and contraceptive effects on amygdala and salience resting- state networks in women with previous affective side effects on the pill. Neuropsychopharmacology 43, 555–563. https://doi.org/10.1038/npp.2017.157. p y p gy p g pp Fava, M., 2004. Daytime sleepiness and insomnia as correlates of depression. J. Clin. Psychiatry 65 (Suppl 16), 27–32. Fox, J., Weisberg, S., 2020. An R Companion to Applied Regression, third ed. Sage, Thousand Oakes, USA. Gingnell, M., Engman, J., Frick, A., Moby, L., Wikstr¨om, J., Fredrikson, M., Sundstr¨om- Poromaa, I., 2013. Oral contraceptive use changes brain activity and mood in women with previous negative affect on the pill-A double-blinded, placebo- controlled randomized trial of a levonorgestrel-containing combined oral contraceptive. Psychoneuroendocrinology 38, 1133–1144. https://doi.org/10.1016/ j.psyneuen.2012.11.006. GIP databank, 2021, Zorginstituut. Accessed on June 2nd, 2021. 〈https://www.gipdata bank.nl/databank?infotype=g&label=00-totaal&tabel=B_01-basis&geg=gebr&item =G03AA〉. Guida, M., Rega, A., Vivone, I., Saccone, G., Sarno, L., Di Carlo, C., Aquino, C.I., Troisi, J., 2020. Variations in sleep associated with different types of hormonal contraceptives. Gynecol. Endocrinol. 36, 166–170. https://doi.org/10.1080/ 09513590.2019.1640204. Disclosures Lamers, F., De Jonge, P., Nolen, W.A., Smit, J.H., Zitman, F.G., Beekman, A.T.F., Penninx, B.W.J.H., 2010. Identifying depressive subtypes in a large cohort study: results from the Netherlands Study of Depression and Anxiety (NESDA). J. Clin. Psychiatry 71, 1582–1589. https://doi.org/10.4088/JCP.09m05398blu. BP has received (non-related) research funding from Boehringer Ingelheim and Jansen Research. All other authors involved in the study do not have any financial or non-financial conflicts to declare. Levine, D.W., Kripke, D.F., Kaplan, R.M., Lewis, M.A., Naughton, M.J., Bowen, D.J., Shumaker, S.A., 2003. Reliability and validity of the Women’s Health Initiative Insomnia Rating Scale. Psychol. Assess. 15, 137–148. https://doi.org/10.1037/ 1040-3590.15.2.137. Appendix A. Supporting information Marra, E., Meijer, S., De Graaf, H., 2020. Changes in young women’s contraceptive use in the Netherlands: findings from three sex under the age of 25 surveys. Genus 76, 1–17. https://doi.org/10.1186/s41118-020-00078-3. Supplementary data associated with this article can be found in the online version at doi:10.1016/j.psyneuen.2021.105390. Nederlandse Vereniging voor Obstetrie en Gynaecologie, 2004. Anovulatie en Kinderwens [WWW Document]. URL 〈https://www.nvog.nl/wp-content/uploads /2017/12/Anovulatie-en-kinderwens-2.0–12-11–2004.pdf〉. Acknowledgements Halbreich, U., Kahn, L.S., 2007. Atypical depression, somatic depression and anxious depression in women: are they gender-preferred phenotypes? J. Affect. Disord. 102, 245–258. https://doi.org/10.1016/j.jad.2006.09.023. l The infrastructure for the NESDA study (www.nesda.nl) is funded through the Geestkracht program of the Netherlands Organisation for Health Research and Development (ZonMw, grant number 10-000- 1002) and financial contributions by participating universities and mental health care organizations (VU University Medical Center, GGZ inGeest, Leiden University Medical Center, Leiden University, GGZ Rivierduinen, University Medical Center Groningen, University of Gro ningen, Lentis, GGZ Friesland, GGZ Drenthe, Rob Giel Onderzoekscen trum). BB is supported by a Veni grant (grant number 91619085, 2018), from the NWO, Netherlands. Hall, K.S., White, K.O.C., Rickert, V.I., Reame, N., Westhoff, C., 2012. Influence of depressed mood and psychological stress symptoms on perceived oral contraceptive side effects and discontinuation in young minority women. Contraception 86, 518–525. https://doi.org/10.1016/j.contraception.2012.04.010. k h´ h fi d d ff d b d h p g j p Hamaker, E.L., Muth´en, B., 2020. The fixed versus random effects debate and how it relates to centering in multilevel modeling. Psychol. Methods 25 (3), 365–379. Joffe, H., Cohen, L.S., Harlow, B.L., 2003. Impact of oral contraceptive pill use on premenstrual mood: predictors of improvement and deterioration. Am. J. Obstet. Gynecol. 189, 1523–1530. https://doi.org/10.1016/S0002-9378(03)00927-X. Keyes, K.M., Cheslack-Postava, K., Westhoff, C., Heim, C.M., Haloossim, M., Walsh, K., Koenen, K., 2013. Association of hormonal contraceptive use with reduced levels of depressive symptoms: a national study of sexually active women in the United States. Am. J. Epidemiol. 178 (9), 1378–1388. Kuznetsova, A., Brockhoff, P.B., Christensen, R.H.B., 2017. lmerTest package: tests in linear mixed effects models. J. Stat. Softw. 82. https://doi.org/10.18637/jss.v082. i13. Baglioni, C., Battagliese, G., Feige, B., Spiegelhalder, K., Nissen, C., Voderholzer, U., Lombardo, C., Riemann, D., 2011. Insomnia as a predictor of depression: a meta- analytic evaluation of longitudinal epidemiological studies. J. Affect. Disord. 135, 10–19. https://doi.org/10.1016/j.jad.2011.01.011. Anderl, C., Li, G., Chen, F.S., 2020. Oral contraceptive use in adolescence predicts lasting vulnerability to depression in adulthood. J. Child Psychol. Psychiatry 61 (2), 148–156. References Nelson, C., 1999. The Composite International Diagnostic Interview (CIDI) web site. Bull. World Health Organ. 77, 614. American Psychiatric Association, 1994. Diagnostic And Statistical Manual of Mental Disorders: DSM-IV. Washington, DC. Novick, J.S., Stewart, J.W., Wisniewski, S.R., Cook, I.A., Manev, R., Nierenberg, A.A., Rosenbaum, J.F., Shores-Wilson, K., Balasubramani, G.K., Biggs, M.M., Zisook, S., Rush, A.J., 2005. Clinical and demographic features of atypical depression in outpatients with major depressive disorder: preliminary findings from STAR*D. J. Clin. Psychiatry 66, 1002–1011. https://doi.org/10.4088/JCP.v66n0807. Anderl, C., Li, G., Chen, F.S., 2020. Oral contraceptive use in adolescence predicts lasting vulnerability to depression in adulthood. J. Child Psychol. Psychiatry 61 (2), 148–156. Paunio, T., Korhonen, T., Hublin, C., Partinen, M., Koskenvuo, K., Koskenvuo, M., Kaprio, J., 2015. Poor sleep predicts symptoms of depression and disability 8 M.W.L. Morssinkhof et al. Psychoneuroendocrinology 133 (2021) 105390 retirement due to depression. J. Affect. Disord. 172, 381–389. https://doi.org/ 10.1016/j.jad.2014.10.002. retirement due to depression. J. Affect. Disord. 172, 381–389. https://doi.org/ 10.1016/j.jad.2014.10.002. patients with experience of adverse mood during treatment with combined oral contraceptives. Contraception 79, 50–55. https://doi.org/10.1016/j. contraception.2008.08.001. Penninx, B.W.J.H., Eikelenboom, M., Giltay, E.J., van Hemert, A.M., Riese, H., Schoevers, R.A., Beekman, A.T.F., 2021. Cohort profile of the longitudinal Netherlands Study of Depression and Anxiety (NESDA) on etiology, course and consequences of depressive and anxiety disorders. J. Affect. Disord. 287. https://do org/10.1016/j.jad.2021.03.026. p Skovlund, C.W., Mørch, L.S., Kessing, L.V., Lidegaard, O., 2016. Association of hormonal contraception with depression. JAMA Psychiatry 73, 1154–1162. https://doi.org/ 10.1001/jamapsychiatry.2016.2387. Toffol, E., Heikinheimo, O., Koponen, P., Luoto, R., Partonen, T.A., 2011. Hormonal contraception and mental health: results of a population-based study. Hum. Reprod. 26 (11), 3085–3093. Prather, A.A., Vogelzangs, N., Penninx, B.W.J.H., 2015. Sleep duration, insomnia, and markers of systemic inflammation: results from the Netherlands Study of Depression and Anxiety (NESDA). J. Psychiatr. Res. 60, 95–102. https://doi.org/10.1016/j. jpsychires.2014.09.018. UN. Population Division, 2019. Contraceptive Use by Method 2019 Data Booklet [WWW Document]. URL 〈https://www.un.org/development/desa/pd/sites/www.un.org.de velopment.desa.pd/files/files/documents/2020/Jan/un_2019_contraceptiveuse bymethod_databooklet.pdf〉. R Core Team, 2020. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. [WWW Document]. URL 〈http://www.r-project.org/index.html〉. Westhoff, C.L., Heartwell, S., Edwards, S., Zieman, M., Stuart, G., Cwiak, C., Davis, A., Robilotto, T., Cushman, L., Kalmuss, D., 2007. Oral contraceptive discontinuation: do side effects matter? Am. J. Obstet. Gynecol. 196 (4), 412-e1. Rush, A.J., Gullion, C.M., Basco, M.R., Jarrett, R.B., Trivedi, M.H., 1996. The inventory of depressive symptomatology (IDS): psychometric properties. Psychol. Med. 26, 477–486. https://doi.org/10.1017/s0033291700035558. References i Wilsmore, B.R., Grunstein, R.R., Fransen, M., Woodward, M., Norton, R., Ameratunga, S., 2013. Sleep habits, insomnia, and daytime sleepiness in a large and healthy community-based sample of New Zealanders. J. Clin. Sleep. Med. 9, 559–566. https://doi.org/10.5664/jcsm.2750. i Schaffir, J., Worly, B.L., Gur, T.L., 2016. Combined hormonal contraception and its effects on mood: a critical review. Eur. J. Contracept. Reprod. Health Care 21, 347–355. https://doi.org/10.1080/13625187.2016.1217327. p g j Worly, B.L., Gur, T.L., Schaffir, J., 2018. The relationship between progestin hormonal contraception and depression: a systematic review. Contraception 97 (6), 478–489. https://doi.org/10.1016/j.contraception.2018.01.010. Schmalenberger, K.M., Tauseef, H.A., Barone, J.C., Owens, S.A., Lieberman, L., Jarczok, M.N., Eisenlohr-Moul, T.A., 2020. How to study the menstrual cycle: practical tools and recommendations. Psychoneuroendocrinology 123, 104895. S h i S h b t S G d J L Ei l h M l T A M lt B d S Zettermark, S., Perez Vicente, R., Merlo, J., 2018. Hormonal contraception increases the risk of psychotropic drug use in adolescent girls but not in adults: A pharmacoepidemiological study on 800 000 Swedish women. PLoS One 13 (3), 0194773. Schweizer-Schubert, S., Gordon, J.L., Eisenlohr-Moul, T.A., Meltzer-Brody, S., Schmalenberger, K.M., Slopien, R., Ditzen, B., 2021. Steroid hormone sensitivity in reproductive mood disorders: on the role of the GABAA receptor complex and stress during hormonal transitions. Front. Med. 7, 479646. Segebladh, B., Borgstr¨om, A., Odlind, V., Bixo, M., Sundstr¨om-Poromaa, I., 2009. Prevalence of psychiatric disorders and premenstrual dysphoric symptoms in
https://openalex.org/W4372294700	https://www.nature.com/articles/s41467-023-38207-z.pdf	English	null	Coordinated regulation of vegetative phase change by brassinosteroids and the age pathway in Arabidopsis	Nature communications	2,023	cc-by	19,078	Article https://doi.org/10.1038/s41467-023-38207-z Coordinated regulation of vegetative phase change by brassinosteroids and the age pathway in Arabidopsis Bingying Zhou 1,2,4, Qing Luo1,2,4, Yanghui Shen2, Liang Wei2, Xia Song2, Hangqian Liao2, Lan Ni3, Tao Shen3, Xinglin Du1, Junyou Han1, Mingyi Jiang3, Shengjun Feng 2 & Gang Wu 2 Vegetative phase change in plants is regulated by a gradual decline in the level of miR156 and a corresponding increase in the expression of its targets, SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes. Gibberellin (GA), jasmonic acid (JA), and cytokinin (CK) regulate vegetative phase change by affecting genes in the miR156-SPL pathway. However, whether other phyto- hormones play a role in vegetative phase change remains unknown. Here, we show that a loss-of-function mutation in the brassinosteroid (BR) biosynthetic gene, DWARF5 (DWF5), delays vegetative phase change, and the defective phenotype is primarily attributable to reduced levels of SPL9 and miR172, and a corresponding increase in TARGET OF EAT1 (TOE1). We further show that GLYCOGEN SYNTHASE KINASE3 (GSK3)-like kinase BRASSINOSTEROID INSENSITIVE2 (BIN2) directly interacts with and phosphorylates SPL9 and TOE1 to cause subsequent proteolytic degradation. Therefore, BRs function to stabilize SPL9 and TOE1 simultaneously to regulate vegetative phase change in plants. After germination, higher plants undergo a juvenile and an adult phase of development before they acquire reproductively competence. The juvenile-to-adult phase transition is referred to as vegetative phase change. In Arabidopsis, vegetative phase change is marked by the production of abaxial trichomes on leaf blades, an increase in the leaf length-to-width (L/W) ratio, an increase in the degree of serration of the leaf margin, and a decrease in cell size1–3. during plant development4–6. Another miRNA, miR172, which exhibits a complementary expression pattern to miR156 during development, functions to promote vegetative phase change by repressing a class of AP2-like genes, including APETALA2 (AP2), TOE1, TOE2, TOE3, SCHLAFMUTZE (SMZ), SNARCHZAPFEN (SNZ), and it is a direct tran- scriptional target of SPL9. miR172 and AP2-like genes act speciﬁcally to regulate leaf epidermal trait development by affecting the expression of GLABRA1 (GL1) during vegetative phase change5,7. The Arabidopsis genome encodes sixteen SPL genes, among which ten are targeted by miR156. These ten SPL genes can be further divided into three groups based on their function in plant development. The ﬁrst group, including SPL2, SPL9, SPL10, SPL11, SPL13, and SPL15, plays roles in both vegetative phase change and ﬂowering;8 SPL3, SPL4, and SPL5 con- stitute the second group to regulate ﬂoral meristem identity;9 SPL6, during plant development4–6. 1College of Plant Sciences, Jilin University, Jilin 130062, China. 2The State Key Laboratory of Subtropical Silviculture, The Key Laboratory of Quality and Safety Control for Subtropical Fruit and Vege-table, Ministry of Agriculture and Rural Affairs, College of Horticultural Science, Zhejiang A&F University, Hangzhou 311300 Zhejiang, China. 3College of Life Sciences, Nanjing Agricultural University, Nanjing, China. 4These authors contributed equally: Bingying Zhou, Qing Luo. e-mail: 20170039@zafu.edu.cn; wugang@zafu.edu.cn A mutant defective in BR biosynthesis exhibits a delayed vege- tative phase change phenotype A mutant defective in BR biosynthesis exhibits a delayed vege- tative phase change phenotype p g p yp The miR156-SPL age pathway is the master regulatory pathway con- trolling vegetative phase change in plants5. However, how different pathways integrate into the age pathway to regulate vegetative phase change remains largely unexplored in plants. In an attempt to identify more factors involved in this pathway, we screened a selfed M2 gen- eration from an EMS-mutagenized wild-type (WT) Col-0 Arabidopsis population. We identiﬁed a mutant exhibiting a delayed vegetative phase change phenotype, which we initially named del2. In short days, WT plants produced abaxial trichomes on leaf 8.3, whereas del2 pro- duced signiﬁcantly later abaxial trichomes on leaf 10.8 (Fig. 1a). Com- pared with WT, del2 had a signiﬁcantly slower leaf initiation rate (Fig. 1b), and its leaves were also much rounder and smaller (Fig. 1a, c), typical characteristics of juvenile traits. In addition, del2 also exhibited pleiotropic defects, including an overall smaller and dwarfed stature with dark green leaves, and developmental retardation (Fig. 1a–c). Till now, three phytohormones, GA, JA, and CK have been shown to regulate vegetative phase change in Arabidopsis, maize, and rice2,16–19. The effect of GA on vegetative phase change is mediated by the DELLA proteins, a family of transcriptional repressors that are negatively regulated by GA20. In the absence of GA, the DELLA proteins interact with the SPL9 protein to interfere with its transcriptional activity21. JA promotes juvenile development possibly through acti- vating the expression of miR156 in maize and rice via unknown mechanisms17,18. Cytokinin regulates vegetative phase change in Ara- bidopsis by affecting gene expression in the miR172/TOE1-TOE2 mod- ule downstream of the SPL genes19. We crossed del2 to Landsberg erecta (Ler) to generate a segre- gating F2 population to map the del2 mutation. We narrowed down the del2 mutation to a region between markers F14I3 and F11F12 on chro- mosome I (Fig. 1d). We started to focus on AT1G50430 (DWF5), a gene required for BR biosynthesis, in this region because mutants in the DWF5 gene are usually smaller in overall stature with dark green leaf color34. Therefore, we sequenced the DWF5 gene in del2. Coordinated regulation of vegetative phase change by brassinosteroids and the age pathway in Arabidopsis Another miRNA, miR172, which exhibits a complementary expression pattern to miR156 during development, functions to promote vegetative phase change by repressing a class of AP2-like genes, including APETALA2 (AP2), TOE1, TOE2, TOE3, SCHLAFMUTZE (SMZ), SNARCHZAPFEN (SNZ), and it is a direct tran- scriptional target of SPL9. miR172 and AP2-like genes act speciﬁcally to regulate leaf epidermal trait development by affecting the expression of GLABRA1 (GL1) during vegetative phase change5,7. The Arabidopsis genome encodes sixteen SPL genes, among which ten are targeted by miR156. These ten SPL genes can be further divided into three groups based on their function in plant development. The ﬁrst group, including SPL2, SPL9, SPL10, SPL11, SPL13, and SPL15, plays roles in both vegetative phase change and ﬂowering;8 SPL3, SPL4, and SPL5 con- stitute the second group to regulate ﬂoral meristem identity;9 SPL6, Previous studies have shown that vegetative phase change is regulated by the evolutionarily conserved miR156-SPL pathway, also known as the age pathway, in plants, in which miR156 functions to promote juvenile development, whereas most of its targets, SPL genes, function to accelerate adult development. miR156 is highly accumu- lated in the juvenile phase, while its abundance decreases gradually as plants age. Correspondingly, the expression of SPL genes increases 1College of Plant Sciences, Jilin University, Jilin 130062, China. 2The State Key Laboratory of Subtropical Silviculture, The Key Laboratory of Quality and Safety Control for Subtropical Fruit and Vege-table, Ministry of Agriculture and Rural Affairs, College of Horticultural Science, Zhejiang A&F University, Hangzhou 311300 Zhejiang, China. 3College of Life Sciences, Nanjing Agricultural University, Nanjing, China. 4These authors contributed equally: Bingying Zhou, Qing Luo. e-mail: 20170039@zafu.edu.cn; wugang@zafu.edu.cn Nature Communications\| (2023) 14:2608 Nature Communications\| (2023) 14:2608 1 https://doi.org/10.1038/s41467-023-38207-z Article belonging to the third group, may be critical for certain physiological processes8. study identiﬁed a PTM pattern in the SPL9 and TOE1 proteins by BIN2 necessary for SPL9 and TOE1 stabilization and normal function, and reveals an unidentiﬁed role of the BR signaling pathway in vegetative phase change by interacting with the age pathway. p miR156 represses SPL gene expression by transcript cleavage and translational repression8,10. In addition to being regulated by miR156, post translational modiﬁcations (PTMs) of the SPL proteins have also been shown to be crucial for their function. O-fucosyltransferase SPINDLY (SPY) interacts with SPL15 directly, and O-glycosylation of SPL15 by SPY inhibits SPL15 activity to regulate developmental transitions11. A mutant defective in BR biosynthesis exhibits a delayed vege- tative phase change phenotype The sequen- cing result indicated that there was a G-to-A substitution at the 1848th base in the DWF5 coding region, which resulted in a replacement of the tryptophon (TGG) by a stop codon (TGA) to cause an early transla- tional termination of the DWF5 protein (Fig. 1e). BRs are a class of plant-speciﬁc steroid hormones that play vital roles in plant growth, development, and stress response22,23. BR is perceived by BRASSINOSTEROID INSENSITIVE1 (BRI) and its co- receptor kinase BRI1-ASSOCIATED RECEPTOR KINASE1 (BAK1) on the cell surface24,25. The BRI1 KINASE INHIBITOR1 (BKI1), a negative reg- ulator of BRI1 signaling, then dissociates from the plasma membrane26. BRI and BAK1 sequentially phosphorylate and activate the BR signaling cascade27. Activated BRI1 phosphorylates two membrane-localized receptor-like cytoplasmic kinases, BRASSINOSTEROID-SIGNALING KINASE1 (BSK1) and CONSTITUTIVE DIFFERENTIAL GROWTH1/ CDG- LIKE (CDG1)28,29, which in turn activates the nucleocytoplasmic phos- phatase BRI1 SUPPRESSOR 1/BSU1-LIKE (BSU1/BSL)30. BSUI then dephosphorylates and inactivates BRASSINOSTEROID INSENSITIVE2 (BIN2), the GSK3-like kinase, leading to its subsequent degradation in a proteasome-dependent manner31,32. The inhibition of BRASSINAZOLE- RESISTANT1 (BZR1) and BRI1-EMS-SUPPRESSOR1 (BES1) or BZR2 by BIN2 is then released, and they are translocated to the nucleus to regulate downstream target genes33. Although BRs have been shown to involve a plethora of different biological processes, whether they play a role in vegetative phase change still remains unknown. To verify if this mutation is responsible for the del2 defective phenotype, we ampliﬁed the genomic sequence of DWF5 containing the promoter, coding, and 3' sequence, and cloned it into an expres- sion vector to generate the pDWF5::DWF5 construct, and transformed the del2 plants. Phenotypic characterization of different primary transformants in short days indicated that the transgenic plants in the del2 background had indistinguishable phenotypes from WT (Fig. 1a). This result suggests that the defective del2 phenotype is truly attri- butable to the mutation in the DWF5 gene. Therefore, we renamed this mutation to dwf5. Since dwf5 is defective in BR biosynthesis, we determined the level of endogenous BRs in dwf5. As expected, the content of brassinolide (BL) in dwf5 was signiﬁcantly lower than that in WT (Fig. 1f). Then we asked if exogenous addition of BR can restore the dwf5 phenotype. We treated WT and dwf5 seedlings with different concentrations of BL, and characterized their vegetative phase change phenotype in short days. Coordinated regulation of vegetative phase change by brassinosteroids and the age pathway in Arabidopsis In rice, OsSPL14 or IDEAL PLANT ARCHITECTURE1 (IPA1), the homolog of Arabidopsis SPL9, interacts with IPA1 INTERACTING PROTIN1 (IPI1) and OsOTUB1 physically, leading to OsSPL14 ubiquiti- nation and subsequent degradation to modulate rice architecture12,13. The phosphorylation status of Ser163 in OsSPL14 alsoaffects its function to balance growth and immunity in rice14. Previously, we identiﬁed some potential phosphorylation sites adjacent to the nuclear locali- zation signal in the Arabidopsis SPL9 protein15, but the function of SPL9 phosphorylation in plant development and how SPL9 is phos- phorylated by upstream factors remain largely unknown. A mutant defective in BR biosynthesis exhibits a delayed vege- tative phase change phenotype 10 nM BL treatment had almost no effect on WT phenotypes, whereas BL treatment of dwf5 plants slightly but signiﬁcantly accelerated the abaxialtrichome production (dwf5-MOCK 10.2 ± 0.6 versus dwf5−10nM BL 9.0 ± 0.7). This result suggests that dwf5 defective phenotype is partially due to the reduced level of BL in plants (Fig. 1g). The inability of BL treatment to fully restore the dwf5 defective phenotype is probably due to the low absorption of exogenously applied BL by plants or other unknown mechanisms. In the case of WT, it is possible that WT already accumulates enough BRs required for normal devel- opment, a further increase in BRs will not alter its normal development. g p g In an attempt to identify more players in the miR156-SPL pathway to regulate vegetative phase change, we performed a forward genetic screen in an M2 population from an ethyl methanesulfonate (EMS)- mutagenized Arabidopsis Columbia-0 (Col-0) seeds. In this work, we identiﬁed a mutant exhibiting a delayed vegetative phase change phenotype, which we named delayed juvenile-to-adult phase transition mutant2 (del2). Map-based cloning indicated that del2 has a mutation in the DWF5 gene encoding the Δ7-sterol reductase, which functions to convert 5-dehydroepisterol to 24-methylenecholesterol (24-MC) in the BR biosynthetic pathway34. Our results indicate that the defective phenotype of dwf5 is mainly attributable to its downregulation of the SPL9 protein and miR172, and a corresponding upregulation of the TOE1 protein. Molecular and biochemical experiments demonstrated that BIN2 physically interacts with SPL9 and TOE1 in vitro and in vivo, and phosphorylates SPL9 and TOE1 to cause their subsequent pro- teolytic degradation and functional sequestration. Therefore, our Nature Communications\| (2023) 14:2608 dwf5 affects the stability and function of SPL9 instead of tran- scripts of genes in the miR156-SPL pathway dwf5 affects the stability and function of SPL9 instead of tran- scripts of genes in the miR156-SPL pathway Next, we asked if the delayed vegetative phase change phenotype of dwf5 is attributable to changes in the expression of genes in the Nature Communications\| (2023) 14:2608 2 2 Fig. 1 \| Loss-of-function mutation in BR biosynthesis delays vegetative phase change in Arabidopsis. a Phenotypic characterization of the del2 mutant. 28-day- old wild type (WT, Col-0), del2, pDWF5::DWF5 del2 2#, 8# transgenic lines were grown in short days. The ﬁrst leaf with abaxial trichomes was scored. Numbers indicate the ﬁrst leaf with abaxial trichomes (n = 25 plants, ±SD). Different letters indicate signiﬁcant difference between genotypes using one-way ANOVA at P < 0.001. Scale bar = 1 cm. b Leaf initiation rate of WT and del2 in short days. Leaf numbers were scored at 4, 8, 12, 16, 20, 24, 28, 32 DAP. DAP, days after planting. Asterisks denote signiﬁcant difference from WT using two-tailed Student’s t-test (P < 0.001, n = 30 plants, ±SD). c The length-to-width (L/W) ratios of leaf 1-11 from 45-day-old WT and del2 in short days. Asterisks denote signiﬁcant difference from region between markers ciw1 and nga280 on chromosome I. in dwf5. Sequencing of DWF5 identiﬁed a G-to-A substitution (shown by the arrow) in the coding region, which caused an termination of the DWF5 gene. Scale bar = 1 KB. f Quantitatio day-old WT and dwf5. Asterisks denote signiﬁcant difference tailed Student’s t-test (P < 0.01, n = 3 biologically independen g Phenotypic characterization of MOCK- and BL-treated 30-da WT and dwf5 were initially grown on 1/2 MS plates containin short days for 10 days, then transferred to 1/2 hogland liquid 10 nM BL in short days. Numbers indicate the ﬁrst leaf with a (n = 15 plants, ±SD). Different letters indicate signiﬁcant diffe Article https://doi.org/10.1038/s414 Article https://doi.org/10.1038/s41467-023-38207-z region between markers ciw1 and nga280 on chromosome I. e The mutation site in dwf5. Sequencing of DWF5 identiﬁed a G-to-A substitution at the 1848th base (shown by the arrow) in the coding region, which caused an early translational termination of the DWF5 gene. Scale bar = 1 KB. f Quantitation of BL levels in 15- day-old WT and dwf5. Asterisks denote signiﬁcant difference from WT using two- tailed Student’s t-test (P < 0.01, n = 3 biologically independent samples, ±SD). dwf5 affects the stability and function of SPL9 instead of tran- scripts of genes in the miR156-SPL pathway These results suggest that SPL9 is subjected to proteasome-dependent degradation, and it is much less stable in dwf5 than in WT. Therefore, BRs function to stabilize SPL9 in vegetative phase change. This con- clusion is also further supported by the result that the addition of BL to pSPL9::3×FLAG-rSPL9 dwf5 plants increased the level of SPL9 greatly at different time points (Fig 2f) Since SPL9 functions as a direct activator of MIR172B5, we asked if the level or the function of SPL9 were affected in dwf5. We ﬁrst determined the level of the SPL9 protein in dwf5. Due to the lack of available SPL9 antibodies, we crossed the pSPL9::3×FLAG-rSPL9 trans- genic line in which a 3×FLAG epitope tag was fused to the miR156 insensitive form of SPL9 under the regulation of its native promoter to dwf55. Using the FLAG antibody, we performed Western blotting ana- lysis to examine the level of the SPL9 protein in the WT and dwf5 background. Western blotting indicated a >4-fold reduction in the SPL9 protein level in dwf5 compared with that in WT (Fig. 2c), sug- gesting that SPL9 was destabilized in dwf5. We further explored the effect of dwf5 on the function of SPL9 by using an inducible expression system based on the posttranscriptional activation of the rat gluco- corticoid receptor (GR)36. GR was fused to the 5' end of miR156 insensitive SPL9 (rSPL9), and this fusion gene was expressed in trans- genic plants under the regulation of its native promoter. pSPL9::GR- rSPL9 was crossed to dwf5, and homozygous pSPL9::GR-rSPL9 dwf5 plants were recovered in the F2 population. Transgenic pSPL9::GR- rSPL9 seeds in WT and dwf5 background were plated on 1/2 MS med- ium, and treated with the synthetic ligand dexamethasone (DEX) or MOCK for 3 h. RNA was extracted and the abundance of MIR172B was assessed by qRT-PCR. As expected, MIR172B expression was sig- niﬁcantly induced by about 2.6-fold upon DEX induction in WT back- ground compared with an about 1.5-fold induction in the dwf5 background (Fig. 2d). Therefore, the incapacity to fully activate the expression of MIR172B demonstrates that the function of SPL9 is par- tially impaired in dwf5, and DWF5 integrates into the miR156-SPL pathway by stabilizing the SPL9 protein and maintaining its function. dwf5 affects the stability and function of SPL9 instead of tran- scripts of genes in the miR156-SPL pathway g Phenotypic characterization of MOCK- and BL-treated 30-day-old WT and dwf5. WT and dwf5 were initially grown on 1/2 MS plates containing 0 or 10 nM BL in short days for 10 days, then transferred to 1/2 hogland liquid medium with 0 or 10 nM BL in short days. Numbers indicate the ﬁrst leaf with abaxial trichomes (n = 15 plants, ±SD). Different letters indicate signiﬁcant difference between genotypes using one-way ANOVA at P < 0.001. Scale bar = 1 cm. All experiments were repeated 3 times biologically. Fig. 1 \| Loss-of-function mutation in BR biosynthesis delays vegetative phase change in Arabidopsis. a Phenotypic characterization of the del2 mutant. 28-day- old wild type (WT, Col-0), del2, pDWF5::DWF5 del2 2#, 8# transgenic lines were grown in short days. The ﬁrst leaf with abaxial trichomes was scored. Numbers indicate the ﬁrst leaf with abaxial trichomes (n = 25 plants, ±SD). Different letters indicate signiﬁcant difference between genotypes using one-way ANOVA at P < 0.001. Scale bar = 1 cm. b Leaf initiation rate of WT and del2 in short days. Leaf numbers were scored at 4, 8, 12, 16, 20, 24, 28, 32 DAP. DAP, days after planting. Asterisks denote signiﬁcant difference from WT using two-tailed Student’s t-test (P < 0.001, n = 30 plants, ±SD). c The length-to-width (L/W) ratios of leaf 1-11 from 45-day-old WT and del2 in short days. Asterisks denote signiﬁcant difference from WT using two-tailed Student’s t-test (P < 0.001, n = 10 plants, ±SD). d The chro- mosomal location of the del2 mutation. The del2 mutation was narrowed down to a Nature Communications\| (2023) 14:2608 3 https://doi.org/10.1038/s41467-023-38207-z Article repression. Therefore, we crossed the miR172 over-expression line Ubi10::172B and toe1 toe2 double mutant to dwf5, respectively. We then characterized the phenotypes of homozygous Ubi10::172B dwf5, toe1 toe2 dwf5, and their corresponding parental lines in short days. dwf5 produced late abaxial trichomes on leaf 11.3, Ubi10::172B and toe1 toe2 produced early abaxial trichomes on leaf 3.8 and 3.6, respectively; whereas Ubi10::172B dwf5 and toe1 toe2 dwf5 produced abaxial tri- chomes on leaf 4.5 and 3.8 (Fig. 3a), signiﬁcantly earlier than dwf5. toe1 toe2 was almost completely epistatic to dwf5 with respect to abaxial trichome production. Restoration of miR172 expression in the dwf5 background could also signiﬁcantly rescue the late vegetative phase change phenotype of dwf5. dwf5 affects the stability and function of SPL9 instead of tran- scripts of genes in the miR156-SPL pathway p g p g To test the signiﬁcance of the downregulation of SPL9 in dwf5, we crossed the pSPL9::rSPL9 line overexpressing a miR156-resistant version of SPL9 under the control of its native promoter to dwf5. We then characterized the vegetative phase change phenotype of the homo- zygous pSPL9::rSPL9 dwf5 line in short days. On average, pSPL9::rSPL9 dwf5 produced slightly but not signiﬁcantly later abaxial trichomes than pSPL9::rSPL9 (1.4 ± 0.7 versus 1.0 ± 0.0), but remarkably earlier than dwf5 did (Fig. 3a). A detailed phenotypic characterization of pSPL9::rSPL9 dwf5 plants revealed an interesting phenotype: 100% of pSPL9::rSPL9 plants produced abaxial trichomes on leaf 1, whereas 75.7%, 15.7%, and 8.6% of pSPL9::rSPL9 dwf5 plants produced abaxial trichomes on leaf 1, 2, and 3, respectively (Fig. 3b). Moreover, pSPL9::rSPL9 dwf5 plants also had a signiﬁcantly reduced number of abaxial trichomes on the ﬁrst leaf than pSPL9::rSPL9 plants (Fig. 3c). Last but not least, the ﬁrst leaves of pSPL9::rSPL9 dwf5 plants had a signiﬁcantly smaller L/W ratio than those of pSPL9::rSPL9 plants, which is similar to that of WT (Fig. 3d). These genetic data demonstrate that DWF5 is required for SPL9 to promote vegetative phase change, and the function of SPL9 is sequestered in dwf5, which is consistent with the molecular data as shown in Fig. 2d. We also generated double mutant between dwf5 and spl9-4. spl9-4 dwf5 had an enhanced vegetative phase change phenotype than spl9-4 and dwf5 with respect to abaxial trichome production (Fig. 3a). Together with the result that SPL9 is reduced in dwf5, these results suggest that DWF5 functions to regulate vegetative phase change through SPL9 dependent and independent pathways. To further understand if the reduced level of the SPL9 protein is attributable to the reduced stability of SPL9 in dwf5, we treated pSPL9::3×FLAG-rSPL9 plants with MG132 and/or cycloheximide (CHX). As expected, the SPL9 protein level was lower in dwf5 than that in WT in the DMSO-treated samples, CHX treatment alone led to a further reduction in SPL9 in dwf5 than in WT. However, MG132 treatment along or in combination with CHX inhibited SPL9 degradation (Fig. 2e). These results suggest that SPL9 is subjected to proteasome-dependent degradation, and it is much less stable in dwf5 than in WT. Therefore, BRs function to stabilize SPL9 in vegetative phase change. dwf5 affects the stability and function of SPL9 instead of tran- scripts of genes in the miR156-SPL pathway This con- clusion is also further supported by the result that the addition of BL to pSPL9::3×FLAG-rSPL9 dwf5 plants increased the level of SPL9 greatly at different time points (Fig. 2f). p ys ca y te acts t S BIN2, a GSK3-like kinase, acts as one of the central hubs in the BR signaling pathway to negatively regulate BR signaling by interacting with and phosphorylating its substrates37–39. To investigate if the reduced level of SPL9 in dwf5 is mediated by BIN2, we ﬁrst tested if SPL9 interacts with BIN2 using a yeast two-hybrid system. We fused the full-length of SPL9 coding sequence (CDS) to the GAL4 activation domain to generate the prey vector AD-SPL9, and fused the BIN2 CDS to the GAL4 DNA binding domain to generate the bait vector BD-BIN2. These two vectors were then co-transformed into the yeast competent cells, and plated on DDO (SD/-Leu/-Trp) and QDO (SD/-Leu/-Trp/-Ade/- His) media. As shown in Fig. 4a, BIN2 interacted with SPL9 in the yeast dwf5 affects the stability and function of SPL9 instead of tran- scripts of genes in the miR156-SPL pathway These results, together with the down- regulation of miR172 in dwf5, suggest that miR172 functions down- stream of DWF5 with respect to leaf epidermal trait development during vegetative phase change, and the reduction of miR172 in dwf5 is partially responsible for the late abaxial trichome phenotype of dwf5. A gradual change in leaf shape constitutes an important morphological marker associated with vegetative phase change. In contraryto distinct abaxial trichome phenotypes as manifested by dwf5, Ubi10::172B dwf5, and toe1 toe2 dwf5, leaf shape of Ubi10::172B dwf5 and toe1 toe2 dwf5 resembled that of dwf5 to a greater extent. This result suggests that dwf5 is epistatic to miR172 and TOE1/TOE2 with respect to leaf shape development during vegetative phase change, and BRs contribute to leaf shape development by affecting genes other than miR172 and TOE1/TOE2. This is also in accordance with our previous result that miR172 and TOE1/TOE2 only contribute to leaf epidermal trait devel- opment in vegetative phase change5. miR156-SPL pathway. We measured the expression of some key genes in the miR156-SPL pathway using RNA from 12-day-old seedlings of WT and dwf5 mutant in short days. Quantitative reverse transcription-PCR (qRT-PCR) revealed that levels of mature miR156, SPL3, SPL9, SPL13, TOE1, and TOE2 were similar between WT and dwf5 (Fig. 2a, b). How- ever, the abundance of MIR172B and mature miR172 was both reduced remarkably in dwf5 (Fig. 2b). It is not surprising to see that the expression of the downstream TOE1/TOE2 in dwf5 remained indis- tinguishable from that in WT because miR172 represses AP2-like genes mainly by translational repression35. These results imply that the delayed vegetative phase change phenotype of dwf5 might result from the reduced levels of miR172. miR156-SPL pathway. We measured the expression of some key genes in the miR156-SPL pathway using RNA from 12-day-old seedlings of WT and dwf5 mutant in short days. Quantitative reverse transcription-PCR (qRT-PCR) revealed that levels of mature miR156, SPL3, SPL9, SPL13, TOE1, and TOE2 were similar between WT and dwf5 (Fig. 2a, b). How- ever, the abundance of MIR172B and mature miR172 was both reduced remarkably in dwf5 (Fig. 2b). It is not surprising to see that the expression of the downstream TOE1/TOE2 in dwf5 remained indis- tinguishable from that in WT because miR172 represses AP2-like genes mainly by translational repression35. These results imply that the delayed vegetative phase change phenotype of dwf5 might result from the reduced levels of miR172. dwf5 affects the stability and function of SPL9 instead of tran- scripts of genes in the miR156-SPL pathway Since SPL9 functions as a direct activator of MIR172B5, we asked if the level or the function of SPL9 were affected in dwf5. We ﬁrst determined the level of the SPL9 protein in dwf5. Due to the lack of available SPL9 antibodies, we crossed the pSPL9::3×FLAG-rSPL9 trans- genic line in which a 3×FLAG epitope tag was fused to the miR156 insensitive form of SPL9 under the regulation of its native promoter to dwf55. Using the FLAG antibody, we performed Western blotting ana- lysis to examine the level of the SPL9 protein in the WT and dwf5 background. Western blotting indicated a >4-fold reduction in the SPL9 protein level in dwf5 compared with that in WT (Fig. 2c), sug- gesting that SPL9 was destabilized in dwf5. We further explored the effect of dwf5 on the function of SPL9 by using an inducible expression system based on the posttranscriptional activation of the rat gluco- corticoid receptor (GR)36. GR was fused to the 5' end of miR156 insensitive SPL9 (rSPL9), and this fusion gene was expressed in trans- genic plants under the regulation of its native promoter. pSPL9::GR- rSPL9 was crossed to dwf5, and homozygous pSPL9::GR-rSPL9 dwf5 plants were recovered in the F2 population. Transgenic pSPL9::GR- rSPL9 seeds in WT and dwf5 background were plated on 1/2 MS med- ium, and treated with the synthetic ligand dexamethasone (DEX) or MOCK for 3 h. RNA was extracted and the abundance of MIR172B was assessed by qRT-PCR. As expected, MIR172B expression was sig- niﬁcantly induced by about 2.6-fold upon DEX induction in WT back- ground compared with an about 1.5-fold induction in the dwf5 background (Fig. 2d). Therefore, the incapacity to fully activate the expression of MIR172B demonstrates that the function of SPL9 is par- tially impaired in dwf5, and DWF5 integrates into the miR156-SPL pathway by stabilizing the SPL9 protein and maintaining its function. To further understand if the reduced level of the SPL9 protein is attributable to the reduced stability of SPL9 in dwf5, we treated pSPL9::3×FLAG-rSPL9 plants with MG132 and/or cycloheximide (CHX). As expected, the SPL9 protein level was lower in dwf5 than that in WT in the DMSO-treated samples, CHX treatment alone led to a further reduction in SPL9 in dwf5 than in WT. However, MG132 treatment along or in combination with CHX inhibited SPL9 degradation (Fig. 2e). Downregulation of miR172 is partially responsible for the dwf5 vegetative phase change phenotype 15-day-old pSPL9::GR-rSPL9 and pSPL9::GR-rSPL9 dwf5 plants grown on 1/2 MS medium in short days were treated with MOCK or dexamethasone (DEX) for 3 h, and total RNA was isolated for qRT-PCR analysis. Data are means ± SD from a representative experiment with three technical replicates for each sample. Asterisk denotes signiﬁcant difference using one-way ANOVA; p < 0.05; P < 0.0 *P < 0.001. e Destabilization of the SPL9 protein in dwf5 is proteasome dependent.15-day-old pSPL9::3×FLAG-rSPL9 and pSPL9::3×FLAG-rSPL9 dw were treated with 50 µM MG132 and/or 100 mM CHX for 1 h in short days. protein was extracted and detected by Western blotting using an anti-FL anti-ACTIN antibody, respectively. f BL treatment of dwf5 increases SPL9 lation. 15-day-old pSPL9::3×FLAG-rSPL9 dwf5 plants in short days were tre MOCK or 1 µM BL for 1 h, and total protein was extracted and detected by blotting using an anti-FLAG and anti-ACTIN antibody, respectively. Num between two blots denote the relative normalized value for each sample intensity of each sample was ﬁrst normalized to the Rubisco band or the sponding ACTIN band (c, e, f), then the resultant value was normalized ag value of FLAG-rSPL9 (c), FLAG-rSPL9 DMSO (e), and FLAG-rSPL9 dwf5 0 h intensity was determined using Image J. All experiments were repeated 3 biologically. Fig. 2 \| dwf5 destabilizes the SPL9 protein and sequesters its function instead of ff ti g iR156 d SPL g i b Th i l l f iR156 Fig. 2 \| dwf5 destabilizes the SPL9 protein and sequesters its function instead of affecting miR156 and SPL gene expression. a, b The expression levels of miR156, SPL3, SPL9, SPL13 (a), MIR172B, miR172, TOE1 and TOE2 (b) in 12-day-old WT and dwf5 in short days. Data are means ± SD from a representative experiment with two technical replicates for each sample. Asterisk denotes signiﬁcant difference from WT using Student’s t-test at P < 0.01. c dwf5 reduces the SPL9 protein level. The SPL9 protein in pSPL9::3×FLAG-rSPL9 and pSPL9::3×FLAG-rSPL9 dwf5 transgenic lines was detected by Western blotting using an anti-FLAG antibody. The Rubisco protein was stained with ponceau as the protein loading control, WT was served as the negative control. Numbers between two blots denote the relative normalized value for each sample. d dwf5 sequesters SPL9 function to activate the expression of MIR172B. Downregulation of miR172 is partially responsible for the dwf5 vegetative phase change phenotype We explored the possibility of if the reduced level of miR172 is responsible for the delayed vegetative phase change phenotype in dwf5. miR172 represses the expression of a class of AP2-like genes, including TOE1, TOE2, TOE3, AP2, SMZ, and SNZ. Plants doubly mutant for TOE1 and TOE2 resemble the phenotype of miR172 overexpression line5. Even though levels of TOE1/TOE2 transcripts remained similar between WT and dwf5 (Fig. 2b), it is possible that the TOE1/TO2 pro- teins levels are elevated in dwf5 due to the reduced level of miR172 that functions to repress AP2-like gene expression through translational Nature Communications\| (2023) 14:2608 4 Fig 2 \| dwf5 destabilizes the SPL9 protein and sequesters its function instead of denotes signiﬁcant difference using one way ANOVA; p < 0 05; ** Article https://doi.org/10.1038/s41467 Article https://doi.org/10.1038/s41467-023-38207-z Fig. 2 \| dwf5 destabilizes the SPL9 protein and sequesters its function instead of affecting miR156 and SPL gene expression. a, b The expression levels of miR156, S 3 S 9 S 13 ( ) 1 2 i 1 2 TO 1 d TO 2 (b) i 12 d ld T d denotes signiﬁcant difference using one-way ANOVA; p < 0.05; P < 0.01; *P < 0.001. e Destabilization of the SPL9 protein in dwf5 is proteasome- d d 1 d ld S 9 3 AG S 9 d S 9 3 AG S 9 d f l Fig. 2 \| dwf5 destabilizes the SPL9 protein and sequesters its function instead of affecting miR156 and SPL gene expression. a, b The expression levels of miR156, SPL3, SPL9, SPL13 (a), MIR172B, miR172, TOE1 and TOE2 (b) in 12-day-old WT and dwf5 in short days. Data are means ± SD from a representative experiment with two technical replicates for each sample. Asterisk denotes signiﬁcant difference from WT using Student’s t-test at P < 0.01. c dwf5 reduces the SPL9 protein level. The SPL9 protein in pSPL9::3×FLAG-rSPL9 and pSPL9::3×FLAG-rSPL9 dwf5 transgenic lines was detected by Western blotting using an anti-FLAG antibody. The Rubisco protein was stained with ponceau as the protein loading control, WT was served as the negative control. Numbers between two blots denote the relative normalized value for each sample. d dwf5 sequesters SPL9 function to activate the expression of MIR172B. Downregulation of miR172 is partially responsible for the dwf5 vegetative phase change phenotype Article https://doi.org/10.1038/s41467-023- Article https://doi.org/10.1038/s41467-023-38207-z ±SD). c Abaxial trichome number on the ﬁrst leaf of pSPL9::rSPL9 and pSPL9::rSPL9 dwf5. Asterisks denote signiﬁcant difference from pSPL9::rSPL9 using two-tailed Student’s t-test at P < 0.001 (n = 10 plants, ±SD). d Leaf length/ width ratio of the ﬁrst leaf from 45-day-old WT, dwf5, pSPL9::rSPL9, and pSPL9::rSPL9 dwf5. Different letters indicate signiﬁcant difference between genotypes using one-way ANOVA at P < 0.01 (n = 10 plants, ±SD). All experi- ments were repeated 3 times biologically. ±SD). c Abaxial trichome number on the ﬁrst leaf of pSPL9::rSPL9 and pSPL9::rSPL9 dwf5. Asterisks denote signiﬁcant difference from pSPL9::rSPL9 using two-tailed Student’s t-test at P < 0.001 (n = 10 plants, ±SD). d Leaf length/ width ratio of the ﬁrst leaf from 45-day-old WT, dwf5, pSPL9::rSPL9, and pSPL9::rSPL9 dwf5. Different letters indicate signiﬁcant difference between genotypes using one-way ANOVA at P < 0.01 (n = 10 plants, ±SD). All experi- ments were repeated 3 times biologically. Fig. 3 \| Genetic interaction of SPL9 and miR172 with dwf5. a Phenotype of 25- day-old WT, dwf5, spl9-4, spl9-4 dwf5, pSPL9::rSPL9, pSPL9::rSPL9 dwf5, Ubi10::MIR172B, Ubi10::172B dwf5, toe1 toe2, and toe1 toe2 dwf5 plants grown in short days. Numbers indicate the ﬁrst leaf with abaxial trichomes (n = 25 plants, ±SD). Different letters indicate signiﬁcant difference between genotypes using one-way ANOVA at P < 0.01. Scale bar = 1 cm. b The percentage of leaf position with abaxial trichomes in pSPL9::rSPL9 and pSPL9::rSPL9 dwf5 (n = 90 plants, Ubi10::MIR172B, Ubi10::172B dwf5, toe1 toe2, and toe1 toe2 dwf5 plants grown in short days. Numbers indicate the ﬁrst leaf with abaxial trichomes (n = 25 plants, ±SD). Different letters indicate signiﬁcant difference between genotypes using one-way ANOVA at P < 0.01. Scale bar = 1 cm. b The percentage of leaf position with abaxial trichomes in pSPL9::rSPL9 and pSPL9::rSPL9 dwf5 (n = 90 plants, demonstrated that BIN2 and SPL9 interact with each other in vivo in Arabidopsis cells. To conﬁrm if BIN2 interacts with SPL9 in vitro, we performed a protein pull-down assay. We ﬁrst expressed the Glu- tathione S-Transferase (GST), GST-BIN2, and 6×His-SPL9 proteins in E. coli, then we used glutathione-agarose beads to bind the GST and GST- BIN2 proteins, and incubated them with His-SPL9 for 4 h in vitro. Last, we collected and boiled the beads to perform Western blotting using an anti-His and an anti-GST antibody, respectively. Downregulation of miR172 is partially responsible for the dwf5 vegetative phase change phenotype 15-day-old pSPL9::GR-rSPL9 and pSPL9::GR-rSPL9 dwf5 plants grown on 1/2 MS medium in short days were treated with MOCK or dexamethasone (DEX) for 3 h, and total RNA was isolated for qRT-PCR analysis. Data are means ± SD from a representative experiment with three technical replicates for each sample. Asterisk denotes signiﬁcant difference using one-way ANOVA; p < 0.05; P < 0.01; *P < 0.001. e Destabilization of the SPL9 protein in dwf5 is proteasome- dependent.15-day-old pSPL9::3×FLAG-rSPL9 and pSPL9::3×FLAG-rSPL9 dwf5 plants were treated with 50 µM MG132 and/or 100 mM CHX for 1 h in short days. The total protein was extracted and detected by Western blotting using an anti-FLAG and anti-ACTIN antibody, respectively. f BL treatment of dwf5 increases SPL9 accumu- lation. 15-day-old pSPL9::3×FLAG-rSPL9 dwf5 plants in short days were treated with MOCK or 1 µM BL for 1 h, and total protein was extracted and detected by Western blotting using an anti-FLAG and anti-ACTIN antibody, respectively. Numbers between two blots denote the relative normalized value for each sample. The intensity of each sample was ﬁrst normalized to the Rubisco band or the corre- sponding ACTIN band (c, e, f), then the resultant value was normalized again to the value of FLAG-rSPL9 (c), FLAG-rSPL9 DMSO (e), and FLAG-rSPL9 dwf5 0 h (f). Band intensity was determined using Image J. All experiments were repeated 3 times biologically. Nature Communications\| (2023) 14:2608 5 5 Fig. 3 \| Genetic interaction of SPL9 and miR172 with dwf5. a Phenotype of 25- day-old WT, dwf5, spl9-4, spl9-4 dwf5, pSPL9::rSPL9, pSPL9::rSPL9 dwf5, Ubi10::MIR172B, Ubi10::172B dwf5, toe1 toe2, and toe1 toe2 dwf5 plants grown in short days. Numbers indicate the ﬁrst leaf with abaxial trichomes (n = 25 plants, ±SD). Different letters indicate signiﬁcant difference between genotypes using one-way ANOVA at P < 0.01. Scale bar = 1 cm. b The percentage of leaf position with abaxial trichomes in pSPL9::rSPL9 and pSPL9::rSPL9 dwf5 (n = 90 plants, ±SD). c Abaxial trichome number on the ﬁrst leaf of pSPL9::rSPL9 and pSPL9::rSPL9 dwf5. Asterisks denote signiﬁcant difference from pSPL9 using two-tailed Student’s t-test at P < 0.001 (n = 10 plants, ±SD). d Lea width ratio of the ﬁrst leaf from 45-day-old WT, dwf5, pSPL9::rSPL9, a pSPL9::rSPL9 dwf5. Different letters indicate signiﬁcant difference be genotypes using one-way ANOVA at P < 0.01 (n = 10 plants, ±SD). All e ments were repeated 3 times biologically. Downregulation of miR172 is partially responsible for the dwf5 vegetative phase change phenotype Western blotting indicated that BIN2 also interacts with SPL9 in vitro (Fig. 4c). As a two-hybrid system. We then performed a bimolecular ﬂuorescence complementation (BiFC) assay to examine if BIN2 interacts with SPL9 in vivo. BIN2 was fused to the C-terminal of yellow ﬂuorescent protein (YFP) (BIN2-cYFP), and SPL9 was fused to the N-terminal of YFP (SPL9- nYFP). We co-transformed these two vectors into WT protoplast iso- lated using a Tape-Arabidopsis Sandwich method40. YFP ﬂuorescence was observed in the transformed cell nuclei overlapped with the nuclei stained by the Hoechst 33342 solution, whereas no ﬂuorescence was detected in the negative control samples (Fig. 4b). This result Nature Communications\| (2023) 14:2608 6 Article https://doi.org/10.1038/s41467-023-38207-z https://doi.org/10.1038/s41467 further step to conﬁrm the interaction between BIN2 and SPL9 in planta, we performed a coimmunoprecipitation (CoIP) assay. We co- expressed SPL9-FLAG and BIN2-GFP fusion proteins in tobacco (Nicotiana benthamiana) leaves, and incubated the total protein with protein afﬁnity gel conjugated with the anti-FLAG antibody. Then we performed Western blotting to detect the eluate with an anti-FLAG and an anti-GFP antibody, respectively (Fig. 4d). The result showed that SPL9 and BIN2 were coimmunoprecipitated. Taken together, our results demonstrate that BIN2 physically interacts with SPL9 both in vitro and in vivo. Fig. 4 \| BIN2 physically interacts with SPL9. a BIN2 interacts with SPL9 in yeast. BIN2 was fused to pGAD (AD), and SPL9 was fused to pGBK (BD). AD and BD empty vectors were used as the negative controls. DDO (SD/-Leu/-Trp), QDO (SD/-Leu/- Trp/-Ade/-His). b BIN2 interacts with SPL9 in vivo in the nucleus as shown by bimolecular ﬂuorescence complementation (BiFC) assay. BIN2 was fused to cYFP, and SPL9 was fused to nYFP. The recombinant plasmids were transformed into the WT Arabidopsis protoplast. The nucleus was stained with hoechest 33342. Scale bar = 10 μm. c BIN2 interacts with SPL9 in vitro in a pull-down assay. SPL9-His was incubated with GST or GST-BIN2 protein, and the proteins immunoprecipitated with glutathione-agarose beads were detected with an anti-His and an anti-GST antibody, respectively. d BIN2 interacts with SPL9 in vivo in an CoIP assay. Total protein was extracted from Nicotiana benthamiana leaves inﬁltrated with Agro- bacterium containing 35 S::3×FLAG/35 S::3×FLAG-rSPL9, and 35 S::BIN2-GFP/ 35 S::3×FLAG-rSPL9 combinations, and incubated with anti-FLAG beads. Coimmu- noprecipitated proteins were detected with an anti-FLAG and an anti-GFP antibody, respectively. All experiments were repeated 3 times biologically. BIN2-mediated phosphorylation of SPL9 is required for its function in vegetative phase change g p g Since BIN2 physically interacts with SPL9, it is possible that BIN2 phosphorylates SPL9 to cause its subsequent degradation or func- tional sequestration. Protein sequence analysis of SPL9 uncovered a GSK3 kinase classical phosphorylation motif of (T/S)-X-X-X-(T/S) (T/S denotes Thr/Ser; X denotes any other amino acids) in the conserved SBP domain (Fig. 5a). To investigate if SPL9 is phosphorylated by BIN2 in vitro, we puriﬁed the bacterium-expressed GST-BIN2 and 6×His- SPL9 fusion proteins, and conducted an in vitro kinase assay in an SDS- PAGE gel containing the phos-tag reagent. Western blotting using an anti-His antibody showed a lagged 6×His-SPL9 band in the phos-tag gel (Fig. 5b), suggesting that SPL9 was phosphorylated by BIN2 in vitro. To robustly establish that SPL9 is a substrate of BIN2, we performed an in vitro kinase assay. We puriﬁed the bacterium-expressed GST-BIN2, 6×His-SPL9 (T) protein in which the 102nd and 106th amino acids are the wild-type threonine, and 6×His-SPL9 (A) in which both the 102nd and 106th threonines were replaced with an alanine. When GST-BIN2 was incubated with 6×His-SPL9 (T) and radiolabeled ATP, autoradiographs showed two bands with the top band corresponding to the auto- phosphorylated GST-BIN2 and the lower band corresponding to the phosphorylated His-SPL9 (Fig. 5c). However, when His-SPL9 (A) was incubated with GST-BIN2, the lower band corresponding to His-SPL9 in the autoradiograph almost disappeared (Fig. 5c). This result further demonstrates that SPL9 is a substrate of BIN2, and the motif of T-P-K-V- T in the SPL9 protein is critical for its phosphorylation by BIN2. BIN2 physically interacts with TOE1 In short days, the bin2-3 bil1 bil2 mutant produced abaxial trichomes on leaf 10.0, which was signiﬁcantly later than WT with abaxial tri- chomes on leaf 6.6 (Supplementary Fig. 3a). This result seems to be contradictory to the elevated level SPL9 in bin2-3 bil1 bil2 since SPL9 functions to promote vegetative phase change. This puzzle prompted us to ask if BIN2 also functions to regulate genes other than SPL9 to regulate vegetative phase change. Because loss-of-function mutations in TOE1 and TOE2 could almost fully restore the late abaxial trichome phenotype of dwf5 (Fig. 3a); therefore, we started to investigate if BIN2 could also interact with TOE1. Article Article We then asked if the reduced level of SPL9 in dwf5 is due to the action of the elevated level of BIN2 since dwf5 alleles are defective in the Δ7 reduction step in the BR biosynthetic pathway34. We ﬁrst examined if the level of BIN2 is elevated in dwf5. To do this, we fused the BIN2 CDS sequence to eGFP, and put it under the control of the BIN2 native promoter to generate the pBIN2::eGFP-BIN2 construct. We then transformed WT, and generated a stable transgenic pBIN2::eGFP- BIN2 line with a single T-DNA insertion in the progeny. We also crossed this line to dwf5 to generate a homozygous pBIN2::eGFP-BIN2 dwf5 line. Western blotting using an anti-GFP antibody indicated that the BIN2 protein level was elevated in the dwf5 background in contrast to the WT background (Supplementary Fig. 1a). To further understand if the elevated level of BIN2 contributes to the dwf5 vegetative phase change phenotype, we germinated WT and dwf5 seeds on 1/2 MS medium supplemented with 10 mM LiCl, a GSK3-like inhibitor41,42, and char- acterized their corresponding phenotypes in short days. Both mock and LiCl-treated WT produced abaxial trichomes on about leaf 8.0, whereas LiCl treatment signiﬁcantly accelerated the production of abaxial trichomes in dwf5 (LiCl 9.4 ± 0.7 versus Mock 11.7 ± 0.7) (Sup- plementary Fig. 1b). These results suggest that the delayed vegetative phase change phenotype in dwf5 is partially attributable to the ele- vated level of the BIN2 protein. function, whereas non-phosphorylation form of SPL9 signiﬁcantly enhanced its function to promote vegetative phase change. To understand how SPL9 phosphorylation affects vegetative phase change phenotype and the protein levels, we ﬁrst generated homo- zygous lines with a single T-DNA insertion in progenies from these transgenic lines, and determined the transcript level of SPL9 in dif- ferent homozygous lines. We then characterized the phenotype of these lines with comparable SPL9 transcript levels (Fig. 5e, f). Among them, T-A 2# and T-A 6# accumulated relatively more SPL9 protein than T-T 1# and T-T 10#, whereas T-D 4#, T-D 8# and T-D 50# had the lowest level of the SPL9 protein (Fig. 5g), which is also consistent with the phenotypic characterization result that the rSPL9-DPKVD trans- genic plants produced much later abaxial trichomes on leaves 3–5 with much rounder ﬁrst leaves (Fig. 5e, h). Article Within rSPL9-APKVA (T-A) and rSPL9-TPKVT (T-T) transgenic plants that produced abaxial tri- chomes on leaf 1, rSPL9-APKVA (T-A) produced the ﬁrst leaves with a slightly bigger L/W ratio than rSPL9-TPKVT (T-T) did (Fig. 5e, h). These results imply that BIN2-mediated phosphorylation of the TPKVT motif in the conserved SBP domain destabilizes the SPL9 protein and sequesters its function, and this mode of regulation is critical for SPL9 function to promote vegetative phase change. To understand how SPL9 is phosphorylated by BIN2 in vivo, we performed a liquid chromatography-mass spectrometry (LC-MS/MS) analysis. We ﬁrst transformed the wild-type Wassilewskija (Ws) and the bin2-3bil1bil2 mutant protoplast with the pSPL9::3×FLAG-rSPL9 con- struct, then we immunoprecipitated the SPL9 protein and analyzed the status of SPL9 phosphorylation with LC-MS/MS. LC-MS/MS indicated that the 102nd threonine was phosphorylated in Ws-0, but not in the bin2-3bil1bil2 mutant (Supplementary Fig. 2a). Interestingly, the 102nd threonine is exactly located in the GSK3 kinase classical phosphor- ylation motif of T-P-K-V-T in the conserved SBP domain (Fig. 5a, Sup- plementary Fig. 2a)15,39. Since BIN2 functions to destabilize SPL9, we expect that loss-of-function mutations in the BIN2 family members would result in an elevated level of the SPL9 protein. Therefore, we determined the level of SPL9 in bin2-3 bil1 bil2 and WT using the protoplast transformation method. As expected, the level of SPL9 was signiﬁcantly elevated by about 2.5-fold in bin2-3 bil1 bil2 in contrast to that in WT (Supplementary Fig. 2b), this result is also consistent with the LC-MS/MS analysis result that the T-P-K-V-T motif in SPL9 was phosphorylated in WT, but not in bin2-3 bil1 bil2 (Supplemen- tary Fig. 2a). Downregulation of miR172 is partially responsible for the dwf5 vegetative phase change phenotype incubated with GST or GST-BIN2 protein, and the proteins immunoprecipitated with glutathione-agarose beads were detected with an anti-His and an anti-GST antibody, respectively. d BIN2 interacts with SPL9 in vivo in an CoIP assay. Total protein was extracted from Nicotiana benthamiana leaves inﬁltrated with Agro- bacterium containing 35 S::3×FLAG/35 S::3×FLAG-rSPL9, and 35 S::BIN2-GFP/ 35 S::3×FLAG-rSPL9 combinations, and incubated with anti-FLAG beads. Coimmu- noprecipitated proteins were detected with an anti-FLAG and an anti-GFP antibody, respectively. All experiments were repeated 3 times biologically. Fig. 4 \| BIN2 physically interacts with SPL9. a BIN2 interacts with SPL9 in yeast. BIN2 was fused to pGAD (AD), and SPL9 was fused to pGBK (BD). AD and BD empty vectors were used as the negative controls. DDO (SD/-Leu/-Trp), QDO (SD/-Leu/- Trp/-Ade/-His). b BIN2 interacts with SPL9 in vivo in the nucleus as shown by bimolecular ﬂuorescence complementation (BiFC) assay. BIN2 was fused to cYFP, and SPL9 was fused to nYFP. The recombinant plasmids were transformed into the WT Arabidopsis protoplast. The nucleus was stained with hoechest 33342. Scale bar = 10 μm. c BIN2 interacts with SPL9 in vitro in a pull-down assay. SPL9-His was performed Western blotting to detect the eluate with an anti-FLAG and an anti-GFP antibody, respectively (Fig. 4d). The result showed that SPL9 and BIN2 were coimmunoprecipitated. Taken together, our results demonstrate that BIN2 physically interacts with SPL9 both in vitro and in vivo. further step to conﬁrm the interaction between BIN2 and SPL9 in planta, we performed a coimmunoprecipitation (CoIP) assay. We co- expressed SPL9-FLAG and BIN2-GFP fusion proteins in tobacco (Nicotiana benthamiana) leaves, and incubated the total protein with protein afﬁnity gel conjugated with the anti-FLAG antibody. Then we 7 Nature Communications\| (2023) 14:2608 7 https://doi.org/10.1038/s41467-023-38207-z BIN2-mediated phosphorylation of SPL9 is required for its function in vegetative phase change Data are me from a representative experiment with three technical replicates for eac Different letters indicate signiﬁcant difference using one-way ANOVA a All experiments were repeated 3 times biologically. Numbers between indicate the relative normalized value for each sample. The intensity o sample was ﬁrst normalized to its corresponding ACTIN, then the resul was normalized again to the value of T-T 1#. The band intensity was de using image J. h Leaf length/width ratio of the ﬁrst leaf from 40-day-ol 10#, T-A 2#, 6#, T-D 4#, 8#, and 50# plants. Different letters indicate s difference between genotypes using one-way ANOVA at P < 0.001 (n = Fig. 5 \| BIN2 phosphorylates SPL9 and the phosphorylation site in SPL9 is required for its normal function to promote vegetative phase change a A Fig. 5 \| BIN2 phosphorylates SPL9 and the phosphorylation site in SPL9 is required for its normal function to promote vegetative phase change. a A schematic diagram of the SPL9 protein. The TPKVT motif is the typical conserved phosphorylation recognition motif of the GSK3 kinase. The threonine in red indi- cates the phosphorylated site. b SPL9 is phosphorylated by BIN2 in vitro. SPL9-His wasincubated with GSTor BIN2-GSTat30 °C for 1 h. Protein was separated in a SDS- PAGE gel containing phos-tag regent and detected by anti-GST and anti-His anti- bodies. SPL9 (P), phosphorylated SPL9 with slower gel mobility shift. c In vitro kinase assay. Kinase assays were performed with puriﬁed GST-BIN2 and His-SPL9 (T) or mutant His-SPL9 (A). d Pie charts show phenotypic distribution of the ﬁrst leaf with abaxial trichomes in the primary rSPL9-APKVA (T-A), rSPL9-TPKVT (T-T), and rSPL9-DPKVD (T-D) transgenic plants. About 70 primary independent trans- formants were characterized. e Phenotypic characterization of representative transgenic plants transformed with T-A, T-T, and T-D with comparable levels of the SPL9 transcript. 21-day-old T-T 1#, 10#, T-A 2#, 6#, T-D 4#, 8#, and 50# lines grown Fig. 5 \| BIN2 phosphorylates SPL9 and the phosphorylation site in SPL9 is in short days were used for phenotypic analysis. Numbers indicate the ﬁrst leaf with abaxial trichomes (n = 20 plants, ±SD). Different letters indicate signiﬁcant difference between genotypes using one-way ANOVA at P < 0.001. Scale bar = 1 cm. f, g SPL9 transcript level (f) and protein level (g) in 14-day-old T-T 1#, 10#, T-A 2#, 6#, T-D 4#, 8#, and 50# transgenic lines in short days. BIN2-mediated phosphorylation of SPL9 is required for its function in vegetative phase change p p p y y To investigate the function of SPL9 phosphorylation by BIN2 in vegetative phase change, we generated constructs expressing a miR156-insensitive form of pSPL9::3×FLAG-rSPL9 (rSPL9-TPKVT) with a wild-type BIN2 classical phosphorylation form, a mutated BIN2 non- phosphorylation form of pSPL9::3×FLAG-rSPL9 (rSPL9-APKVA), and a constitutive BIN2 phosphorylation form of pSPL9::3×FLAG-rSPL9 (rSPL9-DPKVD) under the regulation of the SPL9 native promoter, and transformed these constructs into Col-0 WT. We ﬁrst char- acterized the vegetative phase change phenotype of >70 different primary transgenic lines from each construct in short days. About 96% of rSPL9-APKVA, 65% of rSPL9-TPKVT, and 33% of rSPL9-DPKVD plants produced abaxial trichomes on leaf 1, respectively (Fig. 5d). Furthermore, about 38% of rSPL9-DPKVD plants produced abaxial trichomes on leaves later than 4, which was signiﬁcantly higher than 4% in rSPL9-APKVA and 14% in rSPL9-TPKVT, suggesting that the constitutive phosphorylation form of SPL9 signiﬁcantly impaired its We ﬁrst analyzed the TOE1 protein sequence. Interestingly, there is a GSK3 classical phosphorylation motif of T-K-L-V-T in TOE1 (Fig. 6a). We then tested if TOE1 physically interacts with BIN2 using the yeast two-hybrid system. We generated the prey vector AD-TOE1 and the bait vector BD-BIN2, and co-transformed into the yeast competent cells, and plated on DDO and QDO media. As shown in Fig. 6b, yeast cells only grew on the QDO media when AD-TOE1 and BD-BIN2 were co- transformed, suggesting that BIN2 interacted with TOE1 in the yeast two-hybrid system. We then performed a BiFC assay to examine if BIN2 also interacts with TOE1 in vivo. BIN2 was fused to the C-terminal of YFP (BIN2-cYFP), and TOE1 was fused to the N-terminal of YFP (TOE1- nYFP). We co-transformed different combinations of vectors into the Nature Communications\| (2023) 14:2608 8 Article https://doi.org/10.1038/s41467-023-38207-z 2 phosphorylates SPL9 and the phosphorylation site in SPL9 is or its normal function to promote vegetative phase change. a A diagram of the SPL9 protein. The TPKVT motif is the typical conserved ation recognition motif of the GSK3 kinase. The threonine in red indi- hosphorylated site. b SPL9 is phosphorylated by BIN2 in vitro. SPL9-His ted with GSTor BIN2-GSTat30 °C for 1 h. Protein was separated in a SDS- ontaining phos-tag regent and detected by anti-GST and anti-His anti- 9 (P), phosphorylated SPL9 with slower gel mobility shift. c In vitro y. Kinase assays were performed with puriﬁed GST-BIN2 and His-SPL9 nt His-SPL9 (A). BIN2-mediated phosphorylation of SPL9 is required for its function in vegetative phase change d Pie charts show phenotypic distribution of the ﬁrst baxial trichomes in the primary rSPL9-APKVA (T-A), rSPL9-TPKVT (T-T), DPKVD (T-D) transgenic plants. About 70 primary independent trans- in short days were used for phenotypic analysis. Numbers ind with abaxial trichomes (n = 20 plants, ±SD). Different letters i difference between genotypes using one-way ANOVA at P < 0 cm. f, g SPL9 transcript level (f) and protein level (g) in 14-day-o 2#, 6#, T-D 4#, 8#, and 50# transgenic lines in short days. Da from a representative experiment with three technical replicat Different letters indicate signiﬁcant difference using one-way All experiments were repeated 3 times biologically. Numbers indicate the relative normalized value for each sample. The in sample was ﬁrst normalized to its corresponding ACTIN, then was normalized again to the value of T-T 1#. The band intensi using image J. h Leaf length/width ratio of the ﬁrst leaf from 4 Fig. 5 \| BIN2 phosphorylates SPL9 and the phosphorylation site in SPL9 is required for its normal function to promote vegetative phase change. a A schematic diagram of the SPL9 protein. The TPKVT motif is the typical conserved phosphorylation recognition motif of the GSK3 kinase. The threonine in red indi- cates the phosphorylated site. b SPL9 is phosphorylated by BIN2 in vitro. SPL9-His wasincubated with GSTor BIN2-GSTat30 °C for 1 h. Protein was separated in a SDS- PAGE gel containing phos-tag regent and detected by anti-GST and anti-His anti- bodies. SPL9 (P), phosphorylated SPL9 with slower gel mobility shift. c In vitro kinase assay. Kinase assays were performed with puriﬁed GST-BIN2 and His-SPL9 (T) or mutant His-SPL9 (A). d Pie charts show phenotypic distribution of the ﬁrst leaf with abaxial trichomes in the primary rSPL9-APKVA (T-A), rSPL9-TPKVT (T-T), and rSPL9-DPKVD (T-D) transgenic plants. About 70 primary independent trans- formants were characterized. e Phenotypic characterization of representative transgenic plants transformed with T-A, T-T, and T-D with comparable levels of the SPL9 transcript. 21-day-old T-T 1#, 10#, T-A 2#, 6#, T-D 4#, 8#, and 50# lines grown in short days were used for phenotypic analysis. Numbers indicate the ﬁrst leaf with abaxial trichomes (n = 20 plants, ±SD). Different letters indicate signiﬁcant difference between genotypes using one-way ANOVA at P < 0.001. Scale bar = 1 cm. BIN2-mediated phosphorylation of SPL9 is required for its function in vegetative phase change f, g SPL9 transcript level (f) and protein level (g) in 14-day-old T-T 1#, 10#, T-A 2#, 6#, T-D 4#, 8#, and 50# transgenic lines in short days. Data are means ± SD from a representative experiment with three technical replicates for each sample. Different letters indicate signiﬁcant difference using one-way ANOVA at P < 0.01. All experiments were repeated 3 times biologically. Numbers between two blots indicate the relative normalized value for each sample. The intensity of each sample was ﬁrst normalized to its corresponding ACTIN, then the resultant value was normalized again to the value of T-T 1#. The band intensity was determined using image J. h Leaf length/width ratio of the ﬁrst leaf from 40-day-old T-T 1#, 10#, T-A 2#, 6#, T-D 4#, 8#, and 50# plants. Different letters indicate signiﬁcant difference between genotypes using one-way ANOVA at P < 0.001 (n = 10 plants, ±SD). Fig. 5 \| BIN2 phosphorylates SPL9 and the phosphorylation site in SPL9 is equired for its normal function to promote vegetative phase change. a A chematic diagram of the SPL9 protein. The TPKVT motif is the typical conserved phosphorylation recognition motif of the GSK3 kinase. The threonine in red indi- cates the phosphorylated site. b SPL9 is phosphorylated by BIN2 in vitro. SPL9-His wasincubated with GSTor BIN2-GSTat30 °C for 1 h. Protein was separated in a SDS- PAGE gel containing phos-tag regent and detected by anti-GST and anti-His anti- bodies. SPL9 (P), phosphorylated SPL9 with slower gel mobility shift. c In vitro kinase assay. Kinase assays were performed with puriﬁed GST-BIN2 and His-SPL9 T) or mutant His-SPL9 (A). d Pie charts show phenotypic distribution of the ﬁrst eaf with abaxial trichomes in the primary rSPL9-APKVA (T-A), rSPL9-TPKVT (T-T), and rSPL9-DPKVD (T-D) transgenic plants. About 70 primary independent trans- ormants were characterized. e Phenotypic characterization of representative ransgenic plants transformed with T-A, T-T, and T-D with comparable levels of the in short days were used for phenotypic analysis. Numbers indicate the with abaxial trichomes (n = 20 plants, ±SD). Different letters indicate si difference between genotypes using one-way ANOVA at P < 0.001. Scal cm. f, g SPL9 transcript level (f) and protein level (g) in 14-day-old T-T 1# 2#, 6#, T-D 4#, 8#, and 50# transgenic lines in short days. BIN2-mediated phosphorylation of SPL9 is required for its function in vegetative phase change d BIN2 interacts with TOE1 in vitro in a pull-down assay. TOE1-His was incubated with GST or GST-BIN2, and proteins immun pitated with glutathione-agarose beads were detected with an anti-His and GSTantibody, respectively. e BIN2 interacts with TOE1 in vivo in an CoIP ass protein was extracted from Arabidopsis protoplasts containing 35 S::BIN2- 35 S::3×HA-rTOE1, and the 35 S::BIN2-MYC/35 S::3×HA-rTOE1 combination, a bated with beads conjugated with an anti-HA. Coimmunoprecipitated pro were detected with an anti-MYC and an anti-HA antibody, respectively. Al ments were repeated 3 times biologically. Article https://doi.org/10.1038/s41467-023-3 https://doi.org/10.1038/s41467-023-38207-z Article Fig. 6 \| BIN2 physically interacts with TOE1. a A schematic diagram of the TOE1 protein. The TKLVT motif is the classical phosphorylation motif of BIN2. The threonine in red indicates the phosphorylated site. b BIN2 interacts with TOE1 in yeast. BIN2 was fused to pGAD (AD), and TOE1 was fused to pGBK (BD). AD and BD empty vectors were used as the negative controls. c BIN2 interacts with TOE1 in vivo n the nucleus as shown by BiFC assay. BIN2 was fused to cYFP, and TOE1 was fused to nYFP. The recombinant plasmids were transformed into the WT Arabidopsis protoplast. Scale bar = 10 μm. d BIN2 interacts with TOE1 in vitro in a pull-down assay. TOE1-His was incubated with GST or GST-BIN2, and proteins immunopre pitated with glutathione-agarose beads were detected with an anti-His and an an GSTantibody, respectively. e BIN2 interacts with TOE1 in vivo in an CoIP assay. To protein was extracted from Arabidopsis protoplasts containing 35 S::BIN2-MYC, 35 S::3×HA-rTOE1, and the 35 S::BIN2-MYC/35 S::3×HA-rTOE1 combination, and inc bated with beads conjugated with an anti-HA. Coimmunoprecipitated proteins were detected with an anti-MYC and an anti-HA antibody, respectively. All expe ments were repeated 3 times biologically. assay. TOE1-His was incubated with GST or GST-BIN2, and proteins immunopreci- pitated with glutathione-agarose beads were detected with an anti-His and an anti- GSTantibody, respectively. e BIN2 interacts with TOE1 in vivo in an CoIP assay. Total protein was extracted from Arabidopsis protoplasts containing 35 S::BIN2-MYC, 35 S::3×HA-rTOE1, and the 35 S::BIN2-MYC/35 S::3×HA-rTOE1 combination, and incu- bated with beads conjugated with an anti-HA. Coimmunoprecipitated proteins were detected with an anti-MYC and an anti-HA antibody, respectively. All experi- ments were repeated 3 times biologically. Fig. 6 \| BIN2 physically interacts with TOE1. a A schematic diagram of the TOE1 protein. The TKLVT motif is the classical phosphorylation motif of BIN2. BIN2-mediated phosphorylation of SPL9 is required for its function in vegetative phase change Data are means ± SD from a representative experiment with three technical replicates for each sample. Different letters indicate signiﬁcant difference using one-way ANOVA at P < 0.01. All experiments were repeated 3 times biologically. Numbers between two blots indicate the relative normalized value for each sample. The intensity of each sample was ﬁrst normalized to its corresponding ACTIN, then the resultant value was normalized again to the value of T-T 1#. The band intensity was determined using image J. h Leaf length/width ratio of the ﬁrst leaf from 40-day-old T-T 1#, 10#, T-A 2#, 6#, T-D 4#, 8#, and 50# plants. Different letters indicate signiﬁcant difference between genotypes using one-way ANOVA at P < 0.001 (n = 10 plants, ±SD). schematic diagram of the SPL9 protein. The TPKVT motif is the typical conserved phosphorylation recognition motif of the GSK3 kinase. The threonine in red indi- cates the phosphorylated site. b SPL9 is phosphorylated by BIN2 in vitro. SPL9-His wasincubated with GSTor BIN2-GSTat30 °C for 1 h. Protein was separated in a SDS- PAGE gel containing phos-tag regent and detected by anti-GST and anti-His anti- bodies. SPL9 (P), phosphorylated SPL9 with slower gel mobility shift. c In vitro kinase assay. Kinase assays were performed with puriﬁed GST-BIN2 and His-SPL9 (T) or mutant His-SPL9 (A). d Pie charts show phenotypic distribution of the ﬁrst leaf with abaxial trichomes in the primary rSPL9-APKVA (T-A), rSPL9-TPKVT (T-T), and rSPL9-DPKVD (T-D) transgenic plants. About 70 primary independent trans- formants were characterized. e Phenotypic characterization of representative transgenic plants transformed with T-A, T-T, and T-D with comparable levels of the SPL9 transcript. 21-day-old T-T 1#, 10#, T-A 2#, 6#, T-D 4#, 8#, and 50# lines grown Nature Communications\| (2023) 14:2608 9 9 Fig. 6 \| BIN2 physically interacts with TOE1. a A schematic diagram of the TOE1 protein. The TKLVT motif is the classical phosphorylation motif of BIN2. The threonine in red indicates the phosphorylated site. b BIN2 interacts with TOE1 in yeast. BIN2 was fused to pGAD (AD), and TOE1 was fused to pGBK (BD). AD and BD empty vectors were used as the negative controls. c BIN2 interacts with TOE1 in vivo in the nucleus as shown by BiFC assay. BIN2 was fused to cYFP, and TOE1 was fused to nYFP. The recombinant plasmids were transformed into the WT Arabidopsis protoplast. Scale bar = 10 μm. Discussion V i h Vegetative phase change constitutes a critical phase in plant devel- opment, and it is regulated by both endogenous and exogenous cues. Endogenously, it is regulated by the conserved sequential action between miR156 and miR1725. miR156 acts to repress the expression of ten different SPL genes posttranscriptionally to coordinate different aspects of vegetative trait development, especially leaf shape and leaf epidermal trait development. miR159 and some epigenetic modiﬁers act upstream of miR156 to regulate vegetative phase change43–45. Exogenous cues, such as sugar, phosphorus, temperature, photo- period, phytohormone, affect vegetative phase change by modulating the expression of genes in the miR156-SPL-miR172 pathway16–19,21,46–50. However, the mechanism of how these exogenous factors is involved in vegetative phase change remains to be elucidated. In plants, GA promotes, while JA delays, vegetative phase change in Arabidopsis, maize, and rice2,16–18. Only the mechanism of how GA promotes vegetative phase change has been partially elucidated. In this study, we showed that another phytohormone, BRs, also plays an important role in promoting vegetative phase change in Arabidopsis. This is the fourth phytohormone so far shown to regulate vegetative phase change. Plants defective in BR biosynthesis exhibited a delayed vegetative phase change phenotype. This delayed phenotype is mainly attributable to the reduction in the abundance of miR172 as a result of the proteolytic degradation of SPL9,and to a corresponding increase in the level of the TOE1 protein due to the dual negative regulation by both miR172 and BIN2 in dwf5. This result is in well agreement with a previous report that overexpression of miR172 could suppress the defect of bak151. Based on our results from this study, we proposed a model for coordinated regulation of vegetative phase change by BRs and the age pathway (Fig. 8c). In WT, the presence of normal levels of BRs suppresses the activity of BIN2 to phosphorylate SPL9 and TOE1, thereby maintaining a normal level of SPL9 and TOE1 for plants to proceed to vegetative phase change; in dwf5, the reduced levels of BRs lead to an elevated level of BIN2 (Supplementary Fig. 1a), thus increasing the phosphorylated form of SPL9 and TOE1 simultaneously for subsequent proteolytic degradation with the regulation of TOE1 by SPL9-miR172-TOE1 outweighing the BIN2-TOE1 mode. As a result, TOE1 accumulates to a relatively higher level in dwf5 than that in WT to delay vegetative phase change. Phosphorylation of TOE1 by BIN2 is critical for its function in vegetative phase change Phosphorylation of TOE1 by BIN2 is critical for its function in vegetative phase change To investigate the role of BIN2-mediated phosphorylation of TOE1, we again performed an LC-MS/MS analysis by transforming the UBI10::3- xFLAG-rTOE1 construct insensitive to miR172 into the protoplast from Ws and bin2-3bil1bil2, respectively. LC-MS/MS indicated that the 124th threonine in TOE1 was phosphorylated in WT (Fig. 6a, Supplementary Fig. 4a). To further conﬁrm that BIN2 phosphorylates TOE1 in vitro, we performed an in vitro kinase assay. GST-BIN2, 6×His-TOE1 (T) protein in which the 124th amino acid is the wild-type threonine, and 6×His- TOE1 (A) in which the 124th threonine was replaced with an alanine were puriﬁed from bacterium. When GST-BIN2 was incubated with 6×His- TOE1(T) and radiolabeled ATP, autoradiographs showed two bands with the top band corresponding to the auto-phosphorylated GST- BIN2 and the bottom band corresponding to the phosphorylated His- TOE1 (Fig. 7a). However, when His-TOE1 (A) was incubated with GST- BIN2, the intensity of the bottom band corresponding to His-TOE1 in the autoradiograph is greatly reduced (Fig. 7a). This result further conﬁrms that TOE1 is a substrate of BIN2, and the 124th threonine in the TOE1 protein is critical for its phosphorylation by BIN2. To investigate the function of TOE1 phosphorylation by BIN2 in vegetative phase change, we mutated the 124th threonine in the TKLVT motif to AKLVT, and to DKLVT, respectively. Then we generated con- structs expressing the wild-type form of UBI10::3×FLAG-rTOE1 (rTOE1- TKLVT) insensitive to miR172 regulation, and mutated forms of UBI10::3×FLAG-rTOE1 (rTOE1-AKLVT) and UBI10::3×FLAG-rTOE1 (rTOE1- DKLVT) under the regulation of the Arabidopsis Ubiqutin10 promoter, and transformed these constructs into Col-0. We ﬁrst characterized the vegetative phase change phenotype of 20 different primary transgenic lines transformed with each construct in short days. About 70% of rTOE1-AKLVT, 40% of rTOE1-TKLVT, and 0% of rTOE1-DKLVT plants produced abaxial trichomes later than leaf nine (>L9), respec- tively (Fig. 7b), suggesting that constitutive phosphorylation of TOE1 impaired its function signiﬁcantly to repress vegetative phase change. To see how phosphorylation affects TOE1 function and its stability, we ﬁrst generated homozygous lines with a single T-DNA insertion in progenies from different transgenic lines. Next, we determined the transcript level of TOE1 in those transgenic lines, and compared the phenotype of different transgenic lines with comparable levels of TOE1 transcript (Fig. 7c, d). Discussion V i h Therefore, BRs participate in vegetative phase change by PTM of the SPL9 and TOE1 proteins simultaneously via BIN2, and this is achieved by the physical interaction between BIN2 BIN2-mediated phosphorylation of SPL9 is required for its function in vegetative phase change The threonine in red indicates the phosphorylated site. b BIN2 interacts with TOE1 in yeast. BIN2 was fused to pGAD (AD), and TOE1 was fused to pGBK (BD). AD and BD empty vectors were used as the negative controls. c BIN2 interacts with TOE1 in vivo in the nucleus as shown by BiFC assay. BIN2 was fused to cYFP, and TOE1 was fused to nYFP. The recombinant plasmids were transformed into the WT Arabidopsis protoplast. Scale bar = 10 μm. d BIN2 interacts with TOE1 in vitro in a pull-down (Fig. 6d). To further conﬁrm if BIN2 and TOE1 interact with each other in planta, we performed a CoIP assay. We co-expressed TOE1-HA and BIN2-MYC fusion proteins in the protoplast isolated from Arabidopsis leaves, and incubated with agarose beads conjugated with the anti-HA antibody, then detected the eluate with an anti-MYC and an anti-HA antibody, respectively. Western blotting result indicated that TOE1 and BIN2 were coimmunoprecipitated (Fig. 6e). Taken together, our results demonstrate that BIN2 physically interacts with TOE1 both in vitro and in vivo. WT protoplast. YFP ﬂuorescence was observed in the transformed cell nuclei when BIN2-cYFP and TOE1-nYFP were co-transformed but not in all other negative control samples (Fig. 6c). This result demonstrated that BIN2 and TOE1 interact with each other in vivo in Arabidopsis cells. We also performed a protein pull-down assay to test if BIN2 interacts with TOE1 in vitro. We puriﬁed GST, GST-BIN2, and 6×His-TOE1 pro- teins from E. coli, and incubated the GST and GST-BIN2 proteins with His-TOE1 in vitro separately. Western blotting using an anti-His and an anti-GST antibody indicated that BIN2 also interacts with TOE1 in vitro WT protoplast. YFP ﬂuorescence was observed in the transformed cell nuclei when BIN2-cYFP and TOE1-nYFP were co-transformed but not in all other negative control samples (Fig. 6c). This result demonstrated that BIN2 and TOE1 interact with each other in vivo in Arabidopsis cells. We also performed a protein pull-down assay to test if BIN2 interacts with TOE1 in vitro. We puriﬁed GST, GST-BIN2, and 6×His-TOE1 pro- teins from E. coli, and incubated the GST and GST-BIN2 proteins with His-TOE1 in vitro separately. Phosphorylation of TOE1 by BIN2 is critical for its function in vegetative phase change Consistent with the above phenotypic char- acterization result, transgenic lines with rTOE1-AKLVT (T-A) produced abaxial trichomes on about leaf 9.7, transgenic lines with rTOE1-TKLVT (T-T) produced an intermediate vegetative phase change phenotype with abaxial trichomes on about leaf 9.1, whereas transgenic lines with rTOE1-DKLVT (T-D) had the weakest phenotype with abaxial trichomes on about leaf 7.7 (Fig. 7c). To see how the observed phenotype links to the TOE1 protein level and how phosphorylation affects the level of TOE1, we performed Western blotting using an anti-FLAG antibody to determine the level of the TOE1 protein in these transgenic lines. Among different transgenic lines, T-A 9#, 15#, and 16# accumulated relatively more TOE1 protein, T-T 8#, and 15# accumulated an inter- mediate level of the TOE1 protein, whereas T-D 1#, 8#, 10#, and 11# accumulated the lowest level of the TOE1 protein (Fig. 7e). These results imply that BIN2-mediated phosphorylation of the TKLVT motif is critical for TOE1 function to delay vegetative phase change by destabilizing the TOE1 protein and sequestering its function. BIN2-mediated phosphorylation of SPL9 is required for its function in vegetative phase change Western blotting using an anti-His and an anti-GST antibody indicated that BIN2 also interacts with TOE1 in vitro Nature Communications\| (2023) 14:2608 10 https://doi.org/10.1038/s41467-023-38207-z Article contributes to TOE1 protein accumulation and vegetative phase change, we determined the level of TOE1 in the protoplast from dwf5 mutant transformed with UBI10::3×FLAG-rTOE1 insensitive to miR172 and UBI10::3×FLAG-sTOE1 sensitive to miR172,respectively. The pur- pose of using these two constructs is to understand how the absence and presence of miR172 contribute to TOE1 accumulation. In dwf5 transformed with UBI10::3×FLAG-rTOE1 insensitive to miR172 regula- tion, the level of TOE1 was signiﬁcantly reduced by about two-fold compared with WT (Fig. 8a), this is consistent with the expectation that the elevated level of BIN2 kinase activity in dwf5 (Supplementary Fig. 1a) would accelerate the degradation of TOE1. This also implies that the BIN2-TOE1 regulatory mode is important to keep TOE1 from overaccumulation. However, in dwf5 transformed with UBI10::3×FLAG- sTOE1 sensitive to miR172 regulation, the TOE1 protein was elevated about 2.2-fold in dwf5 compared with WT(Fig. 8b). This is well in agreement with the delayed vegetive phase change phenotype of dwf5, and the genetic result that toe1 toe2 is completely epistatic to dwf5 with respect to the production of abaxial trichomes (Fig. 3a). These results suggest that PTM of the TOE1 protein by both miR172 and BIN2 is important to maintain a homeostatic level of TOE1 in order for normal vegetative phase change to take place, and the regulation of TOE1 through the SPL9-miR172 pathway plays a predominant role in con- trast to the regulation by BIN2. Nature Communications\| (2023) 14:2608 Coordinated regulation of TOE1 at the protein level regulates vegetative phase change Our results above suggest that BIN2 functions in a dual manner to regulate vegetative phase change with opposite outcomes for TOE1: one is to interact with SPL9 physically to destabilize it, thus reducing the level of miR172 to increase the TOE1 protein level, the other is to interact with TOE1 downstream of SPL9 to destabilize it directly. To answer the question of how these two modes of regulation by BIN2 Nature Communications\| (2023) 14:2608 11 Article https://doi.org/10.1038/s41467-023-38207-z IN2 phosphorylates TOE1 and the phosphorylation site in TOE1 is for its normal function to delay vegetative phase change. a Kinase r puriﬁed GST-BIN2 and His-TOE1 (T) or His-TOE1 (A) proteins. b Pie charts enotypic distribution of the ﬁrst leaf with abaxial trichomes in the primary KLVT (T-A), rTOE1-TKLVT (T-T), and rTOE1-DPVLT (T-D) transgenic plants. 0 primary independent transformants were characterized. c Phenotypic rization of representative transgenic plants transformed with rTOE1-AKLVT OE1-TKLVT (T-T), and rTOE1-DPVLT (T-D) with comparable levels of the nscript. 21-day-old T-T 8#, 15#, T-A 6#, 16#, T-D 8#, and 10# lines grown in ys were used for phenotypic analysis. Numbers indicate the ﬁrst leaf with ichomes (n = 15 plants, ±SD). Different letters indicate signiﬁcant difference between genotypes using one-way ANOVA at P < 0.01. Scale bar = d, e TOE1 transcript level (d) and protein level (e) in 14-day-old T-T 1#, 8#, 15# 6#, 9#, 15#, 16#, T-D 1#, 8#, 10#, and 11# transgenic lines in short days. Data means ± SD from a representative experiment with three technical replicates each sample. Different letters indicate signiﬁcant difference using one-way AN at P < 0.01. Numbers between two blots indicate the relative normalized valu each sample. The intensity of each sample was ﬁrst normalized to its correspon ACTIN, then the resultant value was normalized again to the value of T-T 1#. band intensity was determined using image J. All experiments were repeated times biologically. Fig. 7 \| BIN2 phosphorylates TOE1 and the phosphorylation site in TOE1 is required for its normal function to delay vegetative phase change. a Kinase assays for puriﬁed GST-BIN2 and His-TOE1 (T) or His-TOE1 (A) proteins. b Pie charts show phenotypic distribution of the ﬁrst leaf with abaxial trichomes in the primary rTOE1-AKLVT (T-A), rTOE1-TKLVT (T-T), and rTOE1-DPVLT (T-D) transgenic plants. About 20 primary independent transformants were characterized. Coordinated regulation of TOE1 at the protein level regulates vegetative phase change c Phenotypic characterization of representative transgenic plants transformed with rTOE1-AKLVT (T-A), rTOE1-TKLVT (T-T), and rTOE1-DPVLT (T-D) with comparable levels of the TOE1 transcript. 21-day-old T-T 8#, 15#, T-A 6#, 16#, T-D 8#, and 10# lines grown in short days were used for phenotypic analysis. Numbers indicate the ﬁrst leaf with abaxial trichomes (n = 15 plants, ±SD). Different letters indicate signiﬁcant Fig. 7 \| BIN2 phosphorylates TOE1 and the phosphorylation site in TOE1 is difference between genotypes using one-way ANOVA at P < 0.01. Scale bar = 1 cm. d, e TOE1 transcript level (d) and protein level (e) in 14-day-old T-T 1#, 8#, 15#, T-A 6#, 9#, 15#, 16#, T-D 1#, 8#, 10#, and 11# transgenic lines in short days. Data are means ± SD from a representative experiment with three technical replicates for each sample. Different letters indicate signiﬁcant difference using one-way ANOVA at P < 0.01. Numbers between two blots indicate the relative normalized value for each sample. The intensity of each sample was ﬁrst normalized to its corresponding ACTIN, then the resultant value was normalized again to the value of T-T 1#. The band intensity was determined using image J. All experiments were repeated 3 times biologically. Nature Communications\| (2023) 14:2608 12 Article https://doi.org/10.1038/s41467-023-38207-z SPL9, as well as the interaction between BIN2 and TOE1. It is of est to see that BIN2 functions in a dual manner to post- criptionally modify SPL9 and TOE1 in the same genetic pathway opposite outcomes for TOE1. During vegetative phase change, 56 expression declines, while the expression of SPL genes ases4,5; likewise, miR172 expression increases, while TOE1 ession declines52 (Fig. 8c). A high level of miR156 at the juvenile e renders a low level of SPL9 to alleviate the repression of TOE1 by 72, leading to over-accumulation of TOE1. Therefore, it ispossible, This also can be inferred from the result that the miR172 of TOE1 was reduced, while the miR172-insensitive for elevated when the GSK3 kinase activity was abolished i (Supplementary Fig. 4b, c). A previous study indicated that S163 in IPA1 a binding speciﬁcity in response to the infection by fung balance between growth and immunity14. Coordinated regulation of TOE1 at the protein level regulates vegetative phase change However, how phosphorylation is regulated by upstream factors rem In this study, we identiﬁed two GSK3 kinase classical ph cle https://doi.org/10.1038/s4146 This also can be inferred from the result that the miR172-sensitive form of TOE1 was reduced, while the miR172-insensitive form of TOE1 was elevated when the GSK3 kinase activity was abolished in bin2-3bil1bil2 (Supplementary Fig. 4b, c). and SPL9, as well as the interaction between BIN2 and TOE1. It is of interest to see that BIN2 functions in a dual manner to post- transcriptionally modify SPL9 and TOE1 in the same genetic pathway with opposite outcomes for TOE1. During vegetative phase change, miR156 expression declines, while the expression of SPL genes increases4,5; likewise, miR172 expression increases, while TOE1 expression declines52 (Fig. 8c). A high level of miR156 at the juvenile phase renders a low level of SPL9 to alleviate the repression of TOE1 by miR172, leading to over-accumulation of TOE1. Therefore, it ispossible, in this scenario, that the BIN2-TOE1 mode is initiated to safeguard a homeostatic level of TOE1 required for normal juvenile development. A previous study indicated that S163 in IPA1 alters the DNA binding speciﬁcity in response to the infection by fungus to sustain a balance between growth and immunity14. However, how this pattern of phosphorylation is regulated by upstream factors remains unknown. In this study, we identiﬁed two GSK3 kinase classical phosphorylation motifs in both SPL9 and TOE1: one is T-P-K-V-T in the conserved SBP domain of SPL9, the other is T-K-L-V-T in TOE1. The status of Nature Communications\| (2023) 14:2608 13 Article https://doi.org/10.1038/s41467-023-38207-z Fig. 8 \| Coordinated regulation of TOE1 posttranscriptionally by the age pathway and BRs during vegetative phase change. a, b The FLAG-rTOE1 protein insensitive to miR172 level in dwf5 (a), the FLAG-sTOE1protein sensitive to miR172 level in dwf5 (b). Ubi10::3×FLAG-TOE1 or Ubi10::3×FLAG-rTOE1 were transformed into the protoplasts from Col-0 and dwf5, respectively. Total protein was extracted from transformed protoplasts and detected by Western blotting using an anti-FLAG and anti-ACTIN antibody, respectively. Numbers between two blots indicate the relative normalized value for each sample. The intensity of each sample was ﬁrst normalized to its corresponding ACTIN, then the resultant value was normalized again to the value of Col-0. The band intensity was determined using image J. All experiments were repeated 3 times biologically with similar results. c A model for BR-mediated vegetative phase change in Arabidopsis. Coordinated regulation of TOE1 at the protein level regulates vegetative phase change 5a); moreover, loss-of-function mutations in BZR1 delayed vegetative phase change (Supplementary Fig. 5b, c). BZR1 plays dual roles in the BR biosynthetic pathway by feedback inhibition of BR biosynthesis and by regulation of downstream growth responsive genes;54,55 moreover, BZR1 also has a BR-signaling independent regulatory role in anther development56. BZR1 was recently shown to physically interact with SPL9 to cooperatively regulate downstream genes57. Therefore, the complicated role of the GSK3 kinase family and BZR1 in vegetative phase change still awaits further investigation. phosphorylation mediated by BIN2 is required for the stability and function of SPL9 and TOE1 to regulate vegetative phase change. However, the fact that 33% of rSPL9-DPKVD plants, in which this motif is in the constant phosphorylated status, still produced abaxial tri- chomes on leaf 1 or 2 (Fig. 5d) suggests that there might exist other phosphorylation sites important for SPL9 stabilization and function. This assumption is in line with previous studies showing that BIN2 can also phosphorylate non-canonical serine and threonine sites, and those phosphorylation sites exist in the SPL9 protein15,39. The genetic interaction analysis between dwf5 and genes in the miR156-SPL pathway indicated that miR172 and toe1/toe2 are epistatic to dwf5, and SPL9 and DWF5 act synergistically with respect to the production of abaxial trichomes. However, the leaf shape of Ubi10::172B dwf5, spl9-4 dwf5, and toe1 toe2 dwf5 resembled more those of dwf5, suggesting that dwf5 is epistatic to Ubi10::172B, spl9-4, and toe1 toe2 with respect to leaf shape. This genetic result also implies that BRs involve vegetative phase change by other yet-to-known fac- tors other than SPL9 and TOE1. Those targets could be different SPL genes that share functional redundancy with SPL9, or could be other genes that play a role in leaf development. g p y p One obvious challenge for our model in the current study is that if the delayed vegetative phase change phenotype of dwf5 is attributable to the elevated level of BIN2, we would assume that BIN2 loss-of- function mutants exhibit a precocious vegetative phase change phe- notype; however, plants triply mutant for the GSK3-like kinase genes, bin2-3 bil1 bil2, exhibited a delayed vegetative phase change pheno- type in contrast to its wild-type Ws (Supplementary Fig. 3a). This is contradictory to our model. Coordinated regulation of TOE1 at the protein level regulates vegetative phase change DWF5 is the rate-limiting enzyme in the BR biosynthetic pathway. In WT, the presence of normal levels of BRs suppresses the activity of BIN2 to phosphorylate SPL9 and TOE1, thereby main- taining a normal level of SPL9 and TOE1 for plants to proceed to vegetative phase change; in dwf5 with a reduced level of BRs, more phosphorylated BIN2 is accu- mulated and activated to destabilize more SPL9 and TOE1 by physical interaction. This leads to the downregulation of miR172 in dwf5. As the action of the SPL9- miR172-TOE1 mode predominates over that of BIN2-TOE1, TOE1 is upregulated in dwf5, thereby reducing the expression of GL1 to delay trichome production. Therefore, dwf5 exhibited a delayed vegetative phase change phenotype. During vegetative phase change, miR156 expression declines, while the expression of SPL genes increases; likewise, miR172 expression increases, while TOE1 expression declines. Due to the high expression of miR156 at the juvenile phase, very low expression of SPL9 alleviates the repression of TOE1 by miR172, leading to over- accumulation of TOE1. In this scenario, the BIN2-TOE1 mode is initiated to safe- guard a homeostatic level of TOE1 required for normal juvenile development. Therefore, BIN2 functions in a dual manner to posttranscriptionally modify SPL9 and TOE1 with opposite outcomes for TOE1. The gradient change in the color of miR156, SPL9, miR172, and TOE1 represents the gradual change in the abundance of these genes during vegetative phase change. In addition to its function to regulate SPL9 and TOE1, BIN2 also regulates vegetative phase change through affecting the interaction between BZR1 and SPL9, or other unknown pathways. c was created with BioRender.com and Microsoft Powerpoint. P phosphorylation. Molecules in gray color represents the degraded proteins. phenotype of bin2-3bil1bil2 could not be explained by its effect on the level of TOE1. Further analysis indicated that the level of GL1, one of the TOE1 targets and a positive regulator of leaf epidermal trichome development, was signiﬁcantly downregulated in bin2-3bil1bil2, while other genes in the miR156-SPL-miR172 pathway remains comparable between Ws and bin2-3bil1bil2 (Supplementary Fig. 3b). This result implies that the GSK3 kinase family also regulates vegetative phase change through affecting genes downstream of TOE1. Another chal- lenge to our model is that we would expect that bzr1-1D, a dominant mutant of BZR1, should exhibit a precocious vegetative phase pheno- type; however, bzr1-1D also exhibited a delayed vegetative phase change phenotype rather than a precious one (Supplementary Fig. Coordinated regulation of TOE1 at the protein level regulates vegetative phase change It was shown that the bin2-3 bil1 bil2 triple mutant accumulates more phosphorylated form of BIN2 substrate, and there might exist additional GSK3-like kinases that function redun- dantly with BIN253. If this is the case, we would anticipate that more SPL proteins are phosphorylated, destabilized and/or sequestered func- tionally to cause a delayed vegetative phase change phenotype. However, LC-MS/MS analysis revealed that SPL9 was not phosphory- lated in bin2-3bil1bil2, instead SPL9 was elevated in bin2-3bil1bil2 (Supplementary Fig. 2a, b), this result negated our assumption. Another possibility is that the GSK3 kinase family regulates vegetative phase change mainly by posttranscriptional modulation of TOE1. However, LC-MS/MS analysis demonstrated that TOE1 is only phos- phorylated in Ws, but not in bin2-3bil1bil2 (Supplementary Fig. 4a). As expected, the level of miR172-insensitive form of TOE1 was elevated by about 2-fold in bin2-3bil1bil2, whereas the miR172-sensitive form of TOE1 is slightly reduced (Supplementary Fig. 4b, c). We can anticipate that a reduced level of TOE1 would yield a precocious vegetative phase change phenotype; however, vegetative phase change was delayed in bin2-3bil1bil2. Therefore, the delayed vegetative phase change In addition to their role in the regulation of vegetative phase change in Arabidopsis, BRs also have a complicated impact on leaf angle, plant height, and inﬂorescence architecture and other yield- related traits23,58–62. Therefore, our result of the interaction of the BR signaling pathway with SPL9 and TOE1 is also important from a prac- tical point of view in regards to molecular breeding for agronomically important crops, especially for rice. IPA1 promotes the establishment of ideal plant architecture63,64, genetic modulation of the BR pathway in combination of IPA1 may provide a way to breed for elite crops with a higher yield in the future. Plasmid construction and generation of transgenic lines Plasmid construction and generation of transgenic lines To generate the pDWF5::DWF5 construct, the genomic sequence of DWF5, containing the promoter, coding, and 3' sequence, was ampli- ﬁed from Col-0 using PCR, and cloned into the KpnI site of pCAM- BIA1305.1. This construct was then introduced into the Agrobacterium strain GV3101 to transform the dwf5 plants using a ﬂoral dipping method. For yeast two hybrid system, the BIN2 ORF (open reading frame) was cloned into the EcoRI and BamHI sites of the GAL4 tran- scriptional activation domain to generate pGAD-BIN2, the SPL9 ORF was cloned into the NdeI and EcoRI sites of the GAL4 DNA-binding domain to generate the pGBK-SPL9 construct, and the TOE1 ORF was cloned into the NdeI and BamHI sites of pGBK to generate the pGBK- TOE1 construct. For Bimolecular Fluorescent Complimentary (BiFC) experiment, the SPL9 ORF was inserted into the XbaI and SalI sites of pXY103 to generate SPL9-nYFP, the BIN2 ORF was cloned into the BamHI and SalI sites of pXY105 to generate the BIN2-cYFP construct, and TOE1 ORF was cloned into the BamHI and SalI sites of pXY103 to generate the TOE1-nYFP construct. To generate the SPL9-HIS, TOE1-HIS and BIN2-GST constructs for pull-down assay, the ORF sequences of SPL9, TOE1 and BIN2 were cloned into pET-30a (+) and pPGH (derived from pGEX4T-2), respectively. pET-30a (+)-SPL9, pET-30a (+)-TOE1, pPGH, and pPGH-BIN2 were then introduced into the Escherichia coli strain Rosetta (DE3). For coimmunoprecipitation (Co-IP) analysis of SPL9 and BIN2, the SPL9 and BIN2 ORF sequences were individually cloned into a modiﬁed pCAMBIA1300 vector with a 3×FLAG or a GFP tag. For Co-IP analysis of TOE1 and BIN2, the TOE1 and BIN2 ORF sequences were individually cloned into a modiﬁed pBWA(V)HS vector with an MYC or a HA tag. The resultant 35 S::3×FLAG-rSPL9, 35 S::BIN2- GFP, 35 S::BIN2-MYC and 35 S::TOE1-HA constructs were then intro- duced into the Agrobacterium strain GV3101 to inﬁltrate tobacco (Nicotiana benthamiana) leaves or were directly transformed into the Arabidopsis protoplasts. To introduce mutations into the conserved T- P-K-V-T motif in SPL9 and T-K-L-V-T motif in TOE1, overlapping PCR was carried out to amplify the full-length miR156-insensitive SPL9 and miR172-insensitive TOE1 coding sequence using primers with intro- duced mutations, the PCR product was then cloned into the BamHI and NcoI sites in the pSPL9::3×FLAG-rSPL9 and UBI10::3×FLAG-rTOE1 construct5. Article Article https://doi.org/10.1038/s41467-023-38207-z Agrobacterium strain GV3101 to transform WT. All primers used for cloning are listed in Supplementary Table. Phenotypic analysis The plant age was measured from the time when seeds were trans- ferred to the growth chamber. Abaxial trichomes were scored during 2–4 weeks after planting using a stereomicroscope. For leaf initiation rate, the number of leaves was observed and scored using a stereo- microscope every 4 days. For leaf shape analysis, fully expanded leaves were removed from plants, attached to cardboard with double-sided tape, ﬂattened with transparent tape, and then scanned with a digital scanner. All of the experiments in this study were repeated three times. BiFC assay y The recombinant SPL9-nYFP, TOE1-nYFP and BIN2-cYFP plasmids were cotransformed into the WT protoplast isolated using a Tape-Arabi- dopsis Sandwich method through the PEG-mediated method64. Leaves were collected from 3- to 4-week-old plants grown in SD condition. The lower epidermal surface was ﬁrst afﬁxed to a strip of Magic tape (3 M, Scotch), then the leaves were pulled away with the Time tape (Time Med) afﬁxed to the upper epidermal surface. The peeled leaves with the Time tape were transferred to the enzyme solution [1.5% (w/v) cellulase R10 (Yakult), 0.4% (w/v) macerozyme R10 (Yakult), 0.4 M mannitol, 10 mM CaCl2, 20 mM KCl, 0.1% BSA and 20 mM MES pH 5.7], and were digested at room temperature for 1–2 h. The digested solu- tion then was ﬁltered through a 75-µm nylon mesh. The protoplasts were centrifuged at 100 × g for 2 min and washed with the W5 buffer (154 mM NaCl, 125 mM CaCl2, 5 mM KCl, 5 mM glucose, and 2 mM MES, pH 5.7) twice, and then incubated on ice for 30 min. For transforma- tion, the protoplasts were centrifuged and resuspended in ice-cold MMG buffer (0.4 M mannitol, 15 mM MgCl2, and 4 mM MES, pH 5.7). 100 µL protoplasts were mixed with 10 µg plasmid each and 120 µL PEG-Ca2+ buffer [40% (w/v) PEG400, 0.2 M mannitol, 100 mM CaCl2]. The mixture was incubated at room temperature for 10 min, and washed twice with the W5 buffer. Transformed protoplasts were resuspended in 1 mL W5 buffer and transferred into 6-well plates and incubated for 12–16 h. Protoplasts were ﬁnally stained with hoechst33342 (Sigma) for 0.5 h, and were observed with a laser scan- ning confocal microscope (Zeiss, LSM510). BL addition experiment Seeds were germinated on 1/2 MS plate with 0 or 10 nM BL (Sigma) in the short-day growth chamber. 10-day-old seedlings were then trans- ferred to the 1/2 Hogland liquid medium supplemented with 0 or 10 nM BL. The Hogland medium was replaced with new one every 3 days until for abaxial trichome and leaf shape analysis. Yeast two-hybrid The bait plasmid pGBK-BIN2 and the prey plasmid pGAD-SPL9, pGAD- TOE1 were co-transformed into yeast strain AH109. The transformants were screened on the SD/-Leu/-Trp (Double Dropout Supplement, DDO) medium by following the procedure as described in Yeast Pro- tocols Handbook (Clontch, PT3024-1). Healthy colonies from DDO medium were chosen and inoculated onto the SD/-Leu/-Trp/-Ade/-His (Quadruple Dropout Supplement, QDO) medium for further analysis. Map based cloning For gene expression analyses, 12-day-old seedlings without cotyledons were collected and stored at −80 °C until use. Tissues were ground into ﬁne powder in liquid nitrogen using a homogenizer. Total RNA was extracted using TRIzol (Ambion) and digested with RNase-free DNase (TaKaRa) according to the manufacturer’s protocol. Digested RNA was quantiﬁed, then reverse transcribed (RT) was done with a PrimerScript II 1st Strand cDNA Synthesis Kit (TaKaRa). Oligo-dT primer and miRNA- speciﬁc primers were used for preparing the ﬁrst-strand cDNA of mRNA and miRNA, respectively. Real-time PCR was performed using diluted cDNA on Step One Plus (ABI) real-time PCR machine. TUBLIN2 and AtSnoR101 were served as the internal controls for mRNAs and miRNAs analyses, respectively. All qRT-PCR primers are listed in Supplementary Table. To map the dwf5 mutation, dwf5 was ﬁrst crossed to Ler. In the selfed F2 population, plants with the same phenotype to dwf5 were chosen for the mapping purpose. DNA from about 100 mutant plants was pooled, and PCR was carried out using makers across different ﬁve chromo- somes to localize the mutation. Fine mapping was done using DNA from individual DNA to narrow down the mutation. Plasmid construction and generation of transgenic lines These constructs were introduced into the Agrobacterium strain GV3101 to transform WT or were directly transformed into the Arabidopsis protoplasts. To generate the pBIN2::eGFP-BIN2 construct, an about 2000-bp BIN2 promoter sequence was ﬁrst ampliﬁed by PCR, then the sequence was inserted into the KpnI and NcoI sites in the pCAMBIA1305.1 vector, and eGFP was fused to the N-terminus of the BIN2 genomic sequence using overlapping PCR, the PCR fragment was ﬁnally cloned into the NcoI and PmlI sites of the vector harboring the BIN2 promoter sequence. This plasmid was introduced into the Plant materials and growth conditions Plant materials and growth conditions g All genetic stocks, except bin2-3 and bin2-3 bil1 bil2, used in this study were in a Columbia-0 (Col-0) genetic background. spl9-4 (CS807258), pSPL9::rSPL9, pSPL9::3×FLAG-rSPL9, pSPL9::GR-rSPL9, Ubi10::172B, and toe1-2 toe2- were seed stocks as described previously5,45. bzr1-1D (CS65987) was obtained from the Arabidopsis Biological Resource Center (ABRC). bin2-3 and bin2-3 bil1 bil2 were kind gifts from Dr. Jianming Li, and they were in the Ws background. The dwf5 mutant was backcrossed to WT ﬁve times before phenotypic characterization. Seeds were grown in a mixture of peat and vermiculite moss at a 1:1 ratio, and left at 4 °C for 2 days before transfer to the growth chamber. Plants were grown in 32-well ﬂats under short-day conditions (10 h light and 14 h dark, 120 µmol/m2/s) at 22 °C. Nature Communications\| (2023) 14:2608 14 In vitro pull-down assay E. coli transformed with different expressing vectors was induced with 0.2 mM isopropylthio-β-galactoside (IPTG), and was incubated in a shaker at 16 °C for 20 h. Puriﬁcation of His-tagged recombinant protein Nature Communications\| (2023) 14:2608 15 https://doi.org/10.1038/s41467-023-38207-z Article were separated in 12% SDS-PAGE gels and then phosphorylation was detected in the dried gels exposed to phosphor screens. The auto- radiograph (Autorad) signal was detected by a Typhoon9410 phos- phor imager. was performed as described in the Ni-NTA Puriﬁcation System (Novex by Life Technology), the GST-tagged protein was puriﬁed according to a protocol described previously65. For SPL9-His and TOE1-His, cells were collected and resuspended in 10 mL native puriﬁcation buffer (50 mM NaH2PO4 pH8.0, 50 mM NaCl, 1 mM PMSF). The cell lysate was sonicated on ice with 7 s ON/7 s OFF at 30% intensity for 10 min and centrifuged at 12000 rpm for 20 min. The supernatant was transferred to a balanced column with Ni-NTA resin (TransGen) and incubated at 4 °C for 4 h with gentle agitation to keep the resin suspended in the lysate solution. The resin was settled by gravity and washed with 8 mL native wash buffer (50 mM NaH2PO4 pH8.0, 50 mM NaCl, 20 mM imi- dazole) 4 more times and ﬁnally was eluted with 1 mL native elution buffer (50 mM NaH2PO4 pH8.0, 50 mM NaCl, 300 mM imidazole). For BIN2-GST, a similar puriﬁcation process was followed except for the buffer and resin used. Cells were resuspended with PBS-L [50 mM NaH2PO4 pH 8.0, 150 mM NaCl, 1 mM EDTA, 0.2% Triton X-100 (v/v), and 1 mM PMSF]. After binding with the lysate, the GST resin (Trans- Gen) was washed with PBS-EW (50 mM NaH2PO4 pH 8.0, 150 mM NaCl, 1 mM EDTA, 1 mM DTT) and eluted with TNGT [50 mM Tris-HCl pH 8.0, 100 mM NaCl, 0.01% Triton X-100 (v/v), 30 mM Reduced glutathione, 1 mM DTT]. For the pull-down assay, the puriﬁed SPL9-His and TOE1- His recombinant proteins were incubated with resin bound with GST or GST-BIN2 at 4 °C for 4 h with gentle rotation. The beads were then collected and boiled in the SDS loading buffer for 10 min after washing ﬁve times with PBS-EW. Protein samples were separated in a 10% SDS- PAGE gel and were examined by immunoblotting using an anti-His (Sangon) and an anti-GST (TransGen) antibody, respectively. Reporting summary Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article. Co-IP assay y Agrobacterium harboring 35 S::3×FLAG-rSPL9, 35 S::BIN2-GFP, and 35 S::3×FLAG constructs was incubated in LB medium containing 50 mg/mL Kanamycin, 50 mg/mL Rifampicin, 10 mM MES pH5.6, and 20 µM Acetosyringone. Cells at the log growth phase were collected and resuspended in the inﬁltration solution (10 mM MES pH5.6, 150 µM AS, 10 mM MgCl2, 0.5% Glucose) to an OD value of 0.8. The Agro- bacterium solution with 35 S::BIN2-GFP was mixed with 35 S::3×FLAG or 35 S::3×FLAG-SPL9 at a 1:1 ratio, respectively. The mixed solution was then used to inﬁltrate tobacco (Nicotiana benthamiana) leaves. 100 µg of each recombinant 35 S::BIN2-MYC and 35 S::TOE1-HA plasmids was co-transformed into the WT protoplast. Total protein was isolated from Nicotiana benthamiana leaves 2 days after inﬁltration or from transformed protoplast with the IP buffer [50 mM HEPES pH 7.5, 150 µM NaCl, 1 mM EDTA, 0.2% Tritron X-100, protease inhibitor cocktail (Sigma)], and was incubated with agarose beads conjugated with an anti-FLAG (Sigma) or an anti-HA (Sigma) antibody at 4 °C for 4 h with gentle agitation. The beads were collected and washed ﬁve times with the IP buffer and eluted with 3×FLAG peptide (Sigma). The immunoprecipitate was boiled for 10 min in the SDS loading buffer and detected by an anti-FLAG (Beyotime), an anti-GFP (Sangon), an anti- MYC (Sangon), and an anti-HA (Sangon) antibody, respectively. Data availability Source data are provided with this paper. Source data are provided with this paper. Liquid chromatography-mass spectrometry (LC-MS/MS) analysis y For the analysis of the phosphorylation site in SPL9 and TOE1 by BIN2, the SPL9-FLAG and TOE1-FLAG fusion proteins were puriﬁed from Ara- bidopsis protoplasts. The recombinant pSPL9::3xFLAG-rSPL9 or Ub10::3xFLAG-rTOE1 plasmid was transformed into Ws and bin2-3bil1bil2 protoplasts, respectively. Total protein was isolated from protoplasts with the IP buffer, and then was incubated with agarose beads con- jugated with an anti-FLAG (Sigma) antibody at 4 °C for 4 h with gentle agitation. The beads were collected and washed ﬁve times with the IP buffer and boiled for 10 min in the SDS loading buffer. Samples were separated in SDS-PAGE gels, then stained with Coomassie brilliant blue. The band of target protein with the correct size was cut with a scalpel for MS analysis. The samples in the gel were treated as described previously66. The dried polypeptide samples were ﬁrst re-dissolved in Nano-HPLC Buffer A (0.1% formic acid-aqueous solution), and separated using the nano-HPLC liquid phase system Easy-NLC1200. The samples were then loaded to an automatic sampler and adsorbed to a Trap col- umn (RP-C18, Thermo Inc.), and separated with an Analysis column (rp-c18, thermo Inc.) at a ﬂow rate of 300nL/min. The hydrolysates were separated by capillary HPLC and analyzed by Q-Exactive mass spectro- metry (Thermo Scientiﬁc). The scanning range of parent ions was set at 300-1600 m/z, and the Data Dependent Acquisition (DDA) scanning mode was employed. The 20 strongest fragment proﬁles (MS2 Scan) were collected after each full scan. Fragmentation was performed using high-energy collision dissociation (HCD, high energy) with NCE energy of 28 and dynamic removal time of 25 s. The resolution of MS1 was 45000 at M/Z 200, the AGC target was 1E6, and the maximum injection time was 50 ms. The resolution of MS2 was 15000, the AGC target was 1E5, and the maximum injection time was 50 ms. The ProteomeDiscover 2.1 software was used for database search. MS/MS spectra were searched using MaxQuant against the uniprot-Arabidopsis thaliana (Mouse-ear cress) [3702] UP000006548 database. The parameters for database search were set as follows: Fixed modiﬁcations: Carbamidomethyl (C); Variable modiﬁcation: Oxidation (M), Acetyl (Protein N-term), and Phospho (STY); Digestion: trypsin; First search peptide tolerance: 20 ppm; Main search peptide tolerance: 4.5 ppm; Max missed: 2. In vitro kinase assay 1. Poethig, R. S. Phase change and the regulation of shoot morpho- genesis in plants. Science 250, 923–930 (1990). The puriﬁed His-SPL9 protein was incubated with GST or GST-BIN2 protein in 20 µl kinase buffer (25 mM Tris-HCl pH7.5, 10 mM MgCl2, 0.5 mM DTT, 1 mM ATP) at 30 °C for 1 h, respectively. Reactions were stopped by adding the protein loading buffer, and were boiled for 10 min. The samples were separated on 8% SDS-PAGE gels with or without 25 µM Phos-tag reagent (NARD Institute, Ltd) and 100 µM MnCl2 (Sigma), and the blot was then detected with an anti-His and an anti-GST antibody, respectively. For in vitro kinase assays, all recom- binant proteins were puriﬁed from Escherichia coli Rosetta. The reac- tion was performed in 20 mL kinase buffer (20 mM Tris, pH 7.5, 100 mM NaCl, 12 mM MgCl2, 10 µM ATP, and 10 µCi of [γ-32P] ATP). The kinase assays were performed at 37 °C for 30 min, and then were incubated with SDS loading buffer at 100 °C for 10 min. The samples 2. Telfer, A., Bollman, K. M. & Poethig, R. S. Phase change and the regulation of trichome distribution in Arabidopsis thaliana. Devel- opment 124, 645–654 (1997). 3. Usami, T., Horiguchi, G., Yano, S. & Tsukaya, H. The more and smaller cells mutants of Arabidopsis thaliana identify novel roles for Squamosa Promoter Binding Protein-Like genes in the control of heteroblasty. Development 136, 955–964 (2009). 4. Wu, G. & Poethig, R. S. Temporal regulation of shoot development in Arabidopsis thaliana by miR156 and its target SPL3. Development 133, 3539–3547 (2006). 5. Wu, G. et al. The sequential action of miR156 and miR172 regulates developmental timing in Arabidopsis. Cell 138, 750–759 (2009). Nature Communications\| (2023) 14:2608 16 Article https://doi.org/10.1038/s41467-023-38207-z 27. Wang, X. et al. Sequential transphosphorylation of the BRI1/BAK1 receptor kinase complex impacts early events in brassinosteroid signaling. Dev. Cell 15, 220–235 (2008). 6. Wang, J. W., Czech, B. & Weigel, D. miR156-regulated SPL tran- scription factors deﬁne an endogenous ﬂowering pathway in Ara- bidopsis thaliana. Cell 138, 738–749 (2009). 7. Xu, Y., Qian, Z., Zhou, B. & Wu, G. Age-dependent heteroblastic development of leaf hairs in Arabidopsis. N. Phytol. 224, 741–748 (2019). 28. Tang, W. et al. BSKs mediate signal transduction from the receptor kinase BRI1 in Arabidopsis. Science 321, 557–560 (2008). 29. Kim, T. W., Guan, S., Burlingame, A. L. In vitro kinase assay & Wang, Z. Y. The CDG1 kinase mediates brassinosteroid signal transduction from BRI1 receptor kinase to BSU1 phosphatase and GSK3-like kinase BIN2. Mol. Cell 43, 561–571 (2011). 8. Xu, M. et al. Developmental functions of miR156-regulated Squa- mosa Promoter Binding Protein-Like (SPL) genes in Arabidopsis thaliana. PLoS Genet. 12, e1006263 (2016). 30. Kim, T. W. et al. Brassinosteroid signal transduction from cell- surface receptor kinases to nuclear transcription factors. Nat. Cell Biol. 11, 1254–1260 (2009). 9. Yamaguchi, A. et al. The microRNA-regulated SBP-Box transcription factor SPL3 is a direct upstream activator of leafy, fruitful, and Apetala1. Dev. Cell 17, 268–278 (2009). 10. Gandikota, M. et al. The miRNA156/157 recognition element in the 3’ UTR of the Arabidopsis SBP box gene SPL3 prevents early ﬂowering by translational inhibition in seedlings. Plant J. 49, 683–693 (2007). 31. Peng, P., Yan, Z., Zhu, Y. & Li, J. Regulation of the Arabidopsis GSK3- like kinase Brassinosteroid-INSENSITIVE 2 through proteasome- mediated protein degradation. Mol. Plant 1, 338–346 (2008). 32. Zhu, J. Y. et al. The F-box protein KIB1 mediates brassinosteroid- induced inactivation and degradation of GSK3-like kinases in Ara- bidopsis. Mol. Cell 66, 648–657.e644 (2017). 11. Mutanwad, K. V., Neumayer, N., Freitag, C., Zangl, I. & Lucyshyn, D. O-glycosylation of SPL transcription factors regulates plant devel- opmental transitions downstream of miR156. bioRxiv https://doi. org/10.1101/618744 (2019). 33. Sun, Y. et al. Integration of brassinosteroid signal transduction with the transcription network for plant growth regulation in Arabi- dopsis. Dev. Cell 19, 765–777 (2010). 12. Wang, S. et al. Non-canonical regulation of SPL transcription factors by a human OTUB1-like deubiquitinase deﬁnes a new plant type rice associated with higher grain yield. Cell Res. 27, 1142–1156 (2017). 34. Choe, S. et al. Lesions in the sterol delta reductase gene of Arabi- dopsis cause dwarﬁsm due to a block in brassinosteroid biosynth- esis. Plant J. 21, 431–443 (2000). 13. Wang, J. et al. Tissue-speciﬁc ubiquitination by IPA1 Interacting Protein1 modulates IPA1 protein levels to regulate plant architecture in rice. Plant Cell 29, 697–707 (2017). 35. Chen, X. A microRNA as a translational repressor of APETALA2 in Arabidopsis ﬂower development. Science 303, 2022–2025 (2004). 14. Wang, J. et al. A single transcription factor promotes both yield and immunity in rice. Science 361, 1026–1028 (2018). 36. Lloyd, A. M., Schena, M., Walbot, V. & Davis, R. W. Epidermal cell fate determination in Arabidopsis: patterns deﬁned by a steroid- inducible regulator. In vitro kinase assay Science 266, 436–439 (1994). 15. Zhang, H. et al. The nuclear localization signal is required for the function of squamosa promoter binding protein-like gene 9 to promote vegetative phase change in Arabidopsis. Plant Mol. Biol. 100, 571–578 (2019). 37. He, J. X., Gendron, J. M., Yang, Y., Li, J. & Wang, Z. Y. The GSK3-like kinase BIN2 phosphorylates and destabilizes BZR1, a positive reg- ulator of the brassinosteroid signaling pathway in Arabidopsis. Proc. Natl Acad. Sci. USA 99, 10185–10190 (2002). 16. Evans, M. M. & Poethig, R. S. Gibberellins promote vegetative phase change and reproductive maturity in maize. Plant Physiol. 108, 475–487 (1995). 38. Hu, Y. & Yu, D. Brassinosteroids Insensitive2 interacts with Abscisic Acid Insensitive 5 to mediate the antagonism of brassinosteroids to abscisic acid during seed germination in Arabidopsis. Plant Cell 26, 4394–4408 (2014). 17. Beydler, B., Osadchuk, K., Cheng, C. L., Manak, J. R. & Irish, E. E. The juvenile phase of maize sees upregulation of stress-response genes and is extended by exogenous jasmonic acid. Plant Physiol. 171, 2648–2658 (2016). 39. Ye, K. et al. Brassinosteroid-Insensitive 2 negatively regulates the stability of transcription factor ICE1 in response to cold stress in Arabidopsis. Plant Cell 31, 2682–2696 (2019). 18. Hibara, K. et al. Jasmonate regulates juvenile-to-adult phase tran- sition in rice. Development 143, 3407–3416 (2016). 40. Wu, F. H. et al. Tape-Arabidopsis sandwich-a simpler Arabidopsis protoplast isolation method. Plant Methods 5, 16 (2009). 19. Werner, S., Bartrina, I. & Schmülling, T. Cytokinin regulates vege- tative phase change in Arabidopsis thaliana through the miR172/ TOE1-TOE2 module. Nat. Commun. 12, 5816 (2021). 41. Klein et al. A molecular mechanism for the effect of lithium on development. Proc. Natl Acad. Sci. USA 93, 8455–8459 (1996). 20. Harberd, N. P., Belﬁeld, E. & Yasumura, Y. The angiosperm gibberellin-GID1-DELLA growth regulatory mechanism: how an “inhibitor of an inhibitor” enables ﬂexible response to ﬂuctuating environments. Plant Cell 21, 1328–1339 (2009). 42. Stambolic, V., Ruel, L. & Woodgett, J. R. Lithium inhibits glycogen synthase kinase-3 activity and mimics wingless signalling in intact cells. Curr. Biol. 6, 1664–1668 (1996). 43. Xu, M., Hu, T., Smith, M. R. & Poethig, R. S. Epigenetic regulation of vegetative phase change in Arabidopsis. Plant Cell 28, 28–41 (2016). 21. Yu, S. et al. Gibberellin regulates the Arabidopsis ﬂoral transition through miR156-targeted SQUAMOSA promoter binding-like tran- scription factors. Plant Cell 24, 3320–3332 (2012). 44. Xu, Y. et al. https://doi.org/10.1038/s41467-023-38207-z https://doi.org/10.1038/s41467-023-38207-z Acknowledgements 49. Lee, H. et al. Genetic framework for ﬂowering-time regulation by ambient temperature-responsive miRNAs in Arabidopsis. Nucleic Acids Res. 38, 3081–3093 (2010). This work is supported by the National Natural Science Foundation of China (31970191, 31770209, 32172595), the Natural Science Foundation of Zhejiang province (LZ22C020003, LY21C150002), start-up funds from Zhejiang Agriculture and Forestry University (2012FR025) to G.W. and S.F.. We thank Dr. Qingfu Ye at Zhejiang University for providing equipment for the 32P experiment and analysis, and Dr. Jianming Li for kindly providing the bin2-1, bin2-3 and bin2-3 bil1 bil2 seeds. 50. Xin, M. et al. Diverse set of microRNAs are responsive to powdery mildew infection and heat stress in wheat (Triticum aestivum L.). BMC Plant Biol. 10, 123 (2010). 51. Kim, B. H., Kwon, Y., Lee, B. H. & Nam, K. H. Overexpression of miR172 suppresses the brassinosteroid signaling defects of bak1 in Arabidopsis. Biochem. Biophys. Res. Commun. 447, 479–484 (2014). In vitro kinase assay Regulation of vegetative phase change by SWI2/SNF2 chromatin remodeling ATPase BRAHMA. Plant Physiol. 172, 2416–2428 (2016). 22. Gudesblat, G. E. & Russinova, E. Plants grow on brassinosteroids. Curr. Opin. Plant Biol. 14, 530–537 (2011). 23. Nolan, T. M., Vukasinovic, N., Liu, D., Russinova, E. & Yin, Y. Brassi- nosteroids: multidimensional regulators of plant growth, develop- ment, and stress responses. Plant Cell 32, 295–318 (2020). 45. Guo, C. et al. Repression of miR156 by miR159 regulates the timing of the juvenile-to-adult transition in Arabidopsis. Plant Cell 29, 1293–1304 (2017). 24. Wang, Z. Y., Seto, H., Fujioka, S., Yoshida, S. & Chory, J. BRI1 is a critical component of a plasma-membrane receptor for plant ster- oids. Nature 410, 380–383 (2001). 46. Yang, L., Xu, M., Koo, Y., He, J. & Poethig, R. S. Sugar promotes vegetative phase change in Arabidopsis thaliana by repressing the expression of MIR156A and MIR156C. Elife 2, e00260 (2013). 25. Nam, K. H. & Li, J. BRI1/BAK1, a receptor kinase pair mediating brassinosteroid signaling. Cell 110, 203–212 (2002). 47. Yu, S. et al. Sugar is an endogenous cue for juvenile-to-adult phase transition in plants. Elife 2, e00269 (2013). 48. Hsieh, L. C. et al. Uncovering small RNA-mediated responses to phosphate deﬁciency in Arabidopsis by deep sequencing. Plant Physiol. 151, 2120–2132 (2009). 26. Wang, X. & Chory, J. Brassinosteroids regulate dissociation of BKI1, a negative regulator of BRI1 signaling, from the plasma membrane. Science 313, 1118–1122 (2006). Nature Communications\| (2023) 14:2608 17 Article Additional information Additional information Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41467-023-38207-z. Additional information Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41467-023-38207-z. 56. Chen, L. G. et al. BZR1 family transcription factors function redun- dantly and indispensably in BR signaling but exhibit BRI1- independent function in regulating anther development in Arabi- dopsis. Mol. Plant 12, 1408–1415 (2019). 57. Wang, L. Y. et al. BZR1 physically interact with SPL9 to regulate the vegetative phase change and cell elongation in Arabidopsis. Int. J. Mol. Sci. 22, 10415 (2021). Correspondence and requests for materials should be addressed to Shengjun Feng or Gang Wu. Correspondence and requests for materials should be addressed to Shengjun Feng or Gang Wu. 58. Sun, S. et al. Brassinosteroid signaling regulates leaf erectness in Oryza sativa via the control of a speciﬁc U-type cyclin and cell proliferation. Dev. Cell 34, 220–228 (2015). Peer review information Nature Communications thanks Yanhai Yin and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. A peer review ﬁle is available. Peer review information Nature Communications thanks Yanhai Yin and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. A peer review ﬁle is available. 59. Yamamuro, C. et al. Loss of function of a rice brassinosteroid insensitive1 homolog prevents internode elongation and bending of the lamina joint. Plant Cell 12, 1591–606 (2000). Author contributions 52. Jung, J. H. et al. The GIGANTEA-regulated microRNA172 mediates photoperiodic ﬂowering independent of Constans in Arabidopsis. Plant Cell 19, 2736–2748 (2007). B.Z., S.F. and G.W. conceived of the studyand wrote the manuscript. B.Z. and L.W. madevectors and did yeast two-hybrid analysis. B.Z. performed the map-based cloning, genetic analysis, qRT-PCR and data analysis. B.Z. and Q.L. carried out MG132 treatment, pull down assay and western blotting. B.Z., Q.L. and Y.S. carried out Co-IP and the kinase assay. B.Z., X.S., and H.L. performed protoplast isolation and transformation. L.N., T.S., X.D., J.H. and M.J. provided materials and assistance in the kinase assay and Co-IP. S.F. and G.W. oversaw the entire study. All authors read and approved the manuscript. 53. Yan, Z., Zhao, J., Peng, P., Chihara, R. K. & Li, J. BIN2 functions redundantly with other Arabidopsis GSK3-like kinases to reg- ulate brassinosteroid signaling. Plant Physiol. 150, 710–721 (2009). 54. He, J. X. et al. BZR1 is a transcriptional repressor with dual roles in brassinosteroid homeostasis and growth responses. Science 307, 1634–1638 (2005). Competing interests The authors declare no competing interests. Additional information Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41467-023-38207-z. Competing interests 55. Wang, Z. Y. et al. Nuclear-localized BZR1 mediates brassinosteroid- induced growth and feedback suppression of brassinosteroid bio- synthesis. Dev. Cell 2, 505–513 (2002). Reprints and permissions information is available at http://www.nature.com/reprints 60. Hong, Z. et al. A rice brassinosteroid-deﬁcient mutant, ebisu dwarf (d2), is caused by a loss of function of a new member of cytochrome P450. Plant Cell 15, 2900–2910 (2003). Publisher’s note Springer Nature remains neutral with regard to jur- isdictional claims in published maps and institutional afﬁliations. 61. Sakamoto, T. et al. Erect leaves caused by brassinosteroid deﬁ- ciency increase biomass production and grain yield in rice. Nat. Biotechnol. 24, 105–109 (2006). Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. 62. Yang, J. et al. Brassinosteroids modulate meristem fate and differ- entiation of unique inﬂorescence morphology in Setaria viridis. Plant Cell 30, 48–66 (2018). 63. Jiao, Y. et al. Regulation of OsSPL14 by OsmiR156 deﬁnes ideal plant architecture in rice. Nat. Genet. 42, 541–544 (2010). 64. Miura, K. et al. OsSPL14 promotes panicle branching and higher grain productivity in rice. Nat. Genet. 42, 545–549 (2010). 65. Schäfer, F., Seip, N., Maertens, B., Block, H. & Kubicek, J. Puriﬁcation of GST-tagged proteins. Methods Enzymol. 559, 127–139 (2015). 66. Gampala, S. S. et al. An essential role for 14-3-3 proteins in brassi- nosteroid signal transduction in Arabidopsis. Dev. Cell 13, 177–189 (2007). © The Author(s) 2023 © The Author(s) 2023 18 Nature Communications\| (2023) 14:2608
https://openalex.org/W2908349810	http://section.iaesonline.com/index.php/IJEEI/article/download/518/333	English	null	Malayalam Handwritten Character Recognition Using AlexNet Based Architecture	Indonesian Journal of Electrical Engineering and Informatics (IJEEI)	2,018	cc-by	5,105	Indonesian Journal of Electrical Engineering and Informatics (IJEEI) Vol. 6, No. 4, December 2018, pp. 393~400 ISSN: 2089-3272, DOI: 10.11591/ijeei.v6i1.518 Indonesian Journal of Electrical Engineering and Informatics (IJEEI) Vol. 6, No. 4, December 2018, pp. 393~400 ISSN: 2089-3272, DOI: 10.11591/ijeei.v6i1.518 Indonesian Journal of Electrical Engineering and Informatics (IJEEI) Vol. 6, No. 4, December 2018, pp. 393~400 ISSN: 2089-3272, DOI: 10.11591/ijeei.v6i1.518  393  393 393 Corresponding Author: Ajay James, Department of Computer Science and Engineering, Government Engineering College, Thrissur, Kerala, 680009, India. Email: ajayjames80@gmail.com Malayalam Handwritten Character Recognition Using AlexNet Based Architecture Ajay James1, Manjusha J2, Chandran Saravanan3 1,2Department of Computer Science and Engineering, Government Engineering College, India 3Department of Computer Science, NIT Durgapur, India Ajay James1, Manjusha J2, Chandran Saravanan3 1,2Department of Computer Science and Engineering, Government Engineering College, India 3Department of Computer Science, NIT Durgapur, India ABSTRACT Article Info Article history: Received Jun 1, 2018 Revised Sep 6, 2018 Accepted Dec 4, 2018 Keyword: AlexNet Convolutional neural network Handwritten character recognition Malayalam character recognition Optical character recognition This research article proposes a new handwritten Malayalam character recognition model based on AlexNet based architecture. The Malayalam language consists of a variety of characters having similar features, thus, differentiating characters is a challenging task. A lot of handcrafted feature extraction methods have been used for the classification of Malayalam characters. Convolutional Neural Networks (CNN) is one of the popular methods used in image and language recognition. AlexNet based CNN is proposed for feature extraction of basic and compound Malayalam characters. Furthermore, Support Vector Machine (SVM) is used for classification of the Malayalam characters. The 44 primary and 36 compound Malayalam characters are recognised with better accuracy and achieved minimal time consumption using this model. A dataset consisting of about 180,000 characters is used for training and testing purposes. This proposed model produces an efficiency of 98% with the dataset. Further, a dataset for Malayalam characters is developed in this research work and shared on Internet. Accepted Dec 4, 2018 Keyword: AlexNet Convolutional neural network Handwritten character recognition Malayalam character recognition Optical character recognition AlexNet Convolutional neural network Handwritten character recognition Malayalam character recognition Optical character recognition Copyright © 2018 Institute of Advanced Engineering and Science. All rights reserved. Copyright © 2018 Institute of Advanced Engineering and Science. All rights reserved. 1. INTRODUCTION Optical Character Recognition (OCR) is the process of recognising handwritten text and printed text. The recognised texts are converted into an encoded format. Four essential steps in character recognition are: a) data pre-processing, b) segmentation, c) feature extraction, and d) classification [1]. The Malayalam script consists of 15 vowels, 36 consonants, and 5 pure consonants as shown in following Figure 1. There are 12 dependent vowels and 144 compound characters as shown in the Figure 2 and Figure 3 respectively. The characters are compounded both vertically and horizontally in Malayalam script. Convolutional Neural Network is used for character recognition as well as for other purposes like gender prediction [2], finding fidelity content of document digitalisation [3], image style recognition [4], plant classification [5], rail surface defect detection [6],and several other applications.CNN has its advantage in image classification and feature extraction due to its special characteristics such as local connectivity strategy and weight sharing strategy [7] which drastically reduces usage of many parameters. Handwritten character recognition is a difficult task, because the writing styles of people differs on a large scale. Apart from the writing styles, other factors like noise and occurrence of skew also increases the difficulty. Unavailability of a standard dataset for Malayalam characters increases the complexity. Several models have been used by researchers to improve the accuracy rate of characters by designing new features, integrating different known features, and using multiple classifiers for classification. Various handcrafted Journal homepage: http://section.iaesonline.com/index.php/IJEEI/index  394 ISSN: 2089-3272 feature extraction methods have been employed to extract features of Malayalam characters, but, all failed to achieve 100% accuracy. Moreover, these conventional methods are time consuming and most of them failed to achieve higher recognition rate. feature extraction methods have been employed to extract features of Malayalam characters, but, all failed to achieve 100% accuracy. Moreover, these conventional methods are time consuming and most of them failed to achieve higher recognition rate. A survey of different feature extraction and classification methods is conducted and presented here. G. Pirlo et al. [8] presented a fuzzy-zoning based classification method for handwritten characters. The Recognition and reliability rate obtained for this fuzzy based zoning method is 93% and 95% respectively. Dileep Kumar Patel et al. [9] used Discrete Wavelet Transform (DWT) and Euclidean distance transform to find similar feature patterns. They noticed the minimum distance between the characters during classification. The accuracy obtained is 90%. 1. INTRODUCTION A neural network based off-line English handwritten character and digit recognition was proposed by Ashok Kumar et al. [10]. ShilpyBansal et al. [11] presented a technique called Neighborhood Foreground Pixels Density for handwritten Gurumukhi character feature extraction. Support Vector Machine (SVM) was used for classification. The accuracy achieved was 91.95%. A novel offline Malayalam handwritten text segmentation method was proposed by Shanjana C et al. [12]. SVM is used for character classification and achieved accuracy of 82%. y DurjoySenMaitra et al. [13] 5-layer CNN model like LeNet-5 reported an accuracy of 99.10% for English and multi script characters. A CNN based Bangla digit recognition was proposed by M.A.H Akhand et al. [14], 98.80% accuracy was obtained in the proposed model. A study on different applications of deep learning was conducted by S.M. Sofiqul Islam et al. [15]. The review evaluates two models of CNN, AlexNet, and Visual Geometric Group (VGG-S) in nine different benchmark datasets. Gurumukhi character recognition using particle swarm optimization and a neural network has been presented by JaspreetKaur et al. [16]. Their results showed PSONN outperforms ANN in recognizing Gurmukhi characters with 100% accuracy for basic characters. A novel approach to integrate two different classifiers for the recognition of off-line Arabic handwritten characters was made by Mohamed Elleuch et al. [17], 94.9% accuracy was reportedin this method. Shenzhen Gu et al. [18] used AlexNet architecture for the detection of a tennis ball in pictures. Ahmed El-Sawy et al. [19] proposed an off-line Arabic handwritten character recognition system using CNN model. A nine-layer CNN was used, and 5.1% misclassification error was reported for the testing data. Darmatasia et al. [20] proposed a CNN based feature extraction model. The overall accuracy, when tested on ten form documents, was obtained as 83.37%. Pranav P et al. [21] worked on Malayalam handwritten character recognition using CNN. The model was compared with LeNet-5 architecture and obtained a higher accuracy result. 95% accuracy was obtained for this method.SVM based character recognition has been proposed by GauriKatiyar et al. [22]. The method emphasizes the advantage of using SVM classifier for character classification. The results show an average accuracy of 95.74% for uppercase characters and 92.19% for lower case characters. AarthnaMaheshwari et al. [23] worked on handwritten English alphabets using PSO algorithm. ANN was used for recognition and obtained an accuracy of 83.8462%. 1. INTRODUCTION y From the above surveys of object recognition methods, it is observed that the character recognition has scope for further research and development. An emerging trend which has shown advancement in recognition rate and reduction in time consumption is CNN. Here, an attempt is made to improve the recognition rate and reduce time consumption of Malayalam handwritten simple and compound characters using AlexNet based CNN model. The primary objective of this research work is to develop a suitable model that efficiently extract features of Malayalam characters and classifies the character. The proposed new model is having higher recognition rate with higher accuracy and minimal training time. Further, a new dataset for Malayalam characters is developed for testing proposed new model and shared in the Internet. Figure 1. Vowels, Consonants and Pure consonants Figure 1. Vowels, Consonants and Pure consonants Figure 1. Vowels, Consonants and Pure consonants IJEEI, Vol. 6, No. 4, December 2018: 393 – 400 395  IJEEI ISSN: 2089-3272 Figure 3. Compound Characters 2. RESEARCH METHOD From the research surveys conducted, the drawbacks of handcrafted feature learning from the works of G. Pirlo [8] and Dileep Kumar Patel [9] are identified. In these methods, different features of the characters need to be extracted manually at each iteration, thereby increasing the cost of computation. To avoid the computational cost and to reduce the manual feature computation process, a convolutional feature extraction technique using neural network model to extract and train the network using different features of the same data is introduced. This improves the efficiency of character recognition and reduces the cost of computation which occurred while using manual feature extraction method. One of the features of CNN is that its recognition rate improves as the training set increases. This requires collecting a large dataset from people of different ages and different sections. Since, Malayalam does not have a large dataset, collecting handwritten character data is the first requirement for this research work. The collected data is then pre-processed in a small amount and augmented to enlarge the size of the dataset. This data isfurther divided into training and test set in the ratio 80:20, respectively. The primary aim of this research work is to automate the feature extraction by using CNN architecture. The CNN model proposed here consists of a 24-layer architecture, like AlexNet, which is used for extracting features of characters and SVM is used for classifying the output characters. 2.3. AlexNet-24 Modeling and Architecture g The next step is building aCNN architecture that effectively trains and tests the data. The different frameworks of CNN are studied to select the best model that helps in solving the recognition problems. The number of layers, type of layers, number of neurons per layer, size of the kernel, stride in each operation, and the type of classifier are selected. Different settings of the network are tested on the various selected network configuration to find the best accuracy. The proposed model used consists of 24 layers with five convolutions, 7 activation layers, five pooling layers, two drop outs, and 5 fully connected layers. The architecture is like that of AlexNet convolutional neural network. The difference lies in the number of ReLU layers and normalization layers used. Also, instead of conventional softmax layer SVM is used for classification. The first layer other than the data input layer is called the convolutional layer. The operation performed by this layer is called convolution operation. Step 1: The input data is represented in the form of pixels of size 227x227x3. ep 1: The input data is represented in the form of pixels of size 227x227x3. p p p p Step 2: A filter of size k x k is selected from the image and is convolved over the entire image pixels. p g g p Step 3: The first layer calculates the match of feature to a patch of the image, which multiplies each pixel in a feature by the corresponding pixel value in the image. p g g p Step 3: The first layer calculates the match of feature to a patch of the image, which multiplies each pixel in a feature by the corresponding pixel value in the image. y p g p g Step 4: Add up the answers and divide by total pixel values. Step 4: Add up the answers and divide by total pixel values. Step 5: In the first layer, if both pixels are black, (-1) x (-1) =1 or if both pixels are white then 1 x 1=1, every matching pixel result in a 1. Step 5: In the first layer, if both pixels are black, (-1) x (-1) =1 or if both pixels are white then 1 x 1=1, every matching pixel result in a 1. The third dimension 3 represents the RGB colour channels of the image. 2.2. Augmenting and Pre-processing data The data collected are cropped and characters are separated into 36 different labels. Each character image is of size 86 x 86 pixels. This dataset is augmented to develop a larger dataset. The augmentation is performed by applying affine transformation. The affine transformation includes translation, rotation, and scaling. The affine transformations preserve edge points, shapes, curves etc. So the basic shapes of characters remain the same. Rotation is used for augmenting the dataset, 8 distinct degrees of rotations are used (-2, 2, 4, -4, 8, -8). Following Figure 5(a) shows the character “nna” with eight different degrees of rotation. After augmentation, these dataset images are preprocessed. Negative of the character image is created to check whether any pixel discontinuities are present in the created dataset. Sample negative characters are shown in the following Figure 5(b). The model also predicted the noise present in the image. The final dataset of about 180,000-character images is obtained for the final training and testing. (a) (b) Figure 5. (a) Eight different rotated angles of character, NNA (b) Sample negative of characters, NKA and GMA (b) (b) Figure 5. (a) Eight different rotated angles of character, NNA (b) Sample negative of characters, NKA and GMA 2.1. Collecting Character data set In this proposed method, 36 compound characters and 44 basic characters of Malayalam language are considered. Since Malayalam characters do not have a standardised dataset, the 44 primary characters are collected from a new Malayalam character dataset called P-ARTS KAYYEZHUTU [24] dataset. The 36 compound characters are collected manually from people of different age groups. Grids of size 14 x 8 are printed in A4 papers, and character set is written in these columns of the grid. Following Figure 4 shows the architecture of the proposed system. Malayalam Handwritten Character Recognition Using AlexNet based Architecture (Ajay James) Figure 4. Architecture of the proposed system Figure 4. Architecture of the proposed system Malayalam Handwritten Character Recognition Using AlexNet based Architecture (Ajay James) Malayalam Handwritten Character Recognition Using AlexNet based Architecture (Ajay James)  ISSN: 2089-3272 396 2.3. AlexNet-24 Modeling and Architecture In our experiment input size is 227x227x3, kernel k = 11, with a stride of 4 and zero-padding. The first convolution operation Conv1 yields a feature map with 96 features. The output size of a convolution layer is calculated as follows: 𝐶= (W−K+2P) S (1) 𝐶= (W−K+2P) S (1) where, C is the output layer size, W is the input height / length, K is the kernel size, and P represent padding and S represent stride value. e, C is the output layer size, W is the input height / length, K is the kernel size, and P represent padding represent stride value. p A general convolution operation is represented as: h(t)g(t) = (hg)(t) (2) (2) h(t)g(t) = (hg)(t) h(t)g(t) = (hg)(t) ol. 6, No. 4, December 2018: 393 – 400 IJEEI, Vol. 6, No. 4, December 2018: 393 – 400 397 IJEEI ISSN: 2089-3272  Let h and g are two functions, whose convolution is written as hg. This is the integral of the product of two functions after one is reversed and shifted. The convolution is a type of integral transform as shown below: ℎ∗𝑔(𝑡) = ∫ℎ(𝜏)𝑔(𝑡−𝜏)𝑑𝜏 ∞ −∞ = ∫ ℎ(𝑡−𝜏)𝑔(𝜏)𝑑𝜏 ∞ −∞ (3) ℎ∗𝑔(𝑡) = ∫ℎ(𝜏)𝑔(𝑡−𝜏)𝑑𝜏 ∞ −∞ = ∫ ℎ(𝑡−𝜏)𝑔(𝜏)𝑑𝜏 ∞ −∞ (3) here, t need not represent time domain and this operation is defined as a weighted average of the functionℎ(𝜏) at moment t where 𝑔(−𝜏) is weighting shifted by amount t. Following Figure 6 shows image of a compound character before and after convolution operation. here, t need not represent time domain and this operation is defined as a weighted average of the functionℎ(𝜏) at moment t where 𝑔(−𝜏) is weighting shifted by amount t. Following Figure 6 shows image of a compound character before and after convolution operation. The next necessary layer is called activation function layer. This layer mainly helps to introduce some non-linearity to the network. Also, it helps to convert the outputs of neurons in previous layer as the input to the neurons in the next layer that aids in preventing linear mapping. Max-pooling finds the maximum value in a proposed region. A max-pool layer MP1, with a kernel size of 3 x 3 with stride 2 is used here. This reduces the input size to 112 x 112 x 48. Following Figure 6 shows the sample image before and after the convolution operation. 2.3. AlexNet-24 Modeling and Architecture The second convolution layer Conv2, with kernel 5 x 5 x 48, stride 1 and padding 2 produces a feature map of size 256. Figure 6. Image before and after convolution operation Figure 6. Image before and after convolution operation ReLU is applied after each convolution operation, and the output fed as input to max-pool layer 2. Max-pool MP2 with kernel 3 x 3 and stride two is supplied as input to next convolution layer Conv3. Following Figure 7(a) shows the sample feature map or activation map obtained for the characters after convolution operation. Following Figure 7(b) shows the different features that are obtained after the convolution operation of Malayalam character “AH”. (a) (b) Figure 7. (a) Sample feature map obtained for character (b) Different features of character “AH” extracted by the model (a) (b) (b) (a) Figure 7. (a) Sample feature map obtained for character (b) Different features of character “AH” extracted by the model 3. RESULTS AND ANALYSIS The compound character dataset consists of 36-character classes. This dataset is tested against two other architecture other than AlexNet-24 architecture, namely LeNet-5 with seven-layer architecture and architecture with 152 layers called ResNet. Following Table 1 shows the comparison between three CNN models with two different datasets, raw data, and pre-processed data. The LeNet-5 model produces an accuracy of 85.30% to the raw dataset and 87.54% to the pre-processed dataset. The LeNet-5 architecture consists of seven layers, and it takes a long time for executing the data of about 180,000-character images. The ResNet network consists of 152-layer architecture and there occur the problem of vanishing gradient and degradation. It produces better accuracy rates than LeNet-5 but lesser than the proposed AlexNet-24. The first parameter is initial learning rate. The learning rate indicates the time required for the neural networks to learn different features. If it is low the time in learning increases, whereas if it is high the learning time is reduced but the prediction accuracy decreases. The learning rate is set as 0.001. Other parameters are mini-batch size and epoch. The above Table 2 shows the different mini-batch size used with corresponding accuracy obtained. Mini-batch size is the amount of data that needs to be used for a single iteration. The value after comparison is considered as 128, which produces the highest accuracy in short time. Epoch determines the number of forward and backward pass required for iterations. Here, different epoch values such as 5, 10, 20, and 30 are tried and this value is set to be 20. The two different datasets collected are tested separately using the three models, i.e LeNet-5, ResNet, andAlexNet-24. The input images of 36 compound characters are tested and number of classifications is recorded. The compound characters are classified with the basic character data. The following Table 3 shows that the accuracy obtained for both the basic characters collected from P-ARTS Kayyezhuthu dataset and tested with those of compound characters, AlexNet-24 shows a higher average accuracy level. The Mean Square Error (MSE) rate is 1.58%. Following Table 4 shows the characters, their corresponding misclassified classes, and their percentage of misclassification. The confusion matrix is used for evaluation that produces an accurate level of correct recognition and misclassified character percentage. The average accuracy level obtained is above 98.4%. 2.4. Classification and Model training g Support Vector Machine (SVM) is used for classification instead of Soft-max classifier of CNN for two main reasons. First, SVM reduces the over fitting problem that occurs in CNN. Drop out layer [25] is used for handling the misclassifications. By appropriately tuning the margin control parameter λ over fitting problem is reduced. The λ is selected using cross-validation methods. Secondly, Softmax classifier is made for 1000 class problem, whereas to deal with lesser number of classes, in this research work 80 classes, thus, SVM is more suitable. The goal of SVM is to find the optimum separating hyper plane that maximizes the margin of training data. A multi-class SVM is used here for training the data. It works as follows: Malayalam Handwritten Character Recognition Using AlexNet based Architecture (Ajay James)  398 ISSN: 2089-3272 For a training set (x1, y1), (x2, y2) .... (xn, yn) with labels yi in [1...g], it finds the solution of the following optimization problem during training. 𝑚𝑖𝑛 1 2 ∑𝑊1 ∗𝑊2 𝑔 1 + 𝑎 𝑛∑ᶓ1 𝑛 1 (4) s.t. for all y in [1...g] [x1 w ] >= [xi • wy] + 100 (y1, y) - ξ1 s.t. for all y in [1...g] [xn w ] >= [xi • wy] + 100* (yn, y) - ξ1 𝑚𝑖𝑛 1 2 ∑𝑊1 ∗𝑊2 𝑔 1 + 𝑎 𝑛∑ᶓ1 𝑛 1 (4) s.t. for all y in [1...g] [x1 w ] >= [xi • wy] + 100* (y1, y) - ξ1 s.t. for all y in [1...g] [xn w ] >= [xi • wy] + 100* (yn, y) - ξ1 where, a is the regularization parameter which adjusts the margin size and training error. Δ(yn,y) is the loss function. If yn=y then it returns 0 else 1. where, a is the regularization parameter which adjusts the margin size and training error. Δ(yn,y) is the loss function. If yn=y then it returns 0 else 1. In general, a two-class classifier is built over a feature vector (x, y) which consists of input features and class of the datum. At test time classifier chooses the class. 𝑦= 𝑎𝑟𝑔𝑚𝑎𝑥 𝑌1𝑊1 (5) 𝑦= 𝑎𝑟𝑔𝑚𝑎𝑥 𝑌1𝑊1 (5) Finally, the classifier classifies these data using 80 classes. As mentioned, FC7 layer consist of 4096 connections of neurons and about 122,880 different features of each character are used as training features. That is about 4096 x 122,880 features are fed as input to the SVM classifier. Gradient features of each character are considered to calculate the feature vector values. SVM uses feature vector values for classification. 4. CONCLUSION Recognising Malayalam handwritten characters is one of the challenging research areas. In general, the handwritten characters vary in writing style, from person to person, thus, an automated feature extraction process makes the Malayalam character recognition easier. Compound characters of Malayalam are most prone to misclassification with their basic characters. A dataset consisting of 90,000 basic characters are collected from P-ARTS Kayyezhuthu dataset, and 100,000 compound characters are newly developed in this research work manually using different augmentation methods. In this research experiment an overall training accuracy of 99.9% and a testing accuracy of 98.42% are achieved. Several combinations are tried out withvarious functions and methods in this research work. These methods are selected by accuracy and processing time for executing the data. A comparison of the proposed new model AlexNet-24 architecture is made with two other existing recent models LeNet-5 and ResNet. Also, a complete Malayalam character dataset is newly developed by these basic and compound characters made available through P-ARTS Kayyezhuthu drive. The experimental results show that the proposed model AlexNet-24 outperforms other recent existing models LeNet-5 and ResNet. 3. RESULTS AND ANALYSIS The model is tested several times with different iterations, and a different number of test images are used. A consistent accuracy level of 98.42% is obtained in most of the cases. The experiment is carried out in MATLAB 2017a. IJEEI, Vol. 6, No. 4, December 2018: 393 – 400 Table 1. Comparison of raw data set with Pre-processed data set CNN Raw data accuracy Pre-processed data accuracy LeNet-5 85.30% 87.54% ResNet 96.33% 97.13% AlexNet-24 96.42% 98.42% Table 1. Comparison of raw data set with Pre-processed data set CNN Raw data accuracy Pre-processed data accuracy LeNet-5 85.30% 87.54% ResNet 96.33% 97.13% AlexNet-24 96.42% 98.42% Table 1. Comparison of raw data set with Pre-processed data set CNN Raw data accuracy Pre-processed data accuracy LeNet-5 85.30% 87.54% ResNet 96.33% 97.13% AlexNet-24 96.42% 98.42% IJEEI, Vol. 6, No. 4, December 2018: 393 – 400 399 IJEEI  ISSN: 2089-3272 Table 2. Accuracy Vs training time with Different batch size Mini batch size Accuracy Training time (s) 16 98.21 1208 32 98.29 1190 64 98.34 1233 128 98.40 0956 256 98.39 1075 Table 2. Accuracy Vs training time with Different batch size Mi i b h i A T i i i ( ) Table 3. Comparison of three methods using both two datasets CNN Models Accuracy of data set from P-ARTS Kayyezhuthu Accuracy of compound characters collected LeNet-5 85.25% 87.54% AlexNet-24 98.74% 98.42% ResNet 94.65% 97.13% Table 4. Misclassification table [1] Alex Krizhevsky, IlyaSutskever, "Geoffrey E. Hinton, ImageNet Classification with Deep Convolutional Neural Networks," Proceedings of the 25th International Conference on Neural Information Processing Systems, Lake Tahoe, NV, pp. 1097-1105, Dec 2012. REFERENCES [1] Alex Krizhevsky, IlyaSutskever, "Geoffrey E. Hinton, ImageNet Classification with Deep Convolutional Neural Networks," Proceedings of the 25th International Conference on Neural Information Processing Systems, Lake Tahoe, NV, pp. 1097-1105, Dec 2012. [2] Angel Morera, Ángel Sánchez, José Francisco Vélez, and Ana Belén Moreno., "Gender and Handedness Prediction from Offline Handwriting Using Convolutional Neural Networks," HindawiComplexity, vol. 2, 2018. [3] LI Pengchao, PENG Liangrui, WEN Juan., "Rejecting Character Recognition Errors Using CNN Based Confidence Estimation," Chinese Journal of Electronics, vol. 25(3), 2016. from Offline Handwriting Using Convolutional Neural Networks," HindawiComplexity, vol. 2, 2018. [3] LI Pengchao, PENG Liangrui, WEN Juan., "Rejecting Character Recognition Errors Using CNN Based Confidence Estimation," Chinese Journal of Electronics, vol. 25(3), 2016. Malayalam Handwritten Character Recognition Using AlexNet based Architecture (Ajay James) ] Angel Morera, Ángel Sánchez, José Francisco Vélez, and Ana Belén Moreno., "Gender and Handedness Predicti from Offline Handwriting Using Convolutional Neural Networks," HindawiComplexity, vol. 2, 2018. ] LI Pengchao, PENG Liangrui, WEN Juan., "Rejecting Character Recognition Errors Using CNN Based Confiden Estimation," Chinese Journal of Electronics, vol. 25(3), 2016. , , pp , [2] Angel Morera, Ángel Sánchez, José Francisco Vélez, and Ana Belén Moreno., "Gender and Handedness Prediction from Offline Handwriting Using Convolutional Neural Networks," HindawiComplexity, vol. 2, 2018. REFERENCES [1] Alex Krizhevsky, IlyaSutskever, "Geoffrey E. Hinton, ImageNet Classification with Deep Convolutional Neural Networks," Proceedings of the 25th International Conference on Neural Information Processing Systems, Lake Tahoe, NV, pp. 1097-1105, Dec 2012. [2] Angel Morera, Ángel Sánchez, José Francisco Vélez, and Ana Belén Moreno., "Gender and Handedness Prediction from Offline Handwriting Using Convolutional Neural Networks," HindawiComplexity, vol. 2, 2018. [3] LI P h PENG Li i WEN J "R j i Ch R i i E U i CNN B d C fid REFERENCES Malayalam Handwritten Character Recognition Using AlexNet based Architecture (Ajay James)  400 ISSN: 2089-3272 ISSN: 2089-3272 [4] Tiancheng Sun, Yulong Wang, Jian Yang, and Xiaolin Hu., "Convolution Neural Networks With Two Pathways for Image Style Recognition," IEEE Transactions on Image Processing, vol. 26(9), Sep 2017. g y g , g g, ( ), p ] HulyaYalcin, SalarRazavi., "Plant Classification using Convolutional Neural Networks, "Fifth Internation Conference on Agro-Geoinformatics (Agro-Geoinformatics), IEEE, 2016. [6] Lidan SHANG, " Qiushi YANG, Jianing WANG, Shubin LI, WeiminLEI, "Detection of Rail Surface Defects Based on CNNImage Recognition and Classification," International Conference on Advanced Communications Technology (ICACT)(0975–8887), Feb 2018. gy [7] Li Chen, Song Wang, Wei Fan, Jun Sun, Satoshi Naoi., "Beyond Human Recognition: A CNN-Based Framework for Handwritten Character Recognition," 3rdIAPR Asian Conference on Pattern Recognition, 2015. [8] G.Pirlo and D. Impedovo, "Fuzzy-Zoning-Based Classification for Handwritten Characters," IEEE Transactions on Fuzzy Systems, vol. 19(4), Aug 2011. [9] Dileep Kumar Patel, Tanmoy Som, Sushil Kumar, Yadav, Manoj Kumar Singh., "Handwritten Character Recognition using Multi-Resolution Technique and Euclidean Distance Metric," Journal of Signal and Information Processing, vol. 3, pp. 208-214, 2012. [10] Ashok Kumar, Pradeep Kumar Bhatia., "Offline Handwritten Character Recognition Using Improved Back- Propagation Algorithm," Proceedings of 2ndInternational Conference on Emerging Trends in Engineering and Management, ICETEM, 2013. g [11] Shilpy Bansal, Mamta Garg, Munish Kumar., "A Technique for Offline Handwritten Character Recognition," IJCAT International Journal of Computing and Technology, vol. 1(2), pp. 2415-1521, Mar 2014. [12] Shanjana C., Ajay James., "Offline Recognition of Malayalam Handwritten Text," 8th International Conference Interdisciplinarity in Engineering INTER-ENG 2014, Tirgu-Mures Romania, pp. 22-28, 2014. [13] Durjoy Sen Maitra, Ujjwal Bhattacharya, and Swapan K. Parui., "CNN Based Common Approach to Handwritten Character Recognition of Multiple Scripts," 13thInternational Conference on Document Analysis and Recognition (ICDAR), 2015. [14] M.A.H Akhand, Mahtab Ahamed, M.M. HafizurRahman., "Multiple Convolutional Neural Network Training for Bangla Handwritten Numeral Recognition," International Conference on Computer & Communication Engineering, 2016. [15] S.M. Sofiqul Islam, Shanto Rahman, Emon Kumar Dey., "Application of Deep Learning to Computer Vision: A Comprehensive Study," 5thInternational Conference on Informatics, Electronics and Vision (ICIEV), 2016. [16] Jaspreet Kaur, B. S. Dhaliwal, S. S. Gill., "Offline Handwritten Gurmukhi Character Recognition using Particle Swarm Optimized Neural Network," IJCA Proceedings on International Conference on Advances in Emerging Technology (ICAET) 2016 (12), pp. 35-41, Sep 2016. REFERENCES [17] Mohamed Elleuch, Rania Maalej, and Monji Kherallah., "A New Design Based-SVM of the CNN Classifier Architecture with Dropout for Offline Arabic Handwritten Recognition," The International Conference on Computational Science (ICCS), vol. 80, 2016. [18] Shenzhen Gu, Lu Ding, Yue Yang, and Xinyi Chen., "A New Deep Learning Method Based on AlexNet Model and SSD Model for Tennis Ball Recognition," IEEE 10thInternational Workshop on Computational Intelligence and Applications,Hiroshima, Japan, pp. 11-12, Nov 2017. [19] Ahmed El-Sawy, Mohamed Loey, Hazem EL-Bakry., "Arabic Handwritten Characters Recognition using Convolutional Neural Network," WSEASTransactions on Computer Research, E-ISSN. 2415-1521, vol. 5, 2017. [20] Darmatasia, Mohamad Ivan Fanany., "Handwriting Recognition on Form Document Using Convolutional Neural Network and Support Vector Machines (CNN-SVM)," Fifth International Conference on Information and Communication Technology (IcoICT), 2017. [21] Pranav P. Nair, Ajay James, C.Saravanan., "Malayalam Handwritten Character Recognition Using Convolutional Neural Network," International Conference on Inventive Communication and Computational Technologies (ICICCT), 2017. [22] Gauri Katiyar, Ankita Katiyar, Shabana Mehfuz., "Off-Line Handwritten Character Recognition System Using Support Vector Machine," American Journal of Neural Networks and Applications, vol. 3(2), pp. 22-28, 2017. pp f pp ( ) pp 3] Aarthna Maheshwari, Khushboo Shah., "Offline Handwritten Character Recognition using MLPNN and PS Algorithm," International Journal of Innovative Science and Research Technology, vol. 3(2), Feb 2018. [23] Aarthna Maheshwari, Khushboo Shah., "Offline Handwritten Character Recognition using MLPNN and PSO Algorithm," International Journal of Innovative Science and Research Technology, vol. 3(2), Feb 2018. [24] https://drive.google.com/drive/folders/0B1eLyjUeuERZWUVIOU9OZm40RHc [24] https://drive.google.com/drive/folders/0B1eLyjUeuERZWUVIOU9OZm40RHc ://drive.google.com/drive/folders/0B1eLyjUeuERZWUVIO [25] N. Srivastava, G. Hinton, Alex Krizhevsky, IlyaSutskever, RuslanSalakhutdinov., "Dropout: A Simple way to prevent Neural Network from Overfitting," Journal of Machine Learning Research, vol. 15, pp. 1929-1958, 2014. IJEEI, Vol. 6, No. 4, December 2018: 393 – 400
https://openalex.org/W3119140885	https://karolinum.cz/data/clanek/8263/Theol_10_1_0023.pdf	English	null	Benedictional Ecclesiology: Renewal of the Church from Worship Experiences?	Acta Universitatis Carolinae. Theologica	2,020	cc-by	7,147	AUC AUC THEOLOGICA 2020 – Vol. 10, No. 1 Pag. 23–37 © 2020 The Authors. 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 author and source are credited. ( g , ), 3 Cf. the examples from different epochs of liturgical history in Jürgen Bärsch and Bene- dikt Kranemann in coop. with Winfried Haunerland and Martin Klöckener, eds., Ge- schichte der Liturgie in den Kirchen des Westens: Rituelle Entwicklungen, theologische Konzepte und kulturelle Kontexte. 2 Bände (Münster: Aschendorff, 2018); on the mani- fold development processes cf. Albert Gerhards and Benedikt Kranemann, eds., Dyna- mik und Diversität des Gottesdienstes: Liturgiegeschichte in neuem Licht (Quaestiones Disputatae 289) (Freiburg/Br.: Herder, 2018). ( g , ), 2 On the connection between lex orandi and lex credendi thus invoked, cf. Julia Knop, Ecclesia orans: Liturgie als Herausforderung für die Dogmatik (Freiburg/Br.: Her- der, 2012); Julia Knop, ‘Liturgie als Thema und Herausforderung der Dogmatik: zur systematisch-theologischen Relevanz des Gottesdienstes,’ in Sakramentale Feier und theologia prima: der Vollzug der Liturgie als Anfang und Mitte der Theologie, Kloster- neuburger Symposion 2018 (Pius-Parsch-Studien 16), eds. Andreas Redtenbacher and Markus Schulze (Freiburg/Br.: Herder, 2019), 53–73. was revised for publication. For a more comprehensive German version see: Julia Knop and Benedikt Kranemann, ‘Benediktionale Ekklesiologie – Welche Kirche baut der Segen auf?’, Segensfeiern in der offenen Kirche: Neue Gottesdienstformen in the- ologischer Reflexion (Quaestiones Disputatae 305), eds. Julia Knop and Benedikt Kranemann (Freiburg/Br.: Herder, 2020), 248–265. ABSTRACT The article was written as a conversation between Dogmatics and Litur- gical Studies. It asks what kind of self-understanding of the church becomes visible in the blessing ceremonies that have been newly adopted in recent years. In the background, there are considerations that such liturgical celebrations shape the image of the Church in time and society. At the same time, it is discussed how such new liturgical celebrations develop traditional concepts of ecclesiology. The bless- ing ceremonies discussed below, such as the blessing of couples on Valentine’s Day or the blessing of same-sex couples, are not regulated by the official Church. They are part of a changing liturgical practice from the ecclesial base. This development of the Church on a practical and theoretical level deserves theological reflection. Here God’s message of salvation is proclaimed in new liturgical formats. In these liturgies, Christians approach a changing society with the liturgy. The theological discussion about such celebrations is gaining importance in German theology. It is also reflected in the dioceses which are opening up to the inner-church reform process. In particular, it plays a role in the Synodaler Weg, which has recently begun in Germany, a process of reform within the Church. Key words Ecclesiology; Blessing; Blessing ceremony; New liturgical formats; Liturgical the- ology DOI: 10.14712/23363398.2020.42 DOI: 10.14712/23363398.2020.42 * The following essay is based on a lecture that was given on 21st March 2019 at a joint conference of the Theological Research College at the University of Erfurt and the Faculty of Catholic Theology of Charles University in Prague at the Dominican Mon- astery in Prague. The topic of the 5th joint conference was: ‘Sacraments in Context. The Theological Disciplines Facing New Questions in the 21st Century’. The text 23 JULIA KNOP AND BENEDIKT KRANEMANN 4 For a description of the history of blessings see: Adolph Franz, Die kirchlichen Be- nediktionen im Mittelalter (Freiburg/Br.: Herder, 1909; reprint Bonn: Nova und ve- tera, 2006); on the history of the corresponding liturgical books and the celebrations described in them, as on a theology of blessing cf. Florian Kluger, Benediktionen: Studien zu kirchlichen Segensfeiern (Studien zur Pastoralliturgie 31) (Regensburg: Pustet, 2011). 5 Biblical theological foundations, models of practice and further theological perspec- tives are put together in Julia Knop and Benedikt Kranemann, eds., Segensfeiern in der offenen Kirche: Neue Gottesdienstformen in theologischer Reflexion (Quaestiones Disputatae 305) (Freiburg/Br.: Herder, 2020). 1. Liturgy as an Expression of Church’s Self-understanding 1. Liturgy as an Expression of Church’s Self-understanding In the liturgy of the Church, what and how the Church believes, and how she understands herself is expressed, practised, and staged. Faith and celebration shape each other mutually; they form the ecclesial self-understanding of an era. The concrete form of the service presents a certain image of the church and at the same time has an effect back on theological theory formation and church concept development. What the church believes and how it expresses this faith in the service is thereby not once and for all (given), but the subject of active shap- ing and historically in flux.2 Non-simultaneity between the local and the universal church, but also between the positions of the different actors and between different theological disciplines is not the excep- tion but the rule, and not a drama. The local church can react more precisely to social and church developments.3 Needs for development in teaching, theory, and liturgy can be grasped more sharply and bet- ter when reflected on the spot than is possible and appears urgent to a distant central office that is pressing for unity in the universal church. Scientific theology and church leadership take on different roles and work with different competencies; pastoral practice, in turn, needs and shapes Christian contemporaneity with its respective culture and society. Theoretically and institutionally perceived so far rather reserved- ly, blessing ceremonies, i.e., ‘benedictions’, have for some time played 24 BENEDICTIONAL ECCLESIOLOGY an increasingly important role in the pastoral ministry of the Cath- olic Church in Germany in a very diverse way. In them, the Church presents itself in a partly unusual, yet creative and affectionate way. There are still traditional blessings in the course of the Church and calendar year or related to unique occasions such as the house bless- ing for moving in.4 However, new forms of celebration are also being developed and tested.5 In this way, not only pastoral workers but also communities as a whole react to social developments in the shaping and perception of human life in partnership and family. They also take into account changing sociological conditions in religion and describe the task of the church and its ritual practice more openly: Blessings are celebrated on a denominational or ecumenical basis. There are also group- and gender-related blessings. ) ( ) 6 Cf. among others Michael Domsgen and Emilia Handke, eds., Lebensübergänge beglei- ten: was sich von Religiösen Jugendfeiern lernen lässt (Leipzig: Evangelische Verlags- anstalt, 2016); Hans Gerald Hödl, Johann Pock and Teresa Schweighofer, eds., Christli- che Rituale im Wandel: Schlaglichter aus theologischer und religionswissenschaftlicher Sicht (Wiener Forum für Theologie und Religionswissenschaft 14) (Göttingen: V&R unipress, 2017); Ulrike Wagner-Rau and Emilia Handke, eds., Provozierte Kasualpra- xis: Rituale in Bewegung (Praktische Theologie heute 166) (Stuttgart: Kohlhammer, 2019); also the contributions in the booklet Diakonische Kirche in säkularer Gesell- schaft, Internationale katholische Zeitschrift Communio 47, no. 3 (2018). 5 , f , ( ) 7 Cf. the basic text of the ad hoc working group ‘Segensfeiern’ Sachbereich 5 ‘Familie’ of the Zentralkomitees der deutschen Katholiken, last modified November 27, 2019, https://bit.ly/32wprku; cf. the annex to this text ‘Segen schenken – Segensfeiern für gleichgeschlechtliche Paare,’ accessed December 30, 2019, https://bit.ly/399XvnZ; also the essays in Stephan Loos, Michael Reitemeyer and Georg Trettin, eds., Mit dem Segen der Kirche?: Gleichgeschlechtliche Partnerschaft im Fokus der Pastoral (Frei- burg/Br.: Herder, 2019); Ewald Volgger and Florian Wegscheider, eds., Benediktion Disputatae 305) (Freiburg/Br.: Herder, 2020). 6 Cf. among others Michael Domsgen and Emilia Handke, eds., Lebensübergänge beglei- ten: was sich von Religiösen Jugendfeiern lernen lässt (Leipzig: Evangelische Verlags- anstalt, 2016); Hans Gerald Hödl, Johann Pock and Teresa Schweighofer, eds., Christli- che Rituale im Wandel: Schlaglichter aus theologischer und religionswissenschaftlicher Sicht (Wiener Forum für Theologie und Religionswissenschaft 14) (Göttingen: V&R unipress, 2017); Ulrike Wagner-Rau and Emilia Handke, eds., Provozierte Kasualpra- xis: Rituale in Bewegung (Praktische Theologie heute 166) (Stuttgart: Kohlhammer, 2019); also the contributions in the booklet Diakonische Kirche in säkularer Gesell- schaft, Internationale katholische Zeitschrift Communio 47, no. 3 (2018). 7 Cf. the basic text of the ad hoc working group ‘Segensfeiern’ Sachbereich 5 ‘Familie’ of the Zentralkomitees der deutschen Katholiken, last modified November 27, 2019, https://bit.ly/32wprku; cf. the annex to this text ‘Segen schenken – Segensfeiern für gleichgeschlechtliche Paare,’ accessed December 30, 2019, https://bit.ly/399XvnZ; also the essays in Stephan Loos, Michael Reitemeyer and Georg Trettin, eds., Mit dem Segen der Kirche?: Gleichgeschlechtliche Partnerschaft im Fokus der Pastoral (Frei- burg/Br.: Herder, 2019); Ewald Volgger and Florian Wegscheider, eds., Benediktion von gleichgeschlechtlichen Partnerschaften (Schriften der Katholischen Privat-Univer- sität Linz 8) (Regensburg: Pustet, 2020). 8 The term ‘rite deaconia’ (Ritendiakonie), which goes back to Paul Michael Zulehner, is used here; cf. Paul M. Zulehner, ‘Ritendiakonie,’ in Die diakonale Dimension der Liturgie (Quaestiones Disputatae 218), eds. Benedikt Kranemann, Thomas Sternberg and Walter Zahner (Freiburg/Br.: Herder, 2006), 271–283; Johannes Pock, ‘Gottes- dienste „im Vorhof der Heiden“?: pastoraltheologische Überlegungen zur Liturgie zwi- schen gefeiertem Mysterium und Ritendiakonie,’ Heiliger Dienst 67 (2013), 246–258. 9 Depending on the liturgy, different ministers and role bearers take on the leadership of a service, which must, however, be at the service of the celebrating community. This does not change the fact that there are always precedents for the liturgy which contradict this. For liturgical-theological reasons, a form of liturgy should be main- tained which shows the Church as a community of faith and which involves all the baptised. Where liturgical orders contradict this, they themselves should be called into question, instead of making theological-legal misunderstandings and problems the norm. g y , g ( ), 9 Depending on the liturgy, different ministers and role bearers take on the leadership of a service, which must, however, be at the service of the celebrating community. This does not change the fact that there are always precedents for the liturgy which contradict this. For liturgical-theological reasons, a form of liturgy should be main- tained which shows the Church as a community of faith and which involves all the baptised. Where liturgical orders contradict this, they themselves should be called into question, instead of making theological-legal misunderstandings and problems the norm. 1. Liturgy as an Expression of Church’s Self-understanding Even people who do not belong to any religious community sometimes ask in a special life situation, for example, after the birth of a child, during adolescence, at the beginning or end of a partnership, to be granted the closeness of God in a blessing ceremony.6 Finally, there are blessing ceremonies for same-sex cou- ples with a long history. The theological debate in science and church is somewhat lagging behind this latter practice7 but is slowly gaining 25 JULIA KNOP AND BENEDIKT KRANEMANN momentum. The form, significance and theological legitimacy of such celebrations are now also being discussed at the diocesan level. momentum. The form, significance and theological legitimacy of such celebrations are now also being discussed at the diocesan level. In all these celebrations, the church acts. This is also the case when no approved liturgical form is taken as a basis, or when we can observe the differences and non-simultaneousness between the differ- ent levels or actors – between pastoral practice and academic reflec- tion, between the moral teaching of the Church and liturgy, between ordination-bound services and those presided over by an unordained person.8 As the prayer of the Church is the prayer of the baptised, the worship of the Church is the worship of the baptised. All the baptised bear the liturgy without prejudice to the specific duties of ministers or the respective roles in the liturgy.9 The close relationship and mutu- al opening up of the Church and liturgy, which has been repeatedly emphasised by liturgical science since the Council, cannot be reduced to sacramental worship services; it applies to every liturgy and is there- fore also applicable to blessings. What self-understanding of the church becomes visible in the men- tioned blessing celebrations of a new and different kind? How do they shape the image of the church in our time and society? To what extent do these blessings – in all their diversity – further develop traditional church self-understanding and traditional ecclesiological concepts? In order to sharpen the question as to what image of the church is shown in the various liturgical celebrations, we will pick out blessings for which rituals have only recently been developed and practised in Germany that have not yet been regulated by Church authorities. 1. Liturgy as an Expression of Church’s Self-understanding Here church development takes place on a practical and theoretical level: 26 BENEDICTIONAL ECCLESIOLOGY in view of new or newly identified needs, new liturgical formats are also emerging in which God’s message of salvation is proclaimed and Christians place themselves at the service of a securely changing soci- ety. An example of this is a blessing ceremony that was held in Erfurt in 2000 and is now widespread in German-speaking countries: the blessing ceremony on Valentine’s Day. Finally, blessing ceremonies for same-sex couples, as they are currently being discussed in the Catholic Church in Germany,10 will be questioned about the church’s self-image that is intended, and practised and traditional church images will be corrected and developed further. In the theological discussion, these and similar celebrations have so far been considered more from their meaning for the respective addressees than from their ecclesiological relevance – wrongly, as it will turn out. The discussion will conclude with reflections from a liturgical as well as a systematic theologi- cal perspective, which will lead to theses that will stimulate further discussion. 10 Finally, in December 2019 the chairman of the German Bishops’ Conference, Car- dinal Reinhard Marx, expressed himself positively about a blessing ‘in the sense of a pastoral accompaniment’ (im Sinne einer seelsorglichen Begleitung); see ‘Marx: Homosexuelle Paare können einen Segen bekommen,’ accessed December 12, 2019, https://bit.ly/30kuCRQ. 11 The celebration was published by the present Auxiliary Bishop of Erfurt, Reinhard Hauke, who designed this celebration. Cf. Reinhard Hauke, Herzlich eingeladen zum Fest des Glaubens …: Projekte für Christen und Nicht-Christen (Leipzig: Benno, 2009), 63–67. Also: Bianka Piontek, ‘Valentinstag in Erfurt: ein Heiliger kehrt zurück in die Kirche,’ in Liturgie mit offenen Türen: Gottesdienst auf der Schwelle zwischen Kirche und Gesellschaft (Beiträge zu Liturgie und Spiritualität 13), eds. Irene Mildenberger and Wolfgang Ratzmann (Leipzig: Evangelische Verlagsanstalt, 2005), 165–170; Tho- mas Klie, ed., Valentin, Halloween & Co.: zivilreligiöse Feste in der Gemeindepraxis (Leipzig: Evangelische Verlagsanstalt, 2006); Stephan Schatzler, Riten und Rituale der Postmoderne: am Beispiel des Bistums Erfurt (München: GRIN, 2012); Birgit Jeg- gle-Merz, ‘Segnungsfeiern am Valentinstag: Eine Initiative aus dem Bistum Erfurt mit weitreichender Ausstrahlung,’ in Segensfeiern in der offenen Kirche: Neue Gottesdienst- formen in theologischer Reflexion, 149–176. 10 Finally, in December 2019 the chairman of the German Bishops’ Conference, Car- dinal Reinhard Marx, expressed himself positively about a blessing ‘in the sense of a pastoral accompaniment’ (im Sinne einer seelsorglichen Begleitung); see ‘Marx: Homosexuelle Paare können einen Segen bekommen,’ accessed December 12, 2019, https://bit.ly/30kuCRQ. 2. Blessing of Couples on Valentine’s Day What ecclesiology is expressed in a blessing ceremony to which couples from very different life situations are publicly invited, people who are ‘on their way in partnership’? The blessing ceremony on Val- entine’s Day was first celebrated in Erfurt in 2000.11 Today it is offered in many places in the German-speaking area, but its rite is neither 27 JULIA KNOP AND BENEDIKT KRANEMANN officially regulated nor necessarily uniform.12 In order to elaborate on the ecclesiology inherent in this celebration of the blessing, a worship service template from 2005 is used. Ecclesiological markers can be found on different levels. The question is: What is the significance of verbal and non-verbal rites? Who is involved, what roles are there? From where can we see ourselves commissioned to give God’s bless- ing to other people? Which forms, which texts, which perspectives are chosen and why? How do you deal with role models from approved forms? How does ecumenical experience come into play? Is a church space used? Is there an invitation by the church and an ecclesiastically legitimised leadership? officially regulated nor necessarily uniform.12 In order to elaborate on the ecclesiology inherent in this celebration of the blessing, a worship service template from 2005 is used. Ecclesiological markers can be found on different levels. The question is: What is the significance of verbal and non-verbal rites? Who is involved, what roles are there? From where can we see ourselves commissioned to give God’s bless- ing to other people? Which forms, which texts, which perspectives are chosen and why? How do you deal with role models from approved forms? How does ecumenical experience come into play? Is a church space used? Is there an invitation by the church and an ecclesiastically legitimised leadership? The celebration of the blessing corresponds to the basic structure that is constitutive for liturgies of Christian churches: the dialog- ical moment of God’s address and the people’s response or, in oth- er words, the moment of the encounter between God and people is clearly expressed. Blessing is or rather shapes a relationship between God and man, as is already clear in the Bible. The celebration of the blessing opens a symbolic space into which people are invited who wish to come before God with their lives. 12 According to the experience of these celebrations, ‘ambiguity and diversity’ and thus tolerance of ambiguity obviously do not contradict being a church. Cf. Thomas Bauer, Die Vereindeutigung der Welt: über den Verlust an Mehrdeutigkeit und Vielfalt (Ditzin- gen: Reclam, 2018). 13 For the biblical assurance of a theology of blessing see Stephan Winter, ‘Theologie und Praxis des Segens,’ in Segensfeiern in der offenen Kirche: Neue Gottesdienstformen in theologischer Reflexion, 37–55. 2. Blessing of Couples on Valentine’s Day The inviting people – Chris- tians – know that they are motivated and empowered to promise God’s blessing to others through biblical tradition and through centuries of blessing practice, but also through everyday religiousness (wishing for the blessing). Behind this is the conviction that all blessings begin with God, that the blessing belongs to the community of God and man, and that the blessing densely expresses the hope of God’s nearness in the lives of people.13 The liturgy takes place in a church room. It is led by a Lutheran pas- tor and a Roman-Catholic priest. Married couples give witness to their partnership. According to the schedule, musicians are also involved. The role of the two clergymen is to lead the ecumenical blessing ser- vice. This includes opening and closing, a prayer at the beginning, the viewing and interpretation of images, an invitation to the Our Father, 28 BENEDICTIONAL ECCLESIOLOGY and the Aaron Blessing as the final blessing. The married couples tell about their relationship, say the intercessions together with the cler- gy, and at the end of the service – after the Aaron blessing – bless the couples who wish to receive this. Occasion, space, actors, language, and sign actions, and even the occasion signal that the church is acting here. The distribution of roles in the celebration is differentiated and not fixed on the ordained. The two ordained persons open and close the service. The reports on the partnership that are important for the cel- ebration, however, are contributed by the couples. Their competence based on life experience is decisive for the performance of this task in the service. Baptised persons perform central liturgical tasks such as the intercessory prayer and the prayer of blessing over the couples independently of a ministry or ordination. The ‘invitation to blessing’ states that it is about ‘a request to God for the success of the partner- ship’. The fact that clergymen and married couples bless equally shows that both have the ability to do so and that this does not depend on an ordination. The prerequisite for being able to give the blessing is bap- tism. It forms the theological foundation on which liturgical action is based. 14 The invitation to the blessing does not speak of baptism, but formulates that the love of God biblically seen becomes visible in his blessing. On the other hand, it estab- lishes a connection between the experience of love and its transmission. From here, references to baptism can be established, especially if one understands baptism as God’s care and love for humankind. 15 Cf. Liturgische Institute Salzburg, ed., Benediktionale: Studienausgabe für die katholischen Bistümer des deutschen Sprachgebietes, erarbeitet von der Internatio- nalen Arbeitsgemeinschaft der Liturgischen Kommissionen im deutschen Sprach- bi t (T i Zü i h Ei i d l [ ] B i 1979) (P t llit i h R ih i 14 The invitation to the blessing does not speak of baptism, but formulates that the love of God biblically seen becomes visible in his blessing. On the other hand, it estab- lishes a connection between the experience of love and its transmission. From here, references to baptism can be established, especially if one understands baptism as God’s care and love for humankind. 15 Cf. Liturgische Institute Salzburg, ed., Benediktionale: Studienausgabe für die katholischen Bistümer des deutschen Sprachgebietes, erarbeitet von der Internatio- nalen Arbeitsgemeinschaft der Liturgischen Kommissionen im deutschen Sprach- gebiet (Trier, Zürich, Einsiedeln [u.a.]: Benziger, 1979) (Pastoralliturgische Reihe in p 17 However, there are inner-church initiatives which should lead to a recognition of such blessing ceremonies by the church leadership, for example, by the Central Committee of German Catholics; see the texts mentioned in footnote 5. An instruc- tive presentation, phenomenological, and theological analysis of concrete worship forms according to which partnership blessings are made in Germany are offered by Dominik Bodenstein and Andreas Krebs, ‘Riten zur Segnung gleichgeschlechtlicher Partnerschaften’, in Segensfeiern in der offenen Kirche: Neue Gottesdienstformen in theologischer Reflexion, 195–210. 2. Blessing of Couples on Valentine’s Day Baptised persons are not only able to speak of God – for exam- ple, by giving a public testimony of their faith – they are also empow- ered to give God’s love and attention to others.14 Linguistically, the blessing service constitutes a community from the beginning. The introductory prayer already in the first sentence speaks of an assembly and emphasises the community aspect. Twice the praying ‘we’ is mentioned. In the doxology, the gathered congre- gation (‘we’) praises God ‘who meets us as a good and lasting com- munity’. The blessing is not an individual event but is connected with a community that relates to God and is defined from God. We encounter various basic liturgical elements which also occur regularly in church blessing services from the Book of Blessings:15 read- 29 JULIA KNOP AND BENEDIKT KRANEMANN ing of Scripture (here, 1 Cor 13:1–11) – (intercessory) prayer – blessing. Incidentally, the testimonies of the couples precede the proclamation and supplication. They illustrate how the act of blessing is carried out against the background of these experiences. Life and faith, experi- ence and tradition meet and interpret each other. The partnership and common life of (married) couples16 are placed under the blessing of God. The request for a blessing is addressed to God. It is presented without restriction or condition and, according to the presentation, is not subject to any institutional restrictions or moral tutelage. It is spo- ken into the individual life situation, which in turn is not rigidly but dynamically perceived. The human desire and need for blessings is taken seriously. ing of Scripture (here, 1 Cor 13:1–11) – (intercessory) prayer – blessing. Incidentally, the testimonies of the couples precede the proclamation and supplication. They illustrate how the act of blessing is carried out against the background of these experiences. Life and faith, experi- ence and tradition meet and interpret each other. The partnership and common life of (married) couples16 are placed under the blessing of God. The request for a blessing is addressed to God. It is presented without restriction or condition and, according to the presentation, is not subject to any institutional restrictions or moral tutelage. It is spo- ken into the individual life situation, which in turn is not rigidly but dynamically perceived. The human desire and need for blessings is taken seriously. Verbindung mit der Zeitschrift „Gottesdienst“). Cf. Kluger, Benediktionen. The follow- ing explanations always refer to the German edition of the Benediktionale. 16 The text is not clear here. Sometimes it talks about couples, sometimes about married couples. Verbindung mit der Zeitschrift „Gottesdienst“). Cf. Kluger, Benediktionen. The follow- ing explanations always refer to the German edition of the Benediktionale. 16 The text is not clear here. Sometimes it talks about couples, sometimes about married couples. 17 However, there are inner-church initiatives which should lead to a recognition of such blessing ceremonies by the church leadership, for example, by the Central Committee of German Catholics; see the texts mentioned in footnote 5. An instruc- tive presentation, phenomenological, and theological analysis of concrete worship forms according to which partnership blessings are made in Germany are offered by Dominik Bodenstein and Andreas Krebs, ‘Riten zur Segnung gleichgeschlechtlicher Partnerschaften’, in Segensfeiern in der offenen Kirche: Neue Gottesdienstformen in theologischer Reflexion, 195–210. 19 Cf. Peter Schallenberg, ‘Homosexualität aus Sicht des katholischen Lehramtes,’ in Mit dem Segen der Kirche?: Gleichgeschlechtliche Partnerschaft im Fokus der Pasto- ral, 71–85; Stephan Goertz, ‘Gleichgeschlechtliche Sexualität und Partnerschaft: moraltheologische Gründe für eine Revision der kirchlichen Lehre,’ in „Wer bin ich, ihn zu verurteilen?“, 86–98; Julia Knop, Beziehungsweise: Theologie der Ehe, Part- nerschaft und Familie (Regensburg: Pustet, 2019), 86–101; Jochen Sautermeister, ‘Segnung gleichgeschlechtlicher Paare: strukturanalytische Beobachtungen zent- raler Argumentationstypen aus theologisch-ethischer Sicht,’ in Mit dem Segen der Kirche: Die Segnung gleichgeschlechtlicher Partnerschaften in der Diskussion, ed. Andreas Krebs and Matthias Ring (Bonn: Alt-Katholischer Bistumsverlag, 2018), 111–122. p y 21 Congregation for Catholic Education, Instruction Concerning the Criteria for the Dis- cernment of Vocations with regard to Persons with Homosexual Tendencies in view of their Admission to the Seminary and to Holy Orders, accessed December 12, 2019, sec. 2, https://bit.ly/3jcyRYD. 22 Cf. Pope Francis, Post-Synodal Apostolic Exhortation Amoris Lætitia of The Holy Father Francis to Bishops, Priests and Deacons, Consecrated Persons, Christian Mar- ried Couples and all the Lay Faithful on Love in the Family, accessed December 12, 2019, sec. 251, https://bit.ly/2WHZ5IX. 20 Sacred Congregation for the Doctrine of the Faith, Persona humana. Declaration on certain questions concerning sexual ethics, accessed December 12, 2019, sec. 8, https://bit.ly/2CH0iZP. 18 Cf. Stephan Goertz, ed., „Wer bin ich, ihn zu verurteilen?“: Homosexualität und katholische Kirche (Katholizismus im Umbruch 3) (Freiburg/Br.: Herder, 2015). 3. Blessing Ceremonies for Same-sex Couples A dynamic, learning, and open church that is on the way with peo- ple also meets same-sex couples in its blessing ceremonies. They are now offered in many places but are officially still under church edict.17 For, as per the frequently encountered reason, the church may not and cannot bless what it morally condemns. On the other hand, those who celebrate such celebrations refer to more recent assessments of homo- sexuality as they are presented by the human sciences and theology, especially exegesis and moral theology.i Blessing ceremonies for same-sex couples are also justifiable, prac- ticable, and meaningful today from a liturgical-theological point of view because these couples ask for God’s blessing for their love rela- tionship, which they want to live out of their faith. The biblical peri- copes in which violent sexual acts between men are condemned do not meet what is understood in the present day as a love relationship 30 BENEDICTIONAL ECCLESIOLOGY between two people of the same sex.18 Homosexuality is seen today, from a human scientific point of view, as a standard variant of human sexuality. The civil law equality of homosexual and heterosexual part- nerships was fully implemented in Germany in 2017. The Institute of Registered Civil Partnerships has already existed since 2001; since 2017, civil marriage is now also possible. Officially, however, homo- sexuality is still described in church morals19 as ‘pathological’20 and ‘objectively disordered’.21Homosexual relationships and actions, as it says in the catechism, ‘are contrary to the natural law. They close the sexual act to the gift of life. They do not proceed from a genuine affective and sexual complementarity. Under no circumstances can they be approved’. (CCC 2357). Whoever is homosexually inclined is called to chastity according to Roman moral teachings (cf. CCC 2349). In his post-synodal apostolic letter Amoris laetitia, which Francis writes in 2016, there are no analogies between heterosexual marriage and homosexual partnership, ‘absolutely no grounds for considering homosexual unions to be in any way similar or even remotely anal- ogous to God’s plan for marriage and family’.22 The social, medical, ethical, and legal re-evaluation of homosexuality does not still find its reflection on the Church’s side as concerns a substantial development , p y j y 22 Cf. 23 Cf. in detail: Thomas Hieke, ‘Segen: Kraftvermittlung und Eintritt in den Heilsraum Gottes: Zehn Thesen zu Segen/Segnen in der christlichen Bibel,’ in Segensfeiern in der offenen Kirche: Neue Gottesdienstformen in theologischer Reflexion, 15–36, and Stephan Winter, ‘Theologie und Praxis des Segens.’ 24 It can be left open here whether such generalising notions of ‘man’ are acceptable. 25 Cf. Hans-Joachim Sander, ‘Das Möbiusband von Segen und Fluch – eine Fundstelle Gottes: Kirche in prekärer Wechselwirkung mit gleichgeschlechtlichen Partnerschaf- ten,’ in Mit dem Segen der Kirche?: Gleichgeschlechtliche Partnerschaft im Fokus der Pastoral, 101–116. 26 Cf. the anthology resulting from a conference organised by various German dioceses: Mit dem Segen der Kirche?: Gleichgeschlechtliche Partnerschaft im Fokus der Pastoral. 23 Cf. in detail: Thomas Hieke, ‘Segen: Kraftvermittlung und Eintritt in den Heilsraum Gottes: Zehn Thesen zu Segen/Segnen in der christlichen Bibel,’ in Segensfeiern in der offenen Kirche: Neue Gottesdienstformen in theologischer Reflexion, 15–36, and Stephan Winter, ‘Theologie und Praxis des Segens.’ , 26 Cf. the anthology resulting from a conference organised by various German di Mit dem Segen der Kirche?: Gleichgeschlechtliche Partnerschaft im Fokus der P 27 Liturgische Kommission des Katholischen Bistums der Alt-Katholiken in Deutsch- land, ed., Die Feier der Partnerschaftssegnung im Katholischen Bistum der Alt-Katholi- ken in Deutschland (Bonn, 2014), 45; cf. also Andreas Krebs, ‘”In vielfältigen Formen wird seine Liebe in uns sichtbar“: zum Stand der Diskussion um die Pluralisierung von Lebensformen im Katholischen Bistum der Altkatholiken in Deutschland,’ in Mit dem Segen der Kirche: Die Segnung gleichgeschlechtlicher Partnerschaften in der Dis- kussion, 11–27. 3. Blessing Ceremonies for Same-sex Couples Pope Francis, Post-Synodal Apostolic Exhortation Amoris Lætitia of The Holy Father Francis to Bishops, Priests and Deacons, Consecrated Persons, Christian Mar- ried Couples and all the Lay Faithful on Love in the Family, accessed December 12, 2019, sec. 251, https://bit.ly/2WHZ5IX. 31 JULIA KNOP AND BENEDIKT KRANEMANN of its teachings and practice, which would correct previous devaluation and discrimination. Now, ‘blessing’ means literally ‘benedictio’, but not a moral eval- uation by a human institution. Blessing is the praise of God for cre- ation and salvation and the request to God to turn to mankind in salva- tion.23 This cannot be denied to the couples at issue here, especially in view of the new human scientific and theological insights mentioned above. To understand homosexuality as a standard variant of human sexuality means theologically to see it as part of the good, God-willed creation. The theology of blessing, as it is formulated in the Book of Blessings, starts out from a fundamental need of the blessing of man and interprets it as a desire for ‘salvation, protection, happiness and fulfilment’ (Benediktionale, PE 1) for one’s own life.24 Where people long for a blessing in order to be granted God’s fundamental promise of creation (cf. Gen 1:31) or to be assured of it, this cannot be denied. For the Church is not a source of blessing, nor an authority able to decide on the good or bad of a person, but she is a sign and instrument (cf. LG 1) of the salvation that God himself is and gives. With regard to an ecclesiology from the liturgy, it should be noted here that the Church allows herself to be called to the service of God and man for the bless- ing, when it is a matter of thanksgiving and praise, interpretation of life by faith and a plea for new life chances, the connection of the life of a couple to the salvific history of God, etc. It does not create the blessing but brings into the word that blessing which is promised.25 Because of the Church’s self-understanding, as the current inner- church discussion shows and as already existing models for such celebrations show, such blessing celebrations must be celebrated as the liturgy of the Church with the corresponding theological and aes- thetic celebration form.26 The church’s appreciation of the respective 32 BENEDICTIONAL ECCLESIOLOGY relationship is expressed in the form of the celebration. 3. Blessing Ceremonies for Same-sex Couples These cele- brations are then the performance of a changed ecclesial approach to different partnership constellations. Through the congregation or com- munity gathered for this celebration and the conduct of the service, the church explicitly expresses its commitment to this couple. In the litur- gy for the blessing of same-sex couples of Old Catholics in Germany, the congregation is asked about its willingness to support the couple on their journey.27 Thus, such a liturgy becomes an act of conversion of the church, which renounces its previous assessment of a certain form of sexuality and in conversion promises its solidarity to those who have been ostracised up to now, and thus it gives witness to God’s promise. Here, a liturgically renewed ecclesiology becomes apparent. 4. Church’s Self-image and Blessing Ceremonies 4. Church’s Self-image and Blessing Ceremonies The blessing ceremonies that are the subject of this discussion express the church in a new, sometimes unusual way. Rehearsed forms and traditional concepts of the church are broken up and questioned. It becomes apparent that the church can also be lived differently. This need not be explicitly addressed in the respective texts – but it can be experienced. How does the church present itself here? It is an inviting church, interested in its fellow human beings regardless of their church affiliation. In these liturgies, Christians act out of the hope of God’s attention to all people and put this prominently in the foreground. It is a church that invites people of very different faiths, church members, and non-denominational groups, to hear and experience God’s benedictio. It does not close itself off in the service but shows itself to be open to the culture in which it lives and which shapes it. The invitation alone is an ecclesiologically interesting fact. It is about a church that proclaims and celebrates its faith with people who do not need to bring any preconditions except a basic trust in the power of God’s blessing. That is why the texts and rites of a blessing 33 JULIA KNOP AND BENEDIKT KRANEMANN ceremony on Valentine’s Day, for example, are kept relatively simple, but at the same time quite dense: a biblical reading, the Lord’s Prayer, the Aaronite blessing, not least the testimonies of the couples. ceremony on Valentine’s Day, for example, are kept relatively simple, but at the same time quite dense: a biblical reading, the Lord’s Prayer, the Aaronite blessing, not least the testimonies of the couples. g p In the presented liturgies, the church also shows a diaconal self-un- derstanding. The respective celebration of the blessing is not primari- ly directed towards the self-preservation of the church; the gaze is not primarily directed ad intra. It is not about stabilisation and consolida- tion of the denominational community. Rather, the service becomes the place and moment of encounter of church, society, and culture. Believers who act in this way show interest in people and their suc- cessful lives. Precisely for this reason, it will have to be said that the Church of Jesus Christ becomes visible here, namely as a church that is constantly changing with the people who live in and with it in dif- ferent ways. 4. Church’s Self-image and Blessing Ceremonies y Moreover, it is the church that renounces definitions and judgments. In contrast to a church’s self-image as a teaching and judging author- ity superior to both the individual and society, the church here shows a new self-image: the celebrations are open to people who want to be blessed and who feel this is good in their present life situation. This church grants God’s blessing to all those people who ask for it. Judging by the texts of, for example, the Valentine’s Day celebrations, but also by the forms circulating for the blessing of homosexual partnerships, official church doctrine plays no role in dealing with a partnership, ways of life, marriage, etc. (although one might ask how the couples who give testimony are chosen). The blessing is put forward as a bless- ing for the people. The texts and drawing acts are then selected. It is about friendship, love, partnership, and the request that God may bless them. The church abstains from moral judgment about these people and their lives and does not set up any corresponding preconditions for the blessing. In this way, it maintains a certain openness in its posi- tion by addressing questions but refrains from giving final answers or giving its answer in the form of a promise, a blessing instead of by formulating a moral doctrine. The church, as it appears in the described blessing celebrations, remains dynamic. The communion which once gathers in a place for Valentine’s Day services is not designed to be permanent and stable, as much as it is naturally possible for appropriate connections to be formed. Even a funeral celebration is probably a frequent, one-time event for the community, from which no constant worship practice or 34 BENEDICTIONAL ECCLESIOLOGY community ties need to follow. In the blessing ceremonies for same-sex couples, church dynamics are shown in the readiness to follow social developments and scientific findings also on the ritual level, even if this makes visible inner-church non-simultaneity of liturgy and moral teaching. Above all the blessing ceremonies on Valentine’s Day present a church which cannot be defined by the boundaries of a denomi- nation, but is ecumenically open. Here the liturgy does not become a place of denominational separation or profile sharpening. The Val- entine’s blessing, for example, is (at least in Erfurt, but may look dif- ferent elsewhere) explicitly ecumenically answered for and shaped. 4. Church’s Self-image and Blessing Ceremonies Baptised believers and believers commissioned by their churches are conceptually responsible; church members and non-denominational churches are equally invited. ‘Church’ is determined interdenomina- tionally; it is a church that is not denominationally appropriated and for which denominational homogeneity is not a precondition for com- mon prayer. Confessional ecclesiologies can be set aside here for one simple reason: there is no need for it here. Even more: such blessing ceremonies offer, at least in Western ecumenism, a good reason to ask which dimensions and elements of faith and worship have long since been shaped and accounted for together. Both examples of couples blessings show a church in which insti- tutional conditions and the importance attached to them are shifting. The Church is experienced as the Church of the baptised; the inner- church difference marked by ordination is not levelled out but is not decisive in these celebrations. Church understanding and worship are not defined here by the ordained ministry. What is decisive is the prom- ise and authorisation rooted in Baptism to proclaim and promise God’s promises to others and to open up spaces of relationship for interper- sonal relationships and for the encounter with God. In the liturgies that have been questioned here about their implicit ecclesiology, (1.) the ‘assembly’ is in the foreground, while the ‘insti- tution’ recedes. The pneumatological foundation of the church, which arises from the variety of charisms, is here particularly formative; Christological conceptions of church and official Christ representation are rather in the background. What is decisive is (2.) the request that people can place their lives under the blessing of God, who is prom- ised to them as the source of all blessings. What the church is, is here (3.) not determined and staged by denominational boundaries and 35 JULIA KNOP AND BENEDIKT KRANEMANN differences, but by the mandate of all the baptised to pass on God’s blessing. differences, but by the mandate of all the baptised to pass on God’s blessing. 28 Cf. Bernhard Körner, Orte des Glaubens – loci theologici: Studien zur theologischen Erkenntnislehre (Würzburg: Echter, 2014). 29 Cf. Peter Hünermann, Dogmatische Prinzipienlehre: Glaube – Überlieferung – The- ologie als Sprach- und Wahrheitsgeschehen (Münster: Aschendorff, 2003), 207–251. ( ) 29 Cf. Peter Hünermann, Dogmatische Prinzipienlehre: Glaube – Überlieferung ologie als Sprach- und Wahrheitsgeschehen (Münster: Aschendorff, 2003), 20 Conclusion The previous remarks referred to the liturgical and pastoral experi- ences which, at least in part, are more advanced than church teaching. This concerns moral theological, liturgical theological, and ecclesio- logical questions, which are partly intensively discussed in theology and the church. How to deal with such non-simultaneousness? Can the liturgical experiences presented also develop argumentative or norma- tive power for the further development of church doctrine? In systematic theology, particularly in the tradition of Melchior Cano, one distinguishes different places of knowledge of faith, the so-called loci theologici.28 It refers to the places or, more precisely, instances to which authority and relevance are attributed in the church when it is about to identify faith convictions and formulate them in a contemporary way. Cano’s system has been adopted and updated many times. In addition to biblical and ecclesiastical tradition, today’s concepts list persons (groups) and ecclesiastical authorities such as the sensus fidei or the ecclesiastical teaching office, various sciences such as theology, philosophy, history, cultural, and human sciences, as well as the experiences and convictions of other Christian denominations and other religions.29 According to this system, theological knowledge arises and develops in dialogue and from the convergence of a plural- ity of respectively unjustifiable church perspectives, none of which can be renounced. These instances include also the service of worship resp. the liturgical experience and prayer practice of the faithful. The church is a complex, dynamic entity with very different actors who, in their role and by virtue of their competence, shape and form the local church. Wherever people gather for worship in response to God’s word, the church becomes visible. This is where the faith consciousness of the church is articulated. Although the local worship practice cannot develop normative power on its own, it is indispensable as concerns 36 BENEDICTIONAL ECCLESIOLOGY the understanding of the place of the Church, its task, and responsibil- ity in the present time and as regards the questions of identifying the direction of the future development of the Church’s teaching and the need of its reformation. It is vital that such discussion is supported. Julia Knop Benedikt Kranemann Katholisch-Theologische Fakultät an der Universität Erfurt Lehrstuhl für Dogmatik , Lehrstuhl für Liturgiewissenschaft Nordhäuser Straße 63 D-99089 Erfurt, Germany E-mail: julia.knop@uni-erfurt.de E-mail: benedikt.kranemann@uni-erfurt.de Julia Knop Benedikt Kranemann Katholisch-Theologische Fakultät an der Universität Erfurt Lehrstuhl für Dogmatik , Lehrstuhl für Liturgiewissenschaft Nordhäuser Straße 63 D-99089 Erfurt, Germany E-mail: julia.knop@uni-erfurt.de E-mail: benedikt.kranemann@uni-erfurt.de 37 37
W2751232060.txt	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0184241&type=printable	en		Comparing demersal megafaunal species diversity along the depth gradient within the South Aegean and Cretan Seas (Eastern Mediterranean)	PloS one	2,017	cc-by	8,222	RESEARCH ARTICLE Comparing demersal megafaunal species diversity along the depth gradient within the South Aegean and Cretan Seas (Eastern Mediterranean) Panagiota Peristeraki1,2, George Tserpes1, Nikolaos Lampadariou3, Kostantinos I. Stergiou4,5 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Institute of Marine Biological Resources and Inland Waters, Hellenic Centre for Marine Research, Heraklion, Crete, Greece, 2 Biology Department, University of Crete, Heraklion, Crete, Greece, 3 Institute of Oceanography, Hellenic Centre for Marine Research, Heraklion, Crete, Greece, 4 Institute of Marine Biological Resources and Inland Waters, Hellenic Centre for Marine Research, Aghios Kosmas, Athens, Greece, 5 Laboratory of Ichthyology, Department of Zoology, School of Biology, Aristotle University of Thessaloniki, Thessaloniki, Greece notap@hcmr.gr OPEN ACCESS Citation: Peristeraki P, Tserpes G, Lampadariou N, Stergiou KI (2017) Comparing demersal megafaunal species diversity along the depth gradient within the South Aegean and Cretan Seas (Eastern Mediterranean). PLoS ONE 12(9): e0184241. https://doi.org/10.1371/journal. pone.0184241 Editor: Carlo Nike Bianchi, Universita degli Studi di Genova, ITALY Received: June 25, 2016 Accepted: August 21, 2017 Published: September 5, 2017 Copyright: © 2017 Peristeraki et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: The data used for this study derived from the Data Collection Framework for the Greek Fisheries, funded by the EU and the Greek Ministry of Rural Development and Food. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Abstract Knowledge on biodiversity patterns of demersal megafaunal species in the Mediterranean and particularly in its eastern basin is still very scarce. In the present study, fine-scale diversity patterns in relation to depth were analyzed for three major megafaunal groups (fish, cephalopods and crustaceans) in three subareas of the eastern Mediterranean (Crete, Cyclades and Dodecanese islands). The analysis was based on data from the Mediterranean International Trawl Survey conducted during 2005–2014 and the relationship between depth and two different diversity measures (species richness and Shannon-Weaver) was examined using Generalized Additive Modeling (GAM) techniques. Species richness of fish decreased with depth in two of the three subareas (Cyclades, Dodecanese), while the opposite was true for crustaceans in all subareas. Cephalopods had higher species richness at intermediate depths, near the shelf break. Significant differences among subareas were found, with Crete showing a distinct species richness-depth pattern, which was more obvious for fish and cephalopods. The differences among subareas were also highlighted based on the occurrence of alien species of Indo-Pacific origin, which were more frequent in Crete. Our results suggested that the importance of depth-related factors in structuring communities was higher for cephalopods and less important for fish, and that Crete showed a distinct diversity-depth relationship, a fact that can be attributed to its specific geographical and oceanographic characteristics. These results support the current GFCM/FAO’s characterization of Crete as a unique geographic subarea. The findings of the study contribute to understanding the causes of underlying diversity patterns and would assist various environmental management actions, particularly those related to the establishment of marine-protected areas. PLOS ONE \| https://doi.org/10.1371/journal.pone.0184241 September 5, 2017 1 / 18 Demersal megafaunal species diversity in the Eastern Mediterranean Competing interests: The authors have declared that no competing interests exist. Introduction Species diversity has important implications for the functioning of ecosystems. Understanding the causes of underlying diversity patterns, as well as their interaction with environmental factors, is important for ecosystem conservation purposes. In the past, numerous publications on aquatic sciences, in different geographic areas, have studied faunal zonation, including the distribution of species along environmental gradients. These studies suggest that physical factors, such as depth, temperature, pressure, hydrographic conditions, oxygen content, sediment type, water mass structure and topography, as well as biological interactions, are possible causes of spatial community changes in marine environments [1–7]. Fishing pressure has been also suggested as a structuring factor for marine communities [8–12]. Among abiotic parameters, depth is characterised as the most important discriminant factor for faunal communities e.g. [9, 13–18, 19]. Various biodiversity aspects have been studied in the Mediterranean Sea e.g. [16, 19–28] but such studies are rather scarce for the eastern Mediterranean. Previous studies conducted on a Mediterranean-wide basis have provided some broad information for some eastern Mediterranean areas, such as the Aegean and Cretan Seas, averaging environmental covariates (e.g. depth, temperature) across larger geographic areas [23–28]. However, such a coarse-scale approach can mask local species-environment interactions that could provide important information about ecological processes influencing species abundance and distribution [29, 30]. Moreover, fine-scale community studies in the Aegean and Cretan Seas are rather scarce and most of them did not consider any specific community attributes. The few existing studies deal with depth-related distributional patterns of demersal megafaunal species, mostly focusing on the identification of “key-species” by depth zone [14, 31–34]. Thus, the lack of fine-scale community studies of demersal megafaunal communities in the eastern Mediterranean is a significant gap in our attempt to better understand species distribution and habitat selection patterns [35]. In the present study, a fine-scale spatial analysis of the changes in demersal megafaunal species diversity in relation to depth is presented. The analysis considers three large faunal groups, fish, cephalopods and crustaceans, and is based on data collected during the Mediterranean International Trawl Survey (MEDITS) carried out between 2005 and 2014 in the South Aegean and Cretan Seas. Materials and methods Ethic statement Data were collected in the framework of the MEDITS project and refer to the South Aegean Sea (eastern Mediterranean). The sampling procedures followed a standardized protocol (for details see [36]), approved by international authorities (EU/DG Mare, FAO/GFCM). The specific surveys (conducted during 2005–2014) comprise a task of the Data Collection Framework for the Greek Fisheries and are accomplished with the permission of the South Aegean and Cretan Prefectures, which are responsible for the protection of the environment in the corresponding areas. The first two authors coordinate and consistently participate in the MEDITS surveys. Study area The South Aegean Sea is a geographically and hydrologically complex area, with deep basins interrupted by shallower island complexes. The general large-scale water circulation pattern of the Aegean is anticlockwise. The more saline eastern Mediterranean waters flow northwards PLOS ONE \| https://doi.org/10.1371/journal.pone.0184241 September 5, 2017 2 / 18 Demersal megafaunal species diversity in the Eastern Mediterranean in the eastern part of the South Aegean, while the less-saline Black Sea waters flow southwards in the western part [37, 38]. The MEDITS sampling scheme in the South Aegean is rather discontinuous as the complex geomorphology of the area has resulted in large deep-water regions (>800 m) that are not included in the sampling protocol (Fig 1). Taking into account the existing oceanographic characteristics and the survey sampling scheme, three subareas were considered: 1. Crete is an isolated island, one of the biggest in the Mediterranean, with a narrow and relatively steep continental shelf (gradient 1.5˚) followed by a steep slope (2˚–4˚). It is surrounded by the deep Ionian, Libyan and Cretan Seas; the latter is the largest in volume and the deepest (2500 m) basin of the Aegean Sea, separating Crete from the Cycladic and Dodecanese island complexes [38]. The Cretan Sea is one of the most oligotrophic areas of the Mediterranean, with very low primary production [39] and the presence of a strong pelagic microbial loop [40], which reduces particle flux to the sediment [41]. For management purposes, Crete is considered by the General Fisheries Commission for the Mediterranean (GFCM) as a separate Geographical Subarea (GSA). 2. Cyclades is a complex of islands located in the middle of the Aegean Sea, comprising the Cyclades Plateau. The latter is a complex marginal platform, shallower than 250 m, with numerous islands. It separates the central from the South Aegean, and the deeper parts of the Plateau (150–250 m) are covered by sediments ranging from calcareous muddy sand to sandy mud [42]. The west edge of the Cyclades complex neighbors the Greek mainland and is influenced by the less-saline Black Sea waters [37, 43]. 3. Dodecanese is an island complex, dispersed in the eastern part of Aegean Sea, along the coast of Minor Asia, with a high variability of depths [38] because of the wide continental shelf at the northern part, interrupted by deep waters at the southern part of the area. Most of the Dodecanese islands are situated near the large shallow bays of the Turkish coast. They are mainly influenced from the more saline eastern Mediterranean waters that flow northwards in the eastern part of Aegean. Dodecanese is separated from the Cyclades complex by deep seas, and is considered the gateway for Lessepsian species, many of which establish populations here before their westward expansion [44, 45]. Such species have been considered an important driver of diversity changes in the Mediterranean [46]. Though no specific estimates for the total fishing pressure in each subarea are available, fishing pressure can be considered higher in Cyclades, followed by Dodecanese and lower in Crete, according to the total number of registered commercial fishing vessels in each area. According to the classification of fishing areas by Sylaios et al., [47], Cyclades is characterized as a fishing area with a moderate to high fishery production, while Dodecanese and Crete are characterized as fishing areas with a low to moderate fishery production. Sampling The study utilized data obtained from the MEDITS experimental surveys conducted in the South Aegean and Cretan Seas. The MEDITS survey is carried out annually in various areas of the Mediterranean Sea following a standardized protocol (see [36] for further details) and its primary goal is to monitor changes regarding the abundance and structure of the demersal megafauna community. The survey design is based on a depth-stratified random sampling scheme considering five depth strata: 10–50 m, 50–100 m, 100–200 m, 200–500 m, and 500– 800 m. Sampling in the South Aegean and Cretan Seas includes a total of 61 fixed sampling PLOS ONE \| https://doi.org/10.1371/journal.pone.0184241 September 5, 2017 3 / 18 Demersal megafaunal species diversity in the Eastern Mediterranean stations, which are distributed from 20 to 800 m according to the surface of the depth strata (Fig 1). For each station (haul), all fish, cephalopod and crustacean specimens were identified to the species level according to FAO species identification keys. In a few cases, specimens were identified only to the genus or family level due to difficulties in identification on board (fish: some Gobius and Myctophidae specimens; crustaceans: individuals of the genera Munida, Pagurus and Inacus and the family Parthenopidae; cephalopods: specimens of a few sepiolidae species and specimens in bad condition of the genus Alloteuthis, which were assigned to the most abundant Alloteuthis species caught at the station). Consequently, the total number and biomass by species was estimated for each haul (see MEDITS manual for details [48]). In the present study, we used data from the surveys carried out during 2005–2014. Given that surveys were not conducted in 2007 and 2009–2013 in the study area, the available data refer to 2005, 2006, 2008 and 2014. Both the sampling scheme and the vessel used for the surveys were the same during the study period Data analysis For each sampling station, two indices by faunistic category (fish, cephalopods and crustaceans) were estimated: (a) species richness, defined as the total number of species captured at Fig 1. Distribution of MEDITS sampling stations (red circles) in the South Aegean and Cretan Seas. https://doi.org/10.1371/journal.pone.0184241.g001 PLOS ONE \| https://doi.org/10.1371/journal.pone.0184241 September 5, 2017 4 / 18 Demersal megafaunal species diversity in the Eastern Mediterranean each station and (b) the Shannon-Weaver (H’) index (S1 Table) [49]: H0 ¼ s X pi lnpi i¼1 where, pi = ni/N, ni = number of individuals in the ith species, N = total number of individuals and s = number of species The index was computed using the “vegan” library under the R language environment [50]. A Generalized Additive Modelling (GAM) approach [51] was used to examine the effect of depth on the above indices by subarea for each faunistic category, separately. The “year” (as a factor) was also tested as a candidate predictor variable in the GAM models to account for accidental sampling inconsistencies among years due, for instance, to bad weather conditions. Hence, the general form of the nested GAM models was: Index ¼ a þ factorðYear Þ þ sðDepth; by ¼ subareaÞ þ e Index ¼ a þ sðDepth; by ¼ subareaÞ þ e where, a is the intercept, s indicates the smoother function of the corresponding independent variable and e is a random error term. The smoother function used was a penalized cubic regression spline and model fitting was accomplished using the “mgcv” library [52, 53] under the R language environment. The procedure automatically selects the degree of smoothing based on the Generalized Cross Validation (GCV) score, which is a proxy for the model predictive performance. However, to avoid dubious relationships, the model was constrained to be at maximum a quartic relationship. Hence, the maximum degrees of freedom for each smoothing term was set to 4 (i.e. k = 5 in the GAM formulation). Based on the diagnostic residual plots, it was found that a quasi-poisson distribution model and a log link function provided a pertinent fit to the data. The choice of the quasi-poisson distribution allowed for accounting for over-dispersion, which is common in biological data [54]. Best model selection was based on minimizing GCV scores and statistical inference was based on the 95% confidence level. Results A total of 245 taxa were recorded in the 244 hauls analyzed. Out of the 245 taxa, 33 were elasmobranchs, 147 osteichthyes, 35 crustaceans and 30 cephalopods. Seven out of the eight alien fish species found were of Indo-Pacific origin, and one, Sphoeroides pachygaster, of Atlantic origin. Five alien species were recorded in Crete, one in Cyclades and four in Dodecanese (Table 1). For all faunistic categories, the total number of recorded taxa was higher in the Dodecanese (S2 Table). However, the median species richness by haul for fish and cephalopods were higher in Cyclades, while the highest median species richness for crustaceans was estimated in the Dodecanese. In all subareas, species richness was higher for fish and lower for crustaceans (Fig 2). Similar patterns were observed for the median Shannon-Weaver index (Fig 3). In all cases, the inclusion of the “year” factor in the GAMs resulted in models with higher or similar GCV scores and the percentage of the deviance explained was only marginally increased (S3 Table). Thus, models without the “year” effect were considered as the best ones. The analysis of deviance of the applied GAM models on species richness indicated that the effect of depth was always significant (Table 2). Regarding the Shannon-Weaver index, depth was significant in all cases, with the exception of fish in Crete and cephalopods in Cyclades PLOS ONE \| https://doi.org/10.1371/journal.pone.0184241 September 5, 2017 5 / 18 Demersal megafaunal species diversity in the Eastern Mediterranean Table 1. Alien fish species recorded by subarea. Origin Alien Species Indo-Pacific Pteragogus pelycus Crete x x Indo-Pacific Siganus luridus Indo-Pacific Siganus rivulatus Atlantic Sphoeroides pachygaster Indo-Pacific Stephanolepis diaspros x Indo-Pacific Torquigener flavimaculosus x Indo-Pacific Upeneus moluccensis x Indo-Pacific Upeneus pori Cyclades Dodecanese x x x x x https://doi.org/10.1371/journal.pone.0184241.t001 (Table 3). For both indices, the variance explained was higher in the case of cephalopods and lower for fish (Tables 2 and 3). The effect of depth (when significant) on the examined indices is presented in Figs 4 and 5. Fig 2. Boxplot of species richness by faunistic category and subarea (CR = Crete, CY = Cyclades, DO = Dodecanese). https://doi.org/10.1371/journal.pone.0184241.g002 PLOS ONE \| https://doi.org/10.1371/journal.pone.0184241 September 5, 2017 6 / 18 Demersal megafaunal species diversity in the Eastern Mediterranean Fig 3. Boxplot of the Shannon-Weaver index by faunistic category and subarea (CR = Crete, CY = Cyclades, DO = Dodecanese). https://doi.org/10.1371/journal.pone.0184241.g003 Changes of fish species richness with depth followed similar patterns in Cyclades and Dodecanese. In both subareas, species richness decreased as depth increased. In the case of Crete, the fish species richness pattern was different, remaining rather stable down to 450 m and increasing thereafter (Fig 4). In all subareas, changes of cephalopod species richness with depth followed a unimodal distribution pattern. However, the depths corresponding to the estimated distribution peaks were not always the same. Although Cyclades and Dodecanese had a similar peak at 250–280 m, the Table 2. Analysis of deviance for the applied GAM models on species richness. Numbers in brackets indicate the variance explained by each model. (CR = Crete, CY = Cyclades, DO = Dodecanese). Model Parameter DF F P-value Fish(28.75%) s(Depth):CRs(Depth):CYs(Depth):DO 3.523.702.95 2.283.8515.99 0.0440.004<0.001 Cephalopods (61.99%) s(Depth):CRs(Depth):CYs(Depth):DO 3.573.253.76 13.7314.1856.86 <0.001<0.001<0.001 Crustaceans(49.54%) s(Depth):CRs(Depth):CYs(Depth):DO 11.181.89 5.1610.5855.63 0.024<0.001<0.001 https://doi.org/10.1371/journal.pone.0184241.t002 PLOS ONE \| https://doi.org/10.1371/journal.pone.0184241 September 5, 2017 7 / 18 Demersal megafaunal species diversity in the Eastern Mediterranean Table 3. Analysis of deviance for the applied GAM models on the Shannon-Weaver index. Numbers in brackets indicate the variance explained by each model. (CR = Crete, CY = Cyclades, DO = Dodecanese). Model Parameter DF F P-value Fish(12.45%) s(Depth):CRs(Depth):CYs(Depth):DO 13.452.13 3.784.435.18 0.0530.001<0.003 Cephalopods(31.81%) s(Depth):CRs(Depth):CYs(Depth):DO 3.071.143.58 4.071.6822.43 <0.007<0.162<0.001 Crustaceans (24.63%) s(Depth):CRs(Depth):CYs(Depth):DO 1.791.281 3.265.5931.87 0.0400.007<0.001 https://doi.org/10.1371/journal.pone.0184241.t003 modal peak in Crete was observed at shallower depths (about 150 m) (Fig 4). The model estimates for crustaceans demonstrated that species richness in all subareas increased with depth (Fig 4). Changes of the Shannon-Weaver index with depth for fish followed different patterns in Cyclades and Dodecanese. In Cyclades, H’ decreased with an increase in depth. In Dodecanese, H’ decreased until 300–350 m and increased thereafter. In Crete, the effect of depth on H’ was not significant (Fig 5). For cephalopods, the changes in H’ with depth followed different patterns in Crete and Dodecanese. In Crete, H’ was rather stable down to 400 m, showing an increasing trend (with relatively high confidence intervals) thereafter. In Dodecanese, H’ showed a unimodal pattern with a peak at about 250 m. In Cyclades, the effect of depth on H’ was not significant (Fig 5). For crustaceans, changes in H’ with depth followed a similar pattern in all subareas, almost like that of species richness (i.e. increasing trend with depth) (Fig 5). Discussion The current study adds important information to our knowledge of depth-related megafauna species diversity, by faunistic category, in three areas of the eastern Mediterranean Sea, where sampling effort is known to be much lower compared to the western Mediterranean [26]. Analysis on a fine spatial scale highlighted differences in the diversity distribution patterns among neighboring areas and faunistic categories. It must be stressed, however, that the results of the present study largely reflect only the summer conditions when the MEDITS surveys take place and thus seasonal effects cannot be taken into account. Though species richness is assumed to be negatively influenced by low primary production and higher SST values [21, 26, 55], in the case of S. Aegean and Cretan Seas, two highly oligotrophic areas with relatively elevated SST values, a high number of taxa (245), compared to other Mediterranean areas, was recorded. In particular, the recorded numbers of elasmobranch (33), osteichthyes (147) and cephalopod (30) species were higher than the corresponding numbers of species that Colloca et al. [16] record in the western coast of Italy, (14, 128 and 26, respectively) in similar depths. The total number of fish species found (180) in the present study is close to the total number (186) recorded for the whole northern Mediterranean by Granger et al. [27]. These findings, together with previous ones [23, 24, 27], question the belief that species diversity is lower in the eastern Mediterranean [21, 26, 56]. However, the total number of cephalopod species in S. Aegean and Cretan Seas was significantly lower than the one found for the whole northern Mediterranean (58) [28], indicating a high variability in cephalopod species distribution over the Mediterranean Sea. Concerning the total number of crustacean species (35), higher numbers are recorded in different areas of W. Italy in similar depths (37 and 53) [16, 19]. Our results also indicated that alien species of Indo-Pacific origin–at least the ones available to bottom trawling–apart from Dodecanese, which is considered their gateway to the Aegean Sea [44, 45], have also extended their distribution to the Cretan Sea–corroborating findings of PLOS ONE \| https://doi.org/10.1371/journal.pone.0184241 September 5, 2017 8 / 18 Demersal megafaunal species diversity in the Eastern Mediterranean Fig 4. GAM-derived significant effects of depth on species richness for three megafauna groups (fish, cephalopod, crustacean) in the three subareas (Crete, Cyclades, Dodecanese). Zero line indicates mean model estimates. Broken lines indicate two standard errors and the relative density of data points is shown by the ‘‘rug” on the x-axis. https://doi.org/10.1371/journal.pone.0184241.g004 previous works [57–61]. However, they have not established remarkable populations in Cyclades, probably due to the influence of the Black Sea less-saline cold waters in that area [37]. Our results suggested that the effect of depth on megafauna species richness was always significant, in agreement with previous studies in various Mediterranean areas [9, 10, 16, 28, 33, 34, 62–65]. Additionally, species diversity remained stable over the study period, suggesting that environmental or anthropogenic pressure, such as climate change, fishery or touristic development, has not dramatically affected community structure during the last decade. Similar findings are reported from studies based on the MEDITS survey data in different Mediterranean areas [24, 27, 28]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0184241 September 5, 2017 9 / 18 Demersal megafaunal species diversity in the Eastern Mediterranean Fig 5. GAM-derived significant effects of depth on the Shannon-Weaver index (H’) for three megafauna groups (fish, cephalopod, crustacean) in the three subareas (Crete, Cyclades, Dodecanese). Zero line indicates mean model estimates. Broken lines indicate two standard errors and the relative density of data points is shown by the ‘‘rug” on the x-axis. https://doi.org/10.1371/journal.pone.0184241.g005 Regarding the pattern of species richness distribution along the depth gradient, clear differences were detected among the three faunistic categories. For fish, several authors commonly report decreasing trends of species richness with depth in various areas of the Mediterranean [33, 34, 65]. A similar pattern was observed in the present study for fish species richness for Dodecanese and Cyclades. However, in Crete, the fish species richness-depth pattern was rather stable until 400 m depth, and increased deeper. The increasing species richness in the middle-slope areas can be related to the narrow shelf and upper slope of Crete, resulting from the strong steepness of the continental margin and the extended surface area of the lower slope [1, 49]. In an area also characterized by a steep and extended lower slope (Latio, W. Italy), PLOS ONE \| https://doi.org/10.1371/journal.pone.0184241 September 5, 2017 10 / 18 Demersal megafaunal species diversity in the Eastern Mediterranean Colloca et al. [16] did not find any significant trend in fish species richness with depth. However, other factors derived from anthropogenic disturbance could influence the fish community structure in Crete, such as coastal activities related to the tourist industry and development or the aggregation of fishing pressure on the narrow area of the shelf and upper slope [32]. Concerning the pattern of cephalopod and crustacean species richness along the depth gradient, our results agree with previous studies in other Mediterranean regions [16, 19, 28]. The observed constant increasing trend with depth of crustacean species richness could be attributed to specific adaptations of crustacean species, such as the great variety of feeding strategies, according to the availability of food resources. Indeed, different feeding guilds of plankton, benthos, benthopelagic, scavenging and detritus feeders (the latter mostly in bathyal depths) are described for crustacean species in other Mediterranean regions [66–68]. This variety of feeding strategies, which allows them to survive in the harsh environmental conditions of the deep sea, where food resources are limited, is most likely the main reason for the observed high species richness. Though the interpretation of the Shannon-Weaver (Η’) index is rather complicated [69], a direct comparison of the diversity-depth patterns derived from both measures used in the present study can provide some useful information about the community structure. For example, in the case of fish, we can assume that, for Cyclades, the species are evenly distributed within each station as both indices show similar distribution patterns. However, at the other two subareas, the observed differences between species richness and Η’ distribution patterns along the depth gradient may indicate the existence of a few dominant species in the intermediate waters of Dodecanese and in the middle-slope areas of Crete. Comparison of the distribution patterns of both indices for cephalopods may indicate that species are quite evenly distributed, especially in the shallow waters of Dodecanese, while a few dominant species may occur on the shelf and upper slope of Crete and Cyclades. Regarding crustacean species, the similarity of species richness and Η’ patterns with depth indicates that species are evenly distributed. The particularly high explained deviance (61.99%) in the GAM models including cephalopod species richness as dependent variable suggests the importance of depth-related factors in structuring cephalopod communities. The observed peak at depths around the shelf break indicates that cephalopod species are favored by higher productivity [6, 70] or other specific environmental and biological factors prevailing in the shelf break zone [16]. As many cephalopods feed in the water column, it is likely that they take advantage of the segregation of planktonic, small pelagic or benthopelagic species, and/or the high diversity of epibenthic communities around the shelf break [16, 70]. In general, the exploitation of several food sources is of great importance in highly oligotrophic environments such as the South Aegean and Cretan Seas [39]. The relatively high deviance explained for crustaceans (49.54%) indicates the importance of depth-related factors in structuring crustacean communities. Effectively, sediment texture, sediment grain size and organic matter, which are directly related to depth [71, 72], play an important role in the distribution of some crustacean species e.g. [73–78]. Indeed, previous studies in the same area have reported a clear bathymetric zonation according to sediment type [50, 74]. For fish, the explained deviance was the lowest (28.75%), indicating that, besides depthrelated factors, other environmental descriptors, such as habitat type and food availability, may influence community structure [39, 76]. In addition, the particular predatory and competitive abilities of fish, combined with their high mobility, allow them to develop a variety of life strategies and thus occupy larger depth ranges. For example, small-size individuals (recruits) of most species inhabit shallower depths than adults [79–83]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0184241 September 5, 2017 11 / 18 Demersal megafaunal species diversity in the Eastern Mediterranean Differences in the species richness pattern with depth were also detected among the three subareas, with more similarities existing between Cyclades and Dodecanese (Fig 4). The decreasing trend of fish diversity with increasing depth, observed in Cyclades and Dodecanese, can be attributed to the scarcity of food resources in deeper waters that can mainly support small organisms well adapted to the conditions of deep-sea environments [84]. Previous studies in the central and eastern Mediterranean show that fish assemblages on the slope are characterized by small-sized fish species, which are mainly pelagic feeders (on mesopelagic prey), with relatively high dominance and reduced species diversity [9, 16, 33, 80]. However, our results, as mentioned before, may indicate the existence of a few dominant species in the intermediate waters of Dodecanese and the middle-slope areas of Crete. The distinct patterns observed in Crete can be partly attributed to its geographic isolation and relatively greater distance from the mainland. In general, the distance of a marine area from the mainland is a critical factor for species diversity [15]. In addition, the specific geomorphological and oceanographic characteristics of Crete [39–41, 85, 86] have contributed to the formation of distinct species diversity patterns. Analogous differences in megafauna diversity or species assemblages between adjacent areas are also reported by other authors [12, 34, 87, 88]. The increase in fish species richness in the deeper waters of Crete could be related to the intermediate and deep layers of the Cretan Sea, which are considered a reservoir for heat, salt and dissolved oxygen [82], with the latter positively influencing species diversity [7]. In addition, the narrow, steep shelf and slope of Crete [38] may also contribute to the increase of species richness since it is known that the rate of species change is higher in steeper slopes [87]. Though fishery pressure in Crete is low [47], it is aggregated at the narrow surface of the shelf [32] and this may reduce the megafauna species diversity in the shallower waters. Cephalopod species richness followed a unimodal distribution pattern along the depth gradient in all areas but differences were observed regarding the observed peaks. In Crete, the peak was observed at depths of around 150 m, while in Cyclades and Dodecanese, the corresponding peaks were observed in deeper waters around 250–280 m. These differences reflect the different depths of the shelf break in the corresponding areas [42, 89], indicating that the specific hydrological conditions prevailing in this transitional area (strong currents, fronts) favor cephalopod species diversity, as mentioned before. The results of a large-scale study on cephalopod diversity in the Mediterranean indicate that the peak of species richness in the whole South Aegean is between 200–300 m [28]. The present fine-scale analysis, however, allowed detecting differences among closely located areas which were masked in the largescale study. Regarding crustaceans, no differences were observed between subareas. Higher median values of species richness and H’ were estimated for Cyclades regarding fish and cephalopod species. For crustaceans, the higher median values were observed in Dodecanese followed by Crete (Figs 2 and 3). Such differences can be attributed to the particular characteristics of the examined areas. For instance, the flow of the nutrient-rich Black Sea waters in the western part of Cyclades [37, 90] may influence community structure and favor species diversity. Moreover, it is likely that competition among species for niche occupation is low in the extended continental shelf of the Cyclades Plateau, thus, ranges of species can overlap, a fact that leads to increased species richness [91]. The medium-level fishing pressure in Cyclades, higher in comparison to the other study subareas [47], may also reduce species competition through reduction of the dominant species, favoring a more evenly distribution of species. Additionally, the high heterogeneity of surface sediments on the Cyclades Plateau may produce numerous different microhabitats and thus high a turnover of species, resulting in high species diversity [4, 92–94]. The surface sediments of the Cyclades Plateau consist mostly of biogenic sand (50– PLOS ONE \| https://doi.org/10.1371/journal.pone.0184241 September 5, 2017 12 / 18 Demersal megafaunal species diversity in the Eastern Mediterranean 70%), with large amounts of coralline algae debris and terrigenous sand attributed to relict palaeobeach deposits (30–40%), and the deeper parts (150–250 m) are covered by sediments ranging from calcareous muddy sand to sandy mud [42]. Sediments in the other two areas have been described as terrigenous in origin, with coarser sediments in the coastal zone, calcareous muddy sand with small amounts of terrigenous silt and fine sand at the shelf break, and a more homogeneous fine sediment texture of hemi-pelagic deposition in the middle slope [78]. The sediment characteristics, particularly the high variability in grain size, is probably the main reason for the higher diversity values of crustaceans at Dodecanese and Crete (Fig 2). Concluding, our results suggest differences in the species diversity–depth patterns depending on the faunal group or subarea studied. Differences were higher between Crete and the other two subareas than between Cyclades and Dodecanese, a fact that could be attributed to the specific geographical and oceanographic characteristics of this particular subarea. This finding supports the characterization by FAO/GFCM that Crete comprises a unique GSA area for fishery management. The analysis of species diversity at a fine spatial scale revealed differences masked in previously contacted large-scale studies. Thus, fine-scale analysis is important for the thorough understanding of factors affecting species diversity and, consequently, the identification of appropriate environmental conservation and management actions. Particularly, the requirements for the establishment of marine-protected areas, as mentioned in article 21 of the Marine Strategy Framework Directive (2008/56/EC), would greatly benefit from such analyses, which would provide essential information for the identification of diversity hot-spot areas. Supporting information S1 Table. Species density and Shannon-Weaver index by haul and faunistic category. (DOCX) S2 Table. List of all species recorded, by subarea and faunistic category. (CSV) S3 Table. GCV scores and percentage deviance explained (in parenthesis) of the nested GAM models. (XLS) Acknowledgments The data used for this study derived from the Data Collection Framework for the Greek Fisheries, funded by the EU and the Greek Ministry of Rural Development and Food. We would like to thank all the colleagues who participated in the MEDITS surveys during 2005–2014 and the crew of the trawler used for the sampling for their work and good collaboration. We also thank the anonymous reviewers for their constructive comments, which helped us improve the quality of our manuscript. Author Contributions Conceptualization: Panagiota Peristeraki. Data curation: Panagiota Peristeraki. Formal analysis: Panagiota Peristeraki, George Tserpes. Investigation: Panagiota Peristeraki. PLOS ONE \| https://doi.org/10.1371/journal.pone.0184241 September 5, 2017 13 / 18 Demersal megafaunal species diversity in the Eastern Mediterranean Methodology: Panagiota Peristeraki, George Tserpes, Nikolaos Lampadariou. Supervision: Panagiota Peristeraki, Nikolaos Lampadariou, Kostantinos I. Stergiou. Visualization: George Tserpes. Writing – original draft: Panagiota Peristeraki. Writing – review & editing: Panagiota Peristeraki, George Tserpes, Nikolaos Lampadariou, Kostantinos I. Stergiou. References 1. Rex MA. Biological accommodation in the deep-sea benthos: comparative evidence on the importance of predation and productivity. Deep Sea Res and Oceanog Abstr. Elsevier. 1976; 23–10: 975–987. 2. Osman RW, Whitlatch R B. Patterns of species diversity: fact or artefact? Paleobiology, 1978; 4–01: 41–54. 3. Moreno CA, Jaramillo E. The role of grazers in the zonation of intertidal macroalgae of the Chilean coasts. Oikos. 1983; 41: 73–76. 4. Etter RJ, Grassle JF. Patterns of species diversity in the deep sea as a function of sediment particle size diversity. Nature. 1992; 360–6404: 576–578. 5. Gage JD. High benthic species diversity in deep-sea sediments: the importance of hydrodynamics. In: Ormond R.E.G., Gage J.D. and Angel M.V. (Editors), Marine Biodiversity: Patterns and Processes. Cambridge University Press, Cambridge, pp. 1997; 148–177 6. Levin L, Etter R, Rex M, Gooday AJ, Smith CR, Pineda J, et al. Environmental influences on regional deep-sea species diversity. Annu Rev Ecol Syst. 2001; 32: 51–93. Available: http://www.jstor.org/ stable/2678635 7. Levin LA.Oxygen minimum zone benthos: adaptation and community response to hypoxia. Oceanogr. Mar Biol. 2003; 41: 1–45 8. Jennings S, Greenstreet SPR, Reynolds JD. Structural changes in an exploited fish community: a consequence of differential fishing effects on species with contrasting life histories. J of Anim Ecol. 1999; 68: 617–627. 9. Labropoulou A, Papaconstantinou C. Effects of fishing on community structure f demersal fish assemblages. Belg J of Zool. 2005; 135: 191–197 10. Masuti E, Reñones O. Demersal resource assemblages in the trawl fishing grounds of the Balearic Islands (western Mediterranean). Sci. Mar. 2005; 69: 167–366. 11. Kaiser MJ, Clarke K., Hinz H, Austen MCV, Somerfield PJ, Karakassis I. Global analysis of response and recovery of benthic biota to fishing. Mar Ecol Prog Ser. 2006; 311: 1–14. 12. Keskin C, Ordines F, Guijarro B, Massuti E. Comparison of fish assemblages between the Sea of Marmara and the Aegean Sea (north–eastern Mediterranean). J Mar Biol Assoc U K. 2011; 91–6: 1307– 1318. 13. Day DS, Pearcy WG. Species associations on benthic fish on the continental shelf and slope off Oregon. J Fish Res, Board of Canada. 1968; 25: 2665–2675. 14. Tserpes G, Peristeraki P, Potamias G, Tsimenides N. Species distribution in the southern Aegean Sea based on bottom-trawl surveys. Aquat Liv Res. 1999; 12: 167–175. 15. Rex MA, Etter RJ. Deep Sea Biodiversity, Pattern and scale. Harvard University Press, 2010; p. 354. 16. Colloca F, Cardinale M, Belluscio a, Ardizzone G. Pattern of distribution and diversity of demersal assemblages in the central Mediterranean Sea. Est Coast Shelf Sci. 2003; 56: 469–480. doi: https:// doi.org/10.1016/S0272-7714(02)00196-8 17. Macpherson E. Species range size distributions for some marine taxa in the Atlantic Ocean. Effect of latitude and depth. Biol J Linn Soc. 2003; 80–3: 437–455. 18. Reynolds JA. Quantifying habitat associations in marine fisheries: a generalization of the Kolmogorov– Smirnov statistic using commercial logbook records linked to archived environmental data. Can J Fish Aqu Sci. 2003; 60–4: 370–378. 19. Fanelli E, Colloca F, Ardizzone G. Decapod crustacean assemblages off the west coast of central Italy (western Mediterranean). Sci Mar. 2007; 71–1: 19–28. 20. Quignard JP, Tomasini JA. Mediterranean fish biodiversity. Biol Mar Med. 2007; 7: 1–66. PLOS ONE \| https://doi.org/10.1371/journal.pone.0184241 September 5, 2017 14 / 18 Demersal megafaunal species diversity in the Eastern Mediterranean 21. Ben Rais Lasram F, Guilhaumon F, Mouillot D. Fish diversity patterns in the Mediterranean Sea: deviations from amid-domain model. Mar Ecol Prog Ser. 2009; 376:253–267. https://doi.org/10.3354/ meps07786 22. Lasram FBR, Hattab T, Halouani G, Romdhane MS, Le Loc’h F. Modeling of Beta Diversity in Tunisian Waters: Predictions Using Generalized Dissimilarity Modeling and Bioregionalisation Using Fuzzy Clustering. PLoS ONE. 2015; 10(7): e0131728. https://doi.org/10.1371/journal.pone.0131728 PMID: 26147371 23. Gaertner J, Bertrand J, Relini G, Papaconstantinou C, Mazouni N, de Sola L, et al. Spatial pattern in species richness of demersal fish assemblages on the continental shelf of the northern Mediterranean Sea: a multiscale analysis. Mar Ecol Prog Ser. 2007; 341: 191–203. https://doi.org/10.3354/ meps341191 24. Gaertner JC, Maiorano P, Mérigot B, Colloca F, Politou CY, Gil De Sola L,et al. Large-scale diversity of slope fishes: pattern in consistency between multiple diversity indices. PLoSOne. 2013; 8: https://doi. org/10. 1371/journal.pone.0066753 25. Coll M, Piroddi C, Steenbeek J, Kaschner K., Ben RaisLasram F, Aguzzi J, et al. () The biodiversity of the Mediterranean Sea: estimates, patterns, and threats. PLoSOne. 2010; 5:e 11842. https://doi.org/ 10.1371/ journal.pone.0011842 PMID: 20689844 26. Coll M, Piroddi C, Albouy C, Ben RaisLasram F, Cheung WWL, Christensen V, et al. The Mediterranean Sea under siege: spatial overlap between marine biodiversity, cumulative threats and marine reserves. Glob Ecol Biog. 2012; 21:465–480. https://doi.org/10.1111/j.1466-8238.2011.00697.x 27. Granger V, Fromentin JM, Bez N, Relini G, Meynard C, Gaertner JC, et al. Large-scale spatio-temporal monitoring highlights hotspots of demersal fish diversity in the Mediterranean Sea. Prog Oceanogr. 2015; 5–130: 65–74. https://doi.org/10.1016/j.pocean.2014.10.002 28. Keller S, Bartolino V, Hidalgo M, Bittero I, Casciaro L, et al. Spatio -Temporal Patterns of Mediterranean Cephalopod Diversity. PLoSOne. 2016; https://doi.org/10.1371/ journal.pone.0146469 PMID: 26760965 29. Bacheler NM, Bailey KM, Ciannelli L, Bartolino V, Chan KS. Density-dependent landscape and climate effects on spawning distribution of walleye Pollock, Theragra chalcogramma. Mar Ecol Prog Ser. 2009; 391: 1–12. 30. Bacheler NM, Ciannelli L, Bailey KM, Bartolino V. Do wall eye pollock exhibit flexibility in where or when they spawn based on variability in water temperature? Deep Sea Res Part II Top Stud Oceanogr. 2012; 65–70:208–216. 31. Tsimenides N, Tserpes G, Machias A, Kallianiotis A. Distribution of fishes on the Cretan shelf. J Fish Biol. 1991; 39: 661–672. 32. Tserpes G, Tzanatos E, Peristeraki P. Spatial management of the Mediterranean bottom-trawl fisheries; the case of the southern Aegean Sea. Hydrob. 2011; 670: 267–274. 33. Kallianiotis A, Sophronidis K, Vidoris P, Tselepides A. Demersal fish and megafaunal assemblages of the Cretan continental shelf and slope (NE Mediterranean): seasonal variation in species density, biomass and diversity. Prog Ocean. 2000; 46: 429–455. 34. Vassilopoulou V, Papaconstantinou C, Bekas P. Demersal fish community patterns in two areas of the Aegean Sea, Greece. It J Zool. 1998; 65-S1, 293–297, https://doi.org/10.1080/11250009809386835 35. Valavanis VD, Pierce GJ, Zuur AF, Palialexis A, Saveliev A, Katara I, et al. Modelling of essential fishhabitat based on remote sensing, spatial analysis and GIS. Hydrobiologia. 2008; 612:5–20. 36. Bertrand J, Gil de Sola L, Papacostantinou C, Relini G, Souplet A. The general specifications of the MEDITS surveys. Sci Mar. 2002; 66: 9–17. 37. Nielsen JN. Hydrography of the Mediterranean and Adjacent Waters, in Report of the Danish Oceanographic Expedition 1908–1910 to the Mediterranean and Adjacent Waters, V. 1, Copenhagen. 1912; pp. 72–191. 38. Lykousis V, Chronis G, Tselepides A, Price NB, Theocharis A, Siokou-Frangou I, et al. Major outputs of the recent multidisciplinary biochemical researches undertaken in the Aegean Sea. J Mar Syst. 2002; 33–34: 313–334 39. Psarra S, Tselepides A, Ignatiades L, Dafnomili E. Primary production estimates in the Cretan Sea. In: Tselepides A, Papadopoulou K-N, Polychronaki T (eds) CINS: Pelagicbenthic coupling in the oligotrophic Cretan Sea, MAST-II Mediterranean Targeted Project. 1996; pp 46–56 40. Danovaro R, Della Croce N, Dell’Anno A, Marrale A, Pusceddu A. Seasonal and spatial distribution of pico-,nano-, and microparticulate matter and bacterioplankton in the Eastern Mediterranean Sea. In: Tselepides A, Papadopoulou K-N, Polychronaki T (eds) CINCS: Pelagic-benthic coupling the oligotrophic Cretan Sea, MAST-II Mediterranean Targeted Project. 1996; pp 69–81 PLOS ONE \| https://doi.org/10.1371/journal.pone.0184241 September 5, 2017 15 / 18 Demersal megafaunal species diversity in the Eastern Mediterranean 41. Chronis G, Stavrakakis S, Danovaro R, Tselepides A, Wassmann P, Duineveld G, et al. Downward fluxes. In: Tselepides A, Papadopoulou KN, Polychronaki T (eds) CINCS: Pelagic-benthic coupling the oligotrophic Cretan Sea, MAST-II Mediterranean Targeted Project. 1996; pp 82–111. 42. Volakis S, Anagnostou C. Modern sedimentation at the North part of the Cyclades Plateau. Proc. 4th Nat. Symp. of Oceanography and Fishery, 1993; Rhodes, 101–104. 43. Ovchinnikov I.M. Circulation in the surface and intermediate layers of the Mediterranean. Oceanology. 1966; 6: 48–59. 44. Pancucci-Papadopoulou MA., Raitsos DE, Corsini-Foca M. Biological invasions and climate warming: implications for south-eastern Aegean ecosystem functioning. J Mar Biol Assoc U K. 2012; 92: 777– 789. 45. Pancucci-Papadopoulou MA., Raitsos DE, Corsini-Foca M. Biological invasions and climate warming. The Marine Biologist, 2013; 8–11. 46. Azzurro E, Moschella P, Maynou F. Tracking Signals of Change in Mediterranean Fish Diversity Based on Local Ecological Knowledge. PLoS ONE. 2011; 6–9: e24885. https://doi.org/10.1371/journal.pone. 0024885 PMID: 21966376 47. Sylaios G.K., Koytroumanidis T., Tsicliras A.C. Ranking and classification of fishing areas using fuzzy models and techniques. Fish Manag Ecol, 2010, 17, 240–253 48. http://www.sibm.it/MEDITS011/docs/Medits_Handbook_2016_version_8_042016.p 49. Hill M.O. Diversity and evenness: a unifying notation and its consequences. Ecol. 1973; 54: 427–473. 50. R Development Core Team. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. 2013; ISBN 3-900051-07-0, URL http://www.R-project.org. 51. Hastie TJ, Tibshirani RJ. Generalized additive models. Chapman and Hall, London, 352 pp. 1990. 52. Wood SN. Modelling and smoothing parameter estimation with multiple quadratic penalties. J R Stat Soc Ser B. 2000; 62: 413–428. 53. Wood SN. Generalized Additive Models: An introduction with R. Florida: Chapman and Hall/CRC, 2006; 391 pp. 54. Harrison XA. Using observation-level random effects to model overdispersion in count data in ecology and evolution. PeerJ. 2014; 2:e616; https://doi.org/10.7717/peerj.616 PMID: 25320683 55. Begon M., Townsend C. R., Harper J. L. Ecology: From individuals to ecosystems. Malden, MA: Blackwell Pub. 2006 56. Mouillot D, Villéger S, Scherer-Lorenzen M, Mason NWH Functional Structure of Biological Communities Predicts Ecosystem Multifunctionality. PLoS ONE 2011; 6(3): e17476. https://doi.org/10.1371/ journal.pone.0017476 https://doi.org/10.1371/journal.pone.0017476 PMID: 21423747 57. Peristeraki P, Lazarakis G, Skarvelis K, Georgiadis M, Tserpes G. Additional records on the occurrence of alien fish species in the eastern Mediterranean Sea. Med Mar Sci. 2007; 7: 61–67 58. Kasapides P, Peristeraki P, Tserpes G, Magoulas A. First record of the Lessepsian migrant Lagocephalus sceleratus (Gmelin 1789) (Osteichthyes: Tetraodondidae) in the Cretan Sea (Aegean, Greece). Aquat Inv. 2007; 2: 71–73. 59. Kasapides P, Peristeraki P, Tserpes G, Magoulas A. A new record of the Lessepsian invasive fish Etrumeus teres (Osteichthyes: Clupeidae) in the Mediterranean Sea (Aegean, Greece). Aquat Inv. 2007; 2–2: 152–154. 60. Golani D, Appelbaum-Golani B, Peristeraki P. Westward range extension of the Lessepsian migrant the Shrimp scad Alepes djedaba (Forsskål, 1775) in the Mediterranean. Ann Ser Hist Nat. 2013; 23–2: 115–118. 61. Skarvelis K,Giannakaki A, Kosoglou G, Peristeraki P. Data on frequency of occurrence ofalien species caught on the coastal fisheries of Crete. Proc. 11th Panhell Symp Ocean Fish. 2015. 62. Company JB, Maiorano P, Tselepides A, Politu C-Y, Plaity W, Rotllant G, Sardà F. Deep-sea decapod crustaceans in the western and central Mediterranean Sea: a preliminary aspects of species distribution, biomass and population structure. Sci Mar. 2004; 68–3:73–86. https://doi.org/10.3989/scimar. 2004.68s373 63. Massuti E, Renones O, Carbonell A, Oliver P. Demersal fish communities exploited on the continental shelf and slope off Majorca (Balearic islands, NW Mediterranean). Vie Milieu. 1996; 46: 45–55. 64. Moranta J, Stefanescu C, Massutı E, Morales-Nin B, Lloris D. Fish community structure and depth related trends on the continental slope of the Balearic Islands (Algerian basin, western Mediterranean). Mar Ecol Prog Ser. 1998; 171: 247–259. 65. Tecchio S, Ramirez-Llodra E, Sarda F, Company JB. Biodiversity of deep-sea megafauna in western and central Mediterranean basins. Sci Mar. 2011; 75(2): 341–350. https://doi.org/10.3989/scimar. 201175n2341 PLOS ONE \| https://doi.org/10.1371/journal.pone.0184241 September 5, 2017 16 / 18 Demersal megafaunal species diversity in the Eastern Mediterranean 66. Cartes JE. Feeding strategies and partition of food resources in deep-water decapod crustaceans in relation to depth (between 400 and 2300 m). J Mar Biol Assoc U K 1998; 78:509–524 67. Cartes JE. Diets of deep-water pandalid shrimps on the western Mediterranean slope. Mar. Ecol Prog Ser. 1993; 96: 49–61. 68. Cartes JE,Carrason M. The influence of trophic variables in the depth-range distribution and zonation rates of deep-sea megafauna: the case of the Western Mediterranean assemblages. Deep-Sea Res. 2004; 51: 263–279. 69. Magurran AE. Measuring biological diversity. Oxford: Blackwell Science Ltd. 2004; 256 p. 70. Navarro J, Coll M, Somes C J, Olson RJ. Trophic niche of squids: Insights from isotopic data in marin systems worldwide. Deep-Sea Res. 2013; II- 95: 93–102 71. Chronis G, Lykousis V, Georgopoulos D, Zervakis V, Stavrakakis S, Poulos S. Suspended particulate matter and nepheloid layers over the southern margin of the Cretan Sea (N.E. Mediterranean): seasonal distribution and dynamics. Prog Ocean. 2000; 46: 163–185 72. Gray JS. The ecology of marine sediments. Cambridge studies in modern biology 2. 1981; Cambridge University Press, Cambridge 73. Atkinson RJA, Taylor AC. Physiological ecology of burrowing decapods. In: A.A. Fincham and P.S. Rainbow (eds.), Aspects of decapod crustacean biology, Symp. Zool. Soc. Lond. 1988; 59: 201–226 74. Macpherson E. Biogeography and community structure of the decapod crustacean fauna off Namibia (southeast Atlantic). J. Crust. Biol. 1991; 11: 401–415. 75. Pinheiro MAA, Fransozo A, Negreiros-Fransozo ML. Distribution patterns of Arenaeus cribarius (Lamarck, 1818) (Crustacea, Portunidae) in Fortaleza Bay, Ubatuba (SP), Brazil. Rev Bras Biol. 1996; 56: 705–716. 76. Comeau M, Conan GY, Maynou F, Robichaud G, Therriault JC,Starr M. Growth, spatial distribution, and abundance of benthic stages of the snow crab (Chionoecetes pilio) in Bonne Bay, Newfoundland, Canada. Can J Fish Aquat Sci. 1998; 55: 262–279. 77. Vaz S, Carpentier A, Coppin F. Eastern English Channel fish assemblages: measuring the structuring effect of habitats on distinct sub-communities.–ICES J Mar Sci. 2007; 64: 271–287. 78. Emylianov EM, Schimkus KM. Geochemistry and sedimentology of the Mediterranean Sea. 1986; Dordrecht D. Reidel. 79. Stefanescu C, Rucabado J, Lloris D. Depth -size trends in the western Mediterranean demersal deepsea fishes. Mar Ecol Prog Ser. 1992; 81: 205–213. 80. Madurell T, Cartes JE, Labropoulou M. Changes in the structure of fish assemblages in a bathyal site oft he Ionian Sea (eastern Mediterranean). Fish Res. 2004; 66: 245–260 81. Bartolino V, Ottavi A, Colloca F, Ardizzone, Domenico Gian, Stefánsson G. Bathymetric preferences of juvenile European hake (Merluccius merluccius). ICES J Mar Sci. 2008; 65: 963–969. Available: http:// icesjms.oxfordjournals.org/content/65/6/963.short. 82. Tserpes G, Politou CY, Peristeraki P, Kallianiotis A, Papaconstantinou C. Identification of hake distribution pattern and nursery grounds in the Hellenic seas by means of generalized additive models. Hydrob. 2008; 612:125–133. https://doi.org/10.1007/s10750-008-9486-x 83. Druon JN, Fiorentino F, Murenu M, Knittweis L, Colloca F, Osio C, et al. Modelling of European hake nurseries in the Mediterranean Sea: an ecological niche approach. Prog Ocean. 2015; 188–204. http:// www.sciencedirect.com/science/article/pii/S0079661114001803 84. Hutchinson GE. Hommage to Santa Rosalia, or, Why are there so many kinds of animals? Am. Nat. 1959; 93: 154–159. 85. Souvermezoglou E, Krasakopoulou E, Pavlidou A. Modifications of the nutrients and oxygen regime between the Cretan Sea and the Eastern Mediterranean (1986–1995). Proc Internat POEM-BC/MTP Symp. Molitg les Bains-France, 1996; 133–136. 86. Georgopoulos D, Theocharis A, Zodiatis G. Intermediate water formation in the Cretan Sea (S. Aegean Sea). Oceanol. Acta. 1989; 12–4: 353–359. 87. Kallianiotis A, Vidoris P, Sylaios G. Fish species assemblages and geographical sub-areas in the North Aegean Sea, Greece. Fish Res. 2004; 68: 171–187. 88. Garofalo G, Fiorentino F, Gristina M, Cusumano S, Sinacori G. Stability of spatial pattern of fish species diversity in the Strait of Sicily (central Mediterranean). Hydrob. 2007; 580:117–124 https://doi.org/10. 1007/s10750-006-0460-1 89. Theocharis A, Georgopoulos D, Lascaratos A, Nittis K. Water masses and circulation in the central region oft he eastern Mediterranean: eastern Ionian, south Aegean and northern Levantine, 1986– 1987. Deep-Sea Res. 1993; II- 40: 1121–1142. PLOS ONE \| https://doi.org/10.1371/journal.pone.0184241 September 5, 2017 17 / 18 Demersal megafaunal species diversity in the Eastern Mediterranean 90. Hopkins TS. Physical processes in the Mediterranean basins. In Estuarine Transport Processes, Kjerfve B (ed.). University of South Carolina Press: Columbia, SC. 1978; 285–286. 91. Terborgh J. Distribution on Environmental Gradients: Theory and a Preliminary Interpretation of Distributional Patterns in the Avifauna of the Cordillera Vilcabamba, Peru Ecology. 1971; 52– 1: 23–40. 92. Levin SA. Dispersion and population interactions. Am. Nat. 1974; 108: 207–228 93. Jorissen F J, De Stigter H C, Widmark JGV. A conceptual model explaining benthic foraminiferal microhabitats. Mar Micropaleont. 1995; 22: 3–15. 94. Hecker B. Variation in megafaunal assemblages on the continental margin south of New England. Deep-Sea Res. 1990; 37: 37–57. PLOS ONE \| https://doi.org/10.1371/journal.pone.0184241 September 5, 2017 18 / 18
https://openalex.org/W4361248939	https://aacr.figshare.com/ndownloader/files/39812924	Khmer	null	Supplementary Tables 1-5, Figures 1-5 from A Comparison of DNA Copy Number Profiling Platforms	null	2,023	cc-by	293	Affymetrix 100K Affymetrix 500K NimbleGen Oligo Oligo Oligo Single Channel Single Channel Dual Channel Affymetrix, Inc Affymetrix, Inc Nimblegen Systems, Inc. 0.5 0.5 2 116 500 1,526 25 25 60 24,509 5,900 2,000 118.7 49.2 36.1 2A) Phase 1 and (2B) Phase 2. 2B Cell Lines in Phase 2 M14 MALME3M MEWO SKMEL2 SKMEL28 SKMEL5 UACC257 UACC62 WM122 WM1366 WM239A WM3211 WM3248 WM793B WM88 WM983C 451LU 501MEL A) Phase 1 and (2B) Phase 2. 2B Cell Lines in Phase 2 M14 MALME3M MEWO SKMEL2 SKMEL28 SKMEL5 UACC257 UACC62 WM122 WM1366 WM239A WM3211 WM3248 WM793B WM88 WM983C 451LU 501MEL SKY. Note WM983C is consisted of 2 major populations with different karyotypes. r→1p11::5q11→5qter),der(1)(12pter→12p11::1p11→1qter), p25→2qter),+der(3)(3qter→3p11::13q11→13qter),-5,del(6)(:p22→qter),+7, qter),der(9)(Yqter→Yq11::9p13→9qter),der(10)(1qter→1q11::10p15→10qter), 2p11→12qter),der(13)(13qter→13p11::1p11→1pter),der(17)(17pter→17q11::1p11→1pter), 2::11p11→11pter),der(18)(18qter→18p11::7p11→7pter), ::12p11→12pter),+der(20)(5pter→5p11::20p11→20qter), ::22p13→22qter) →p10::p10→pter),der(1)(:3q26→3q11::1p11→1qter),der(2)(7q?::2p16→2qter), 11→8qter),del(5)(pter→q21),i(5)(qter→q10::q10→qter),del(6)(:p21→qter), er),-8,der(8)(22qter→22q11::8p11→8qter),der(9)(19qter→19q11::9p11→9qter), pter),der(13)(12qter→12q11::13p11→13qter),der(15)(5pter→5p11::15p11→15qter), q13::8p11→8pter),der(20)(20qter→20p11::1q12→1qter), 21p11→21qter),der(21)(21qter→21p11::6q11→6q16:) er→q10::q10→qter),der(2)(2pter→2q35::X?),der(2)t(2;20)(20?::2p25→2qter), del(8)(:p21→qter),der(9)(9qter→9p13::2q11→2qter),der(11)(20?::11p12→11q23:), 2p13→2qter),der(13)(5pter→5p11::13p11→13qter),+15,+18,del(20)(?), 2::11p12→11pter),+der(20)(20qter→20p12::16p11→16pter),+21 qter→15q22::Xp22→Xqter)x2,Y,-Y,+1,der(1)(6?::1q11→1pter)x2,del(2)(:p16→qter)x2, 2p25→2qter),+der(3)(3pter→3q29::17?::1?),-4,-6,der(6)(4?::6p25→6q27::6?), →pter)x3,+del(7)(qter→q31::q11→pter)x3,+der(7)(7pter→7q36::3?), 0::q10→qter),der(9)(9qter→9p13::17q?::3q?::2p16→2qter)x2,-10,- →12pter),-13,-13,der(13)(7pter→7p11::13p11→13qter),der(15)(7?::15p11→15qter),- 20,+20,+20,+21[cp10]/ ter→15q?22::Xp22→Xqter),der(1)(6?::1q11→1pter),del(2)(:p16→qter), ?::1?),-4,-5,-6,der(6)(6pter→6q21::9?),+7,del(7)(qter→q31::q11→pter)x2, 1:),+der(7)(12?::7q11→7pter),-9,der(9)(9qter→9p13::17q?::3q?::2p16→2qter),-10,-11, ::6?),-12,-13,-14,-16,-17,-18,-19,+20,+20[8] KY. Note WM983C is consisted of 2 major populations with different karyotypes. →1p11::5q11→5qter),der(1)(12pter→12p11::1p11→1qter), 25→2qter),+der(3)(3qter→3p11::13q11→13qter),-5,del(6)(:p22→qter),+7, er),der(9)(Yqter→Yq11::9p13→9qter),der(10)(1qter→1q11::10p15→10qter), p11→12qter),der(13)(13qter→13p11::1p11→1pter),der(17)(17pter→17q11::1p11→1pter), ::11p11→11pter),der(18)(18qter→18p11::7p11→7pter), 12p11→12pter),+der(20)(5pter→5p11::20p11→20qter), :22p13→22qter) p10::p10→pter),der(1)(:3q26→3q11::1p11→1qter),der(2)(7q?::2p16→2qter), 1→8qter),del(5)(pter→q21),i(5)(qter→q10::q10→qter),del(6)(:p21→qter), r),-8,der(8)(22qter→22q11::8p11→8qter),der(9)(19qter→19q11::9p11→9qter), er),der(13)(12qter→12q11::13p11→13qter),der(15)(5pter→5p11::15p11→15qter), 13::8p11→8pter),der(20)(20qter→20p11::1q12→1qter), p11→21qter),der(21)(21qter→21p11::6q11→6q16:) r→q10::q10→qter),der(2)(2pter→2q35::X?),der(2)t(2;20)(20?::2p25→2qter), el(8)(:p21→qter),der(9)(9qter→9p13::2q11→2qter),der(11)(20?::11p12→11q23:), p13→2qter),der(13)(5pter→5p11::13p11→13qter),+15,+18,del(20)(?), ::11p12→11pter),+der(20)(20qter→20p12::16p11→16pter),+21 ter→15q22::Xp22→Xqter)x2,Y,-Y,+1,der(1)(6?::1q11→1pter)x2,del(2)(:p16→qter)x2, p25→2qter),+der(3)(3pter→3q29::17?::1?),-4,-6,der(6)(4?::6p25→6q27::6?), pter)x3,+del(7)(qter→q31::q11→pter)x3,+der(7)(7pter→7q36::3?), :q10→qter),der(9)(9qter→9p13::17q?::3q?::2p16→2qter)x2,-10,- 2pter),-13,-13,der(13)(7pter→7p11::13p11→13qter),der(15)(7?::15p11→15qter),- 0,+20,+20,+21[cp10]/ er→15q?22::Xp22→Xqter),der(1)(6?::1q11→1pter),del(2)(:p16→qter), ::1?),-4,-5,-6,der(6)(6pter→6q21::9?),+7,del(7)(qter→q31::q11→pter)x2, :),+der(7)(12?::7q11→7pter),-9,der(9)(9qter→9p13::17q?::3q?::2p16→2qter),-10,-11, 6?),-12,-13,-14,-16,-17,-18,-19,+20,+20[8] n log2 ratio of listed nd Affymetrix adjustment 1 8 6 3 5 3 d 4n vs. 2n regions of melanoma cell lines a 983C, and (D.) 1n vs. 2n regions in melanom s a m s lines elano vs. 2n regions measured by SKY melanoma cell lines (A.) LU1205, 1n vs. 2n region for melanoma cell line WM1366. lines (A.) LU1205, 2n regions measured by SKY melanoma . 2n region for melanoma cell line WM13 for all 5 phase 1 e region encompassing the CDKN2A locus for all 5 phase 1 highlighted in green. for all 5 phase 1 e region encompassing the CDKN2A locus highlighted in green. gion encompassing the CDKN2A locu hlighted in green.
https://openalex.org/W2157930957	https://europepmc.org/articles/pmc3408375?pdf=render	English	null	The prevalence, patterns and predictors of diabetic peripheral neuropathy in a developing country	Diabetology & metabolic syndrome	2,012	cc-by	6,855	* Correspondence: pkatulanda@yahoo.com 1Diabetes Research Unit, Department of Clinical Medicine, Faculty of Medicine, University of Colombo, Colombo, Sri Lanka 2Oxford Centre for Diabetes, Endocrinology and Metabolism, University of Oxford, Oxford, UK Full list of author information is available at the end of the article Katulanda et al. Diabetology & Metabolic Syndrome 2012, 4:21 http://www.dmsjournal.com/content/4/1/21 Katulanda et al. Diabetology & Metabolic Syndrome 2012, 4:21 http://www.dmsjournal.com/content/4/1/21 Katulanda et al. Diabetology & Metabolic Syndrome 2012, 4:21 http://www.dmsjournal.com/content/4/1/21 METABOLIC SYNDROME DIABETOLOGY & The prevalence, patterns and predictors of diabetic peripheral neuropathy in a developing country Prasad Katulanda1,2, Priyanga Ranasinghe3, Ranil Jayawardena1,4, Godwin R Constantine1, M H Rezvi Sheriff1 and David R Matthews2 RESEARCH Open Access Abstract Prevalence of diabetes mellitus (DM) has reached epidemic proportions in Sri Lanka. Presently there are studies on the community prevalence of distal peripheral neuropathy (DPN) in Sri Lanka. We describe prevalence, patterns and predictors of DPN in patients with DM in Sri Lanka. Data were collected as part of a national study on DM. In new cases DPN was assessed using the Diabetic-Neuropathy-Symptom (DNS) score, while in those with established diabetes both DNS and Toronto-Clinical-Scoring-System (TCSS) were used. A binary logistic-regression analysis was performed with ‘presence of DPN’ as the dichomatous dependent variable and other independent co-variants. The study included 528 diabetic patients (191-new cases), with a mean age of 55.0 ± 12.4 years and 37.3% were males, while 18% were from urban areas. Prevalence of DPN according to DNS score among all patients, patients with already established diabetes and newly diagnosed patients were 48.1%, 59.1% and 28.8% respectively. Prevalence of DPN in those with established DM as assessed by TCSS was 24% and the majority had mild DPN (16.6%). The remainder of the abstract is based on subjects with established DM. The prevalence of DPN in males and female was 20.0% and 26.4% respectively. The mean age of those with and without DPN was 62.1 ± 10.8 and 55.1 ± 10.8 years respectively (p < 0.001). The majority of those with DPN were from rural-areas (75.3%) and earned a monthly income < Sri Lankan Rupees 12,000 (87.6%). In the binary logistic-regression presence of foot ulcers (OR:10.4; 95%CI 1.8–16.7), female gender (OR:6.7; 95%CI 2.0–9.8) and smoking (OR:5.9; 95%CI 1.4–9.7) were the strongest predictors followed by insulin treatment (OR:4.3; 95%CI 1.3–6.9), diabetic retinopathy (OR:2.7; 95%CI 1.3– 5.4), treatment with sulphonylureas (OR:1.8; 95%CI 1.1–3.2), increasing height (OR:1.8; 95%CI 1.2–2.4), rural residence (OR:1.8; 95%CI 1.1–2.5), higher levels of triglycerides (OR:1.6; 95%CI 1.2–2.0) and longer duration of DM (OR:1.2; 95% CI 1.1–1.3). There is a high prevalence of DPN among Sri Lankan adults with diabetes. The study defines the impact of previously known risk factors for development of DPN and identifies several new potential risk factors in an ethnically different large subpopulation with DM. Keywords: Diabetes mellitus, Distal peripheral Neuropathy, Prevalence, Sri Lanka, Developing country © 2012 Katulanda et al.; licensee BioMed Central Ltd. Correspondence: pkatulanda@yahoo.com 1Diabetes Research Unit, Department of Clinical Medicine, Faculty of Medicine, University of Colombo, Colombo, Sri Lanka 2Oxford Centre for Diabetes, Endocrinology and Metabolism, University of Oxford, Oxford, UK Full list of author information is available at the end of the article © 2012 Katulanda 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. Introduction DPN. Ethnic differences and differential environmental exposure to risk factors in the different populations are other proposed mechanisms. Hence studies aimed at de- fining the extent of DPN and the associated risk factors in this vast subpopulation of diabetic patients would help to improve preventive strategies. Diabetes mellitus (DM) has reached epidemic propor- tions worldwide. Historically, diabetes was considered a disease confined to developed countries and affluent people. However, recent estimates suggest that the preve- lence of diabetes is rising globally, particularly in devel- oping countries [1]. Diabetes mellitus has become an important health concern in the South Asian region with an estimated increase in the prevalence of diabetes of over 151% between 2000 and 2030 [1]. Neuropathy is considered the most common micro-vascular complica- tions of both types 1 and 2 diabetes mellitus [2,3]. Neuropathic disorders in diabetes can impair functioning of the central, peripheral and/or autonomic nervous sys- tems [4]. Distal peripheral neuropathy (DPN), also known as diabetic polyneuropathy affects the peripheral nervous system and is by far the most common type of neuropathy seen in DM [5]. The resultant loss of func- tion in peripheral nerves causes loss of protective sen- sations and impairs patient’s ability to perceive incipient or even apparent ulcerations in the feet. DPN is considered a main risk factor for amputation, and hence a significant cause of morbidity in DM [6]. Although a common and important complication of diabetes, neuropathy has not been studied as often or as extensively as macro-vascular complications or retinopathy and nephropathy [7]. In addition, the reported prevalence estimates vary widely between countries, in part due to the difference in sampling methods and lack of consensus on diagnostic criteria [8]. Hence, for comparative purposes it is important to use studies that utilize similar diagnostic criteria. However, the observed variations in prevalence could partly result from ethnic differences in predisposition and differential exposure to risk factors. Sri Lanka is a developing country in the South Asian region with a population of 20.9 million [14]. The preva- lence of DM has reached epidemic proportions in Sri Lanka with recent studies demonstrating that one in every five adults aged > 20 years has either diabetes or pre-diabetes [15]. To our knowledge, there are no pub- lished data regarding the community prevalence of DPN in Sri Lanka. Abstract 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. Katulanda et al. Diabetology & Metabolic Syndrome 2012, 4:21 http://www.dmsjournal.com/content/4/1/21 Page 2 of 8 Study population and sampling Data were collected as part of a wider cross-sectional national study on diabetes conducted in seven of the nine provinces in Sri Lanka between August 2005 and September 2006 (Sri Lanka Diabetes and Cardiovascular Study – SLDCS). In the SLDCS, the research team ran- domly selected 100 clusters (of 50 adults each) to repre- sent seven out of nine provinces in the country. The sample sizes for each province were determined using a probability-proportional to-size (PPS) technique based on total population of each province. Each cluster was selected by a computer-generated random number list from the ‘Village Office Units’ in each province. Voter registration lists were used to randomly select the first household in each cluster and a uniform criterion was used to select the remaining 49 households. Detailed sampling has been previously reported [15]. Relevant data of 5000 non-institutionalized adults are presented here. The ethical approval for the study was obtained from the Ethical Review Committee, Faculty of Medi- cine, University of Colombo, Sri Lanka. Increasing age, longer duration of diabetes and poor glycaemic control are well recognized risk factors for DPN, while cigarette smoking, retinopathy, hypertension, obesity, hyperlipidaemia and microalbuminuria have also been implicated as potential risk markers [9]. Most prevalence and risk factor studies are from western developed countries, while there is a relative scarcity of data from developing countries, particularly from the South-Asian region [10]. However, an estimated 80% of the global population with diabetes lives in developing countries [1]. The South-Asian population in particular is known to have an increased predisposition for the dis- ease [11]. In addition, several studies have demonstrated that the risk of diabetes related amputations and the prevalence of diabetic foot ulcers in UK is significantly lower in diabetic patients of Asian origin when compared to that of diabetic patients of European origin [12,13]. This reduced risk in Asians was found to be related to the lower incidence of peripheral arterial disease and Introduction The present study aims to describe the community prevalence, patterns and predictors of distal peripheral neuropathy among patients with diabetes mellitus in Sri Lanka with a view of identifying differen- tial risk factors, which may lead to improved preventive measures and care for diabetic patients in the South Asia region. Results Out of the 5000 invited subjects, 4477 participated in the study (response rate 89.5%). This report is based on 528 subjects (11.9%) with DM after excluding 6 subjects with incomplete data (191 new cases [36.2%]). Details of subject recruitment are presented in Figure 1. The mean age was 55.0 ± 12.4 years, 37.3% were males and only 3 patients were suffering from Type 1 DM. Majority of the subjects were treated with metformin (61.3%) and sul- phonylureas (48.4%). Glitazones were used in 4.2%, while insulin was administered in 3.9% of the patients. The prevalence of DPN according to the DNS score among all patients, patients with already established diabetes and newly diagnosed patients was 48.1% (n = 254), 59.1% (n = 199) and 28.8% (n = 55) respectively. The most com- mon symptom among newly diagnosed subjects were burning, aching pain or tenderness of feet (n = 30, 15.7%), followed by numbness of feet (n = 24, 12.6%), prickling sensation of feet (n = 22, 11.5%) and unsteadi- ness in walking (n = 20, 10.5%). Height was measured using Harpenden stadiometers (Chasmors Ltd, London, UK) to the nearest 0.1 cm, as the maximum distance to the uppermost position on the head from heels, with the individual standing bare- foot and in full inspiration. Body weight was measured using a SALTER 920 digital weighing scale (SALTER Ltd, Tonbridge, UK) to the nearest 0.1 kg after an overnight fast and with indoor light clothing. BMI was calculated as weight in kilograms divided by height squared in meters (kg.m-2). Waist circumference was measured midway between the iliac crest and the lower rib margin at the end of normal expiration and hip cir- cumference was measured at the widest level over the greater trochanters using a plastic flexible tape to the nearest 0.1 cm. Seated blood pressure was measured after at least a 10-min rest with Omron IA2 digital blood pressure monitors (Omron Healthcare, Singa- pore). Fasting venous blood samples were obtained for glucose andlipid estimation from all participants, details of analysis have been previously described [15]. Data on physical activity were collected using the short ver- sion of the International Physical Activity Question- naire. Subjects in the ‘moderate’/‘high’ physical activity categories were considered as being physical active [22]. Data collection In new cases, DPN was assessed using the validated Dia- betic Neuropathy Symptom (DNS) Score, while in ‘diag- nosed diabetes’ subjects both DNS and the validated Toronto Clinical Scoring System (TCSS) was used [20]. The DNS score derives from the assessment of 4 symp- toms, the presence of each symptom is scored 1. DPN is considered to be present if score is between 1 and 4 [21]. The TCSS produces a score derived from the clin- ical assessment of 6 symptoms, 5 sensory tests, and lower limb reflexes, giving a maximal score of 19. The degree of neuropathy was based on the TCSS score used in a previous study (no neuropathy: ≤5, mild neuropathy: 6–8, moderate neuropathy: 9–11 and severe neuropathy: ≥12) [20]. Sensation was tested on the dorsum and tip of the first toe. Light touch, pain sense, vibration sense and temperature sense were tested using a 10-gram Semmes- Weinsten monofilament, a pinprick, a 128 Hz tuning fork and a cylinder with different temperatures respect- ively. Tendon reflex was tested by striking the Quadri- ceps and Achilles tendons with a reflex hammer. Statistical analyses dilated ophthalmological examination by two independ- ent ophthalmologists, and classified as background, pre- proliferative, proliferative, advanced retinopathy and maculopathy. Nephropathy was defined as the presence of micro-albuminuria (30 – 299 mg/day) or macro- albuminuria (≥300 mg/day), or by review of medical records [19]. y Data were analysed using SPSS v14 (SPSS Inc., Chicago, IL, USA) statistical software package. The significance of the differences between proportions and means was tested using z-test and Student’s t-test or ANOVA re- spectively. A binary logistic regression analysis was per- formed in all patients with ‘presence of DPN’ as the dependent variable (0 = DPN absent; 1 = DPN present) and gender (0 = male; 1 = female), sector of residence (0 = Urban, 1 = Rural), household income (0 = > LKR 25,000; 1 = LKR 12,000 – 25,000; 2 = < LKR 12,000), height, triglyceride levels, duration of diabetes, current smoking (0 = no, 1 = yes), current alcohol consumption (0 = no, 1 = yes), retinopathy (0 = absent, 1 = present), nephropathy (0 = absent, 1 = present), Foot ulcers (0 = ab- sent, 1 = present), Obesity (0 = BMI < 27.5, 1 = BMI ≥ 27.5), hypertension (0 = absent, 1 = present), physical ac- tivity (0 = active, 1 = inactive), drug treatment (0 = met- formin, 1 = sulfonylurea) and treatment with insulin (0 = not treated, 1 = treated) as the independent variables. The variables were selected by a forward selection pro- cedure based on increment of R2. In all statistical ana- lyses p < 0.05 was considered significant. Definitions b Subjects were considered to have ‘diagnosed diabetes’ if they had been previously diagnosed at a government hospital or by a registered medical practitioner. New cases (‘undiagnosed diabetes’) were diagnosed according to the American Diabetes Association [16] and World Health Organization (WHO) criteria [17]. Obesity was defined as a body mass index (BMI) ≥27.5 kg/m2, based on WHO criteria for Asians [18]. Presence/absence of diabetic retinopathy was determined by standardized Katulanda et al. Diabetology & Metabolic Syndrome 2012, 4:21 http://www.dmsjournal.com/content/4/1/21 Page 3 of 8 Results The remainder of this article is based exclusively on subjects with established DM only (n = 337), in whom both symptoms and signs of neuropathy were evaluated. DPN is considered to be present in all subjects with TCSS > 5. In subjects with established DM the preva- lence of DPN according to the TCSS score was 24.0% and a majority was suffering from mild neuropathy (n = 56, 16.6%) (Table 1). In patients with established diabetes the most common symptom was the presence of numbness of feet (n = 125, 37.1%) followed by Katulanda et al. Diabetology & Metabolic Syndrome 2012, 4:21 http://www.dmsjournal.com/content/4/1/21 Page 4 of 8 Total number of subjects invited = 5,000 (50 adults from 100 clusters) Response rate – 89.5% (n = 4,477 ) Prevalence of DM – 11.9% (n = 534) Exclusion of those with incomplete data (n = 6) Number included in study (n = 528) Newly diagnosed DM (n = 191) Established DM (n = 337) Figure 1 Schematic representation of subject recruitment Total number of subjects invited = 5,000 (50 adults from 100 clusters) Prevalence of DM – 11.9% (n = 534) Exclusion of those with incomplete data (n = 6) Number included in study (n = 528) Established DM (n = 337) Figure 1 Schematic representation of subject recruitment was low (<±0.2) for all of the above factors. No signifi- cant difference was observed in the systolic and the dia- stolic blood pressure, total cholesterol, LDL cholesterol and HDL cholesterol (Table 3). A binary logistic regres- sion was used to identify potential risk factors and asso- ciations of DPN in subjects with DM. Presence of foot ulcers, female gender and current smoking were the most significantly and strongly associated factors followed by treatment with insulin, presence of diabetic retinopathy, burning, aching or tenderness of the feet (n = 109, 32.3%), prickling sensation of feet (n = 100, 29.7%) and unsteadiness (n = 86, 25.5%). On clinical examination of subject with established DM, impaired big-toe pin prick sensation was the commonest sensory deficit (n = 134, 39.7%), followed by impaired light touch (n = 129, 38.3%), vibration (n = 88, 26.1%), joint position (n = 56, 16.6%) and temperature sensations (n = 28, 8.3%). Ankle and knee reflexes were absent or diminished in 28.2% (n = 95) and 11.6% (n = 39) of patients respectively. Results The prevalence of DPN among all, male and female patients was 24.0% (n = 81/337), 20.0% (n = 25/125) and 26.4% (n = 56/212) (males vs. females, p > 0.05). The mean age (±SD) of patients with or without DPN was 62.1 ± 10.8 years and 55.1 ± 10.8 years respectively (p < 0.001). Socio demographic characteristics of subjects with or without DPN are presented in Table 2. A major- ity of patients with DPN were residing in rural areas (75.3%) and earned a monthly income of < Sri Lankan Rupees (LKR) 12,000 (87.6%). The mean duration of DM among patients with DPN was 7.8 ± 7.1 years, while among patients without DPN it was 5.8 ± 5.0 years (p < 0.01). The presence of foot ulcers were significantly greater in patients with DPN (n = 11, 64.7%) than those without DPN (n = 6, 35.3%) (p < 0.001). Table 1 Socio-demographic characteristics and prevalence of Distal Peripheral Neuropathy among patients with newly diagnosed and established diabetes Diabetes status Non-diabetic (n = 3943) Newly diagnosed (n = 191) Established (n = 337) Mean age (±SD) 44.9 ± 15.0 51.4 ± 13.5 56.8 ± 11.2 Gender (%)Male 1572 (39.8%) 72 (37.7%) 125 (37.1%) Female 2377 (60.2%) 119 (62.3%) 212 (62.9%) DNS score (%) 3123 (79.2%) 488 (12.4%) 175 (4.4%) 102 (2.6%) 55 (1.4%) 136 (71.2%) 29 (15.2%) 14 (7.3%) 9 (4.7%) 3 (1.6%) 138 (40.9%) 73 (21.7%) 54 (16.0%) 49 (14.5%) 23 (6.8%) TCSS categories No neuropathy (0–5) Mild neuropathy (6–8) Moderate neuropathy (9–11) Severe neuropathy (>11) NA NA NA NA NA NA NA NA 256 (76.0%) 56 (16.6%) 24 (7.1%) 1 (0.3%) * NA – Not applicable. Table 1 Socio-demographic characteristics and prevalence of Distal Peripheral Neuropathy among patients with newly diagnosed and established diabetes Subjects with DPN were significantly taller and they had a lower body weight, BMI, waist circumference, hip circum- ference and elevated levels of triglycerides (Table 3). How- ever, the strength of association (Spearmans’ correlation) Katulanda et al. Diabetology & Metabolic Syndrome 2012, 4:21 http://www.dmsjournal.com/content/4/1/21 Katulanda et al. Diabetology & Metabolic Syndrome 2012, 4:21 http://www.dmsjournal.com/content/4/1/21 Katulanda et al. Results Diabetology & Metabolic Syndrome 2012, 4:21 http://www.dmsjournal.com/content/4/1/21 Page 5 of 8 Table 2 Socio-demographic characteristics of diabetic subjects with or without DPN DPN present DPN absent Age (±SD) 62.1 ± 10.8 55.1 ± 10.8 Gender (%) MaleFemale 25 (30.9%) 56 (69.1%) 100 (39.1%) 156 (60.9%) Sector of Residence (%) Urban Rural 20 (24.7%) 61 (75.3%) 84 (32.8%) 172 (67.2%) Highest level of Education (%) Primary Secondary Tertiary 32 (39.5%) 49 (60.5%) 0 (0.0%) 52 (20.3%) 192 (75.0%) 12 (4.7%) Household monthly income (%) < LKR 12,000 LKR 12,000 – 25,000 > LKR 25,000 71 (87.6%) 8 (9.9%) 2 (2.5%) 174 (68.0%) 51 (19.9%) 31 (12.1%) LKR - Sri Lankan Rupees. Table 2 Socio-demographic characteristics of diabetic subjects with or without DPN scores was 48.1% and 24.0% respectively. The diagnosis of DPN is complicated and requires the assessment of multiple features of neuropathy as DPN affects a variety of nerve fibres. [23]. For full classification the San Anto- nio Conference on diabetic neuropathy recommends the assessment of at least one measure from each of the fol- lowing categories: neuropathic symptoms, clinical exam- ination, electro-diagnostic tests, quantitative sensory tests and autonomic function tests [24]. Thus, the TCSS which incorporates both assessment of symptoms and signs can be considered more accurate for comparative studies. Sri Lankan community-based prevalence of DPN is comparable with that reported from India, Ban- gladesh and UK using similar diagnostic criteria. How- ever, the prevalence of DPN in Sri Lanka is significantly lower than in other regional, developing and developed countries (Table 5), differences in diagnostic methodolo- gies could partially account for this difference. Ethnic differences in predisposition and differential exposure to risk factors are other probable mechanisms. Most stud- ies are based on either in-hospital patients or follow-up patients attending diabetic clinics. Recent reviews have noted that “knowledge on the epidemiology of DPN is compromised by the lack of large population-based stud- ies” [8]. treatment with sulphonylurea, increasing height, rural sector of residence, higher fasting triglyceride levels, lower household income and longer duration of diabetes after adjusting for age (Table 4). However the presence of hypertension, obesity, nephropathy, alcohol consumption and physical inactivity were not significant predictors of DPN in patients with DM. In the present study there was a significant association between the use of insulin and presence of DPN. Results Some have postulated that exogenous insulin therapy in T2DM might be associated with DPN through an ex- acerbation of obesity, fluid retention, hypertension and hyperlipidemia [31]. However, randomized clinical trials have shown that intensive insulin therapy can prevent or delay the development of DPN compared to conven- tional insulin therapy [32]. Another possible explanation for the association of insulin use with the prevalence of Discussion DPN is a common complication of DM. This is the first comprehensive report on DPN among DM patients from a nationally representative community based sample from Sri Lanka. The Western, Southern, Sabaragamuwa, Uva, North-Western, Central and North-Central pro- vinces were included while the Northern and Eastern provinces affected by the war had to be excluded. The prevalence of DPN according to the DNS and the TCSS Table 3 Physical characteristics and biochemical parameters of patients with or without DPN Mean (±SD) DPN present DPN absent Correlation coefficient p value Height (cm) 155.7 (±9.0) 153.4 (±9.5) 0.167 <0.05 Weight (kg) 54.6 (±11.5) 58.6 (±11.2) −0.162 <0.01 Body mass index (kg/m2) 22.8 (±3.6) 24.2 (±4.0) −0.150 <0.01 Waist circumference (cm) 83.2 (±11.7) 86.2 (±10.3) −0.151 <0.05 Hip circumference (cm) 90.9 (±8.9) 93.6 (±8.9) −0.150 <0.05 Waist-hip ratio 0.92 (±0.08) 0.92 (±0.06) −0.033 NS Systolic blood pressure (mmHg) 140 (±25) 138 (±21) 0.030 NS Diastolic blood pressure (mmHg) 78 (±12) 80 (±12) −0.082 NS Total cholesterol (mg/dl) 217.4 (±49.3) 217.1 (±43.2) −0.007 NS LDL cholesterol (mg/dl) 138.8 (±38.5) 142.3 (±44.2) 0.013 NS HDL cholesterol (mg/dl) 47.4 (±9.5) 45.8 (±9.1) 0.063 NS Triglycerides (mg/dl) 162.1 (±97.4) 138.2 (±67.4) −0.133 <0.05 Table 3 Physical characteristics and biochemical parameters of patients with or without DPN Mean (±SD) Page 6 of 8 Katulanda et al. Diabetology & Metabolic Syndrome 2012, 4:21 http://www.dmsjournal.com/content/4/1/21 Katulanda et al. Discussion Diabetology & Metabolic Syndrome 2012, 4:21 http://www.dmsjournal.com/content/4/1/21 Table 4 Binary logistic regression of potential risk factors associations with DPN Odds ratio (95% CI) p values Gender – male female reference 6.7 (2.0 – 9.8) < 0.01 Sector of residence – urban rural reference 1.8 (1.1 – 2.5) <0.05 Household monthly income - > LKR 25,000 LKR 12,000 – 25,000 < LKR 12,000 reference 1.5 (1.0 – 2.0) 1.4 (1.0 – 1.8) <0.05 <0.05 Duration of diabetes 1.2 (1.1 – 1.3) <0.05 Triglycerides 1.6 (1.2 – 2.0) <0.05 Height 1.8 (1.2 – 2.4) <0.05 Current smoking - non-smoker smoker reference 5.9 (1.4 – 9.7) <0.05 Alcohol consumption - no yes reference 3.0 (0.8 – 6.5) NS Retinopathy – absent present reference 2.7 (1.3 – 5.4) <0.01 Nephropathy – absentpresent reference 0.6 (0.1 – 2.5) NS Hypertension - absentpresent reference 0.8 (0.4 – 1.6) NS Obesity - BMI <27.5BMI > =27.5 Reference 1.0 (0.5 – 1.9) NS Foot ulcers – absentpresent Reference 10.4 (1.8 – 16.7) <0.01 Physical activity - inactiveactive Reference 0.9 (0.4 – 2.3) NS Drug treatment - metformin sulphonylurea Reference 1.8 (1.1 – 2.5) <0.05 Insulin – not treated with insulin treated with insulin Reference 4.3 (1.3 – 6.9) <0.05 LKR - Sri Lankan Rupees. Table 4 Binary logistic regression of potential risk factors associations with DPN triglycerides are commonly associated with insulin re- sistance and represent a valuable clinical marker of the metabolic syndrome and the resultant atherogenic po- tential could contribute towards the progression of DPN [36]. In addition, Schwann cell lipid metabolism has also been found to be abnormal in DPN, and hence elevated triglycerides may represent a blood marker of the pathological changes in the myelin structure of nerve [37]. Thus, correction of elevated triglycerides with dietary control or drug treatment may have an ameliorative effect on the development and the progression of DPN. The significant associ- ation observed between DPN and smoking may at least in part be secondary to the vascular effects smok- ing, as there has been increasing evidence of the im- portance of microvascular factors in the pathogenesis of DPN [38]. The presence of DPN was associated with rural sector of residence and lower household income. A possible ex- planation for the phenomenon could be that poor people are less likely to use health services, which might result in delayed diagnosis and poor control of DM [39]. Discussion Diabetology & Metabolic Syndrome 2012, 4:21 http://www.dmsjournal.com/content/4/1/21 Page 7 of 8 Page 7 of 8 Page 7 of 8 Katulanda et al. Diabetology & Metabolic Syndrome 2012, 4:21 http://www.dmsjournal.com/content/4/1/21 with cross-sectional data from other studies such as the Diabetes Control and Complications Trial, where neur- opathy at baseline were significantly more likely in males [42]. Further studies are required to determine if there are true gender related differences in the risk of develop- ing DPN. Colombo, Sri Lanka. 4Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Queensland, Australia. References 1. Wild S, Roglic G, Green A, Sicree R, King H: Global prevalence of diabetes: estimates for the year 2000 and projections for 2030. Diabetes Care 2004, 27(5):1047–1053. 2. Abbott CA, Carrington AL, Ashe H, Bath S, Every LC, Griffiths J, Hann AW, Hussein A, Jackson N, Johnson KE, et al: The North-West Diabetes Foot Care Study: incidence of, and risk factors for, new diabetic foot ulceration in a community-based patient cohort. Diabet Med 2002, 19 (5):377–384. 3. Daousi C, MacFarlane IA, Woodward A, Nurmikko TJ, Bundred PE, Benbow SJ: Chronic painful peripheral neuropathy in an urban community: a controlled comparison of people with and without diabetes. Diabet Med 2004, 21(9):976–982. 3. Daousi C, MacFarlane IA, Woodward A, Nurmikko TJ, Bundred PE, Benbow SJ: Chronic painful peripheral neuropathy in an urban community: a controlled comparison of people with and without diabetes. Diabet Med 2004, 21(9):976–982. 4. American Diabetes Association, American Academy of Neurology: Consensus statement: report and recommendations of the san antonio conference on diabetic neuropathy. american diabetes association american academy of neurology. Diabetes Care 1988, 11(7):592–597. y y 5. Melton LJI, Dyck PJ: Epidemiology: In Diabetic Neuropathy. 2 editionth edition. Philadelphia: W.B. Saunders; 1999. 6. Jeffcoate WJ, Harding KG: Diabetic foot ulcers. Lancet 2003, 361 (9368):1545–1551. 6. Jeffcoate WJ, Harding KG: Diabetic foot ulcers. Lancet 2003, 361 (9368):1545–1551. 7. Stevens MJ, Feldman EL, Greene DA: The aetiology of diabetic neuropathy: the combined roles of metabolic and vascular defects. Diabet Med 1995, 12(7):566–579. 8. Schmader KE: Epidemiology and impact on quality of life of postherpetic neuralgia and painful diabetic neuropathy. Clin J Pain 2002, 18(6):350–354. p gy p q y p p neuralgia and painful diabetic neuropathy. Clin J Pain 2002, 18(6):350–354. 9. Boulton AJ, Cavanagh PR, Rayman G: The foot in diabetes. 4 editionth edition. Hoboken: John Wiley & Sons Ltd; 2006. g p p y 9. Boulton AJ, Cavanagh PR, Rayman G: The foot in diabetes. 4 editionth edition. Hoboken: John Wiley & Sons Ltd; 2006. 9. Boulton AJ, Cavanagh PR, Rayman G: The foot in diabetes. 4 editionth edition. Hoboken: John Wiley & Sons Ltd; 2006. Competing interests The author(s) declare that they have no competing interests. Acknowledgements The National Science Foundation of Sri Lanka was the primary source of funding for the study. The additional support provided from the Oxford Centre for Diabetes Endocrinology and Metabolism, UK and the NIHR Biomedical Research Centre Programme is gratefully acknowledged. Received: 03 March 2012 Accepted: 29 May 2012 Published: 29 May 2012 Received: 03 March 2012 Accepted: 29 May 2012 Published: 29 May 2012 Acknowledgements h l 15. Katulanda P, Constantine GR, Mahesh JG, Sheriff R, Seneviratne RD, Wijeratne S, Wijesuriya M, McCarthy MI, Adler AI, Matthews DR: Prevalence and projections of diabetes and pre-diabetes in adults in Sri Lanka–Sri Lanka Diabetes, Cardiovascular Study (SLDCS). Diabet Med 2008, 25 (9):1062–1069. 16. American Diabetes Association: Report of the expert committee on the diagnosis and classification of diabetes mellitus. Diabetes Care 1997, 20 (7):1183–1197. 16. American Diabetes Association: Report of the expert committee on the diagnosis and classification of diabetes mellitus. Diabetes Care 1997, 20 (7):1183–1197. Conclusions h 10. Morkrid K, Ali L, Hussain A: Risk factors and prevalence of diabetic peripheral neuropathy:a study of type 2 diabetic outpatients in Bangladesh. Int J Diabetes Dev Ctries 2010, 30(1):11–17. The present study defines the impact of previously known risk factors for the development of diabetic per- ipheral neuropathy in the South-Asian population, while in addition identifies several new potential risk factors of importance in this ethnically different large sub popula- tion with diabetes. The examination of these patients in the future may lead to identification of factors which have led to the development of neuropathy or the pro- gress of established neuropathy, which will enable risk reduction strategies to be developed. 11. Mather HM, Keen H: The Southall diabetes survey: prevalence of known diabetes in Asians and Europeans. Br Med J (Clin Res Ed) 1985, 291 (6502):1081–1084. 12. Abbott CA, Garrow AP, Carrington AL, Morris J, Van Ross ER, Boulton AJ: Foot ulcer risk is lower in South-Asian and african-Caribbean compared with European diabetic patients in the U.K.: the North-West diabetes foot care study. Diabetes Care 2005, 28(8):1869–1875. 13. Chaturvedi N, Abbott CA, Whalley A, Widdows P, Leggetter SY, Boulton AJ: Risk of diabetes-related amputation in South Asians vs. Europeans in the UK. Diabet Med 2002, 19(2):99–104. 14. Department of Census and Statistics - Sri Lanka: Census of population and housing. Population, Intercensal growth and average annual rate of growth by district, 1981 and 2001., :. [http://www.statistics.gov.lk/ PopHouSat/PDF/Population/p9p1%20Growth.pdf] Retrieved February 26, 2012. Competing interests The author(s) declare that they have no competing interests. Authors’ contributions PK, GRC, DRM and MHRS made substantial contribution to conception and study design. PK and GRC were involved in data collection. RJ and PR were involved in refining the study design, statistical analysis and drafting the manuscript. RJ, PR, MHRS, GRC and PK critically revised the manuscript. All authors read and approved the final manuscript. g Several variables such as obesity, presence of hyper- tension, high serum cholesterol and alcohol consump- tion identified as predictors of DPN in other populations did not emerge as independent predictors in the present study. According to our results subjects with DPN had a significantly lower body weight, BMI and waist circumference. In addition presence of obesity was not a significant risk factor for DPN in logistic re- gression analysis. Several studies from Asian countries have also reported similar results or no association be- tween obesity and presence of DPN [10]. Hence, further studies are required to define the role of body weight in DPN in the Asian population. We also found little evi- dence for an important role of blood pressure, even though in other studies hypertension emerged as a strong risk factor [43]. The reasons for these discrepant findings are not clear. Our study has several limitations. The “duration of diabetes” as measured in this study might not reflect the true duration of the disease but the time since diagnosis and actual diabetes onset might precede diagnosis by several years. We were also unable to assess the impact of glycaemic control on presence of DPN, as data on HbA1c values were not available for the majority of the population. 17. World Health Organization: Part 1. Diagnosis and Classification of Diabetes Mellitus. In Definition, Diagnosis and Classification of Diabetes Mellitus and its Complications. Geneva: WHO; 1999. Discussion Previ- ous studies have reported that metabolic control of DM was worse in patients with a lower socio-economic sta- tus [40]. In addition, the increased risk in rural sector residents could be due to lack of access to the better health care facilities available to residents in urban areas. The association of increased height with DPN indicates that increased stature has a generalized adverse effect on peripheral nerve function. The increased nerve length in taller people is associated with greater axon surface area. Therefore, persons with longer nerves (and thus a larger total axon surface area) may be at greater risk for neuro- logic impairment when exposed to otherwise equivalent hazards. Greater leg length might also be associated with a prolonged time requirement for the complete regener- ation of any injured nerve [41]. Finally, in the present study female gender was associated with an increased risk of developing DPN. The finding is not in agreement, LKR - Sri Lankan Rupees. DPN in this cohort could be that insulin use indicates beta cell failure in this group of patients and may reflect a later stage in the natural history of diabetes or a greater severity. Another interesting observation is the trend towards DPN protection observed in the patients treated with metformin as opposed to sulphonylureas. It is known that metformin has multiple effects with direct vascular implications, such as improvement in lipid pro- file, prevention of oxidative stress-induced endothelial cell death and direct neuro-protective effects via inhib- ition of oxidative stress-related apoptotic cell death in primary neurons [33]. On the other recent animal stud- ies have shown that blockage of K+ channels in neurons by sulphonylureas may selectively potentiate neurotox- icity [34]. Table 5 Prevalence of DPN in regional and other developed countries Country Prevalence of DPN (%) All Male Female Sri Lanka 24.0% 20.0% 26.4% Bangladesh [10] 19.7% 20.9% 18.7% India [25] 29.0% 27.9% 30.8% Pakistan [26] 39.6% UK [27] 28.5% 28.5% 28.5% China [28] 32.0% Iran [29] 51.7% 49.9% 51.7% North-Africa [30] 41.0% 38.5% 43.9% Table 5 Prevalence of DPN in regional and other developed countries The association of elevated fasting triglycerides with DPN supports the emerging idea that hyper-triglyceri- daemia contributes to the development and the pro- gression of diabetic neuropathy [35]. Elevated serum Katulanda et al. Author details 1 Tesfaye S, Stevens LK, Stephenson JM, Fuller JH, Plater M, Ionescu-Tirgoviste C, Nuber A, Pozza G, Ward JD: Prevalence of diabetic peripheral neuropathy and its relation to glycaemic control and potential risk factors: the EURODIAB IDDM Complications Study. Diabetologia 1996, 39 (11):1377–1384. 23. Mayfield JA, Sugarman JR: The use of the Semmes-Weinstein monofilament and other threshold tests for preventing foot and amputation in persons with diabetes. J Fam Pract 2000, 49(Suppl 11): S17–S29. 24. San Antonio Conference on Diabetic Neuropathy: Consensus statement: report and recommendations of the san antonio conference on diabetic neuropathy. american diabetes association american academy of neurology. Diabetes Care 1988, 11(7):592–597. doi:10.1186/1758-5996-4-21 Cite this article as: Katulanda et al.: The prevalence, patterns and predictors of diabetic peripheral neuropathy in a developing country. Diabetology & Metabolic Syndrome 2012 4:21. doi:10.1186/1758-5996-4-21 Cite this article as: Katulanda et al.: The prevalence, patterns and predictors of diabetic peripheral neuropathy in a developing country. Diabetology & Metabolic Syndrome 2012 4:21. doi:10.1186/1758-5996-4-21 Cite this article as: Katulanda et al.: The prevalence, patterns and predictors of diabetic peripheral neuropathy in a developing country. Diabetology & Metabolic Syndrome 2012 4:21. 25. Dutta A, Naorem S, Singh TP, Wangjam K: Prevalence of peripheral neuropathy in newly diagnosed type 2 diabetics mellitus. International Journal of Diabetes in Developing Countries 2005, 25:30–33. 26. Khuwaja AK, Rafique G, White F, Azam SI: Macrovascular complications and their associated factors among persons with type 2 diabetes in Karachi, Pakistan - a multi - center study. J Pak Med Assoc 2004, 54:60–66. 27. Young MJ, Boulton AJ, MacLeod AF, Williams DR, Sonksen PH: A multicentre study of the prevalence of diabetic peripheral neuropathy in the United Kingdom hospital clinic population. Diabetologia 1993, 36 (2):150–154. 28. Liu F, Bao Y, Hu R, Zhang X, Li H, Zhu D, Li Y, Yan L, Lu J, Li Q, et al: Screening and prevalence of peripheral neuropathy in type 2 diabetic outpatients: a randomized multicentre survey in 12 city hospitals of China. Diabetes Metab Res Rev 2010, 26(6):481–489. 29. Rahimdel A, Afkhami-Ardekani M, Souzani A, Modaresi M, Mashahiri MR: Prevalence of Sensory Neuropathy in Type 2 Diabetic Patients in Iranian Population (Yazd Province). Iranian Journal of Diabetes and Obesity 2009, 1 (1):30–35. 30. Gill G, Gebrekidan A, English P, Wile D, Tesfaye S: Diabetic complications and glycaemic control in remote North Africa. QJM 2008, 101(10):793–798. 31. Author details 1 Savage S, Estacio RO, Jeffers B, Schrier RW: Increased complications in noninsulin-dependent diabetic patients treated with insulin versus oral hypoglycemic agents: a population study. Proc Assoc Am Physicians 1997, 109(2):181–189. 32. Diabetes Control and Complications Trial Research Group: The effect of intensive diabetes therapy on measures of autonomic nervous system function in the Diabetes Control and Complications Trial (DCCT). Diabetologia 1998, 41(4):416–423. 33. Detaille D, Guigas B, Chauvin C: Metformin prevents high-glucose-induced endothelial cell death through a mitochondrial permeability transition- dependent process. Diabetes 2005, 54:2179–2187. 34. Kou J, Klorig DC, Bloomquist JR: Potentiating effect of the ATP-sensitive potassium channel blocker glibenclamide on complex I inhibitor neurotoxicity in vitro and in vivo. Neurotoxicology 2006, 27(5):826–834. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: Submit your next manuscript to BioMed Central and take full advantage of: 35. Wiggin TD, Sullivan KA, Pop-Busui R, Amato A, Sima AA, Feldman EL: Elevated triglycerides correlate with progression of diabetic neuropathy. Diabetes 2009, 58(7):1634–1640. 36. Tesfaye S, Harris N, Jakubowski JJ, Mody C, Wilson RM, Rennie IG, Ward JD: Impaired blood flow and arterio-venous shunting in human diabetic neuropathy: a novel technique of nerve photography and fluorescein angiography. Diabetologia 1993, 36(12):1266–1274. • Convenient online submission 37. Thomas Pk, Ward Jd, Watkins Pj: Diabetic neuropathy. London: Edward Arnold; 1982:109–136. 38. Author details 1 1Diabetes Research Unit, Department of Clinical Medicine, Faculty of Medicine, University of Colombo, Colombo, Sri Lanka. 2Oxford Centre for Diabetes, Endocrinology and Metabolism, University of Oxford, Oxford, UK. 3Department of Pharmacology, Faculty of Medicine, University of Colombo, 17. World Health Organization: Part 1. Diagnosis and Classification of Diabetes Mellitus. In Definition, Diagnosis and Classification of Diabetes Mellitus and its Complications. Geneva: WHO; 1999. 17. World Health Organization: Part 1. Diagnosis and Classification of Diabetes Mellitus. In Definition, Diagnosis and Classification of Diabetes Mellitus and its Complications. Geneva: WHO; 1999. Page 8 of 8 Katulanda et al. Diabetology & Metabolic Syndrome 2012, 4:21 http://www.dmsjournal.com/content/4/1/21 39. Karim F, Tripura A, Gani MS, Chowdhury AM: Poverty status and health equity: evidence from rural Bangladesh. Public Health 2006, 120(3):193–205. 18. WHO Expert Consultation: Appropriate body-mass index for Asian populations and its implications for policy and intervention strategies. Lancet 2004, 363(9403):157–163. 19. Zelmanovitz T, Gerchman F, Balthazar AP, Thomazelli FC, Matos JD, Canani LH: Diabetic nephropathy. Diabetol Metab Syndr 2009, 1(1):10. 40. Weng C, Coppini DV, Sonksen PH: Geographic and social factors are related to increased morbidity and mortality rates in diabetic patients. Diabet Med 2000, 17(8):612–617. 19. Zelmanovitz T, Gerchman F, Balthazar AP, Thomazelli FC, Matos JD, Canani LH: Diabetic nephropathy. Diabetol Metab Syndr 2009, 1(1):10. 20. Perkins BA, Olaleye D, Zinman B, Bril V: Simple screening tests for peripheral neuropathy in the diabetes clinic. Diabetes Care 2001, 24 (2):250–256. 20. Perkins BA, Olaleye D, Zinman B, Bril V: Simple screening tests for peripheral neuropathy in the diabetes clinic. Diabetes Care 2001, 24 (2):250–256. 41. Polydefkis M, Hauer P, Sheth S: The time course of epidermal nerve fibre regeneration: studies in normal controls and in people with diabetes, with and without neuropathy. Brain 2004, 127:1606–1615. 21. Meijer JW, Smit AJ, Sonderen EV, Groothoff JW, Eisma WH, Links TP: Symptom scoring systems to diagnose distal polyneuropathy in diabetes: the Diabetic Neuropathy Symptom score. Diabet Med 2002, 19 (11):962–965. 42. The DCCT Research Group: Factors in development of diabetic neuropathy. Baseline analysis of neuropathy in feasibility phase of Diabetes Control and Complications Trial (DCCT). The DCCT Research Group. Diabetes 1988, 37(4):476–481. 22. IPAQ: Guidelines for Data Processing and Analysis of the International Physical Activity Questionnaire (IPAQ)-Short and Long Forms., :. [www. ipaq.ki.se/scoring.pdf] Revised Novemeber 2005. 43. Katulanda et al. Diabetology & Metabolic Syndrome 2012, 4:21 http://www.dmsjournal.com/content/4/1/21 Author details 1 Ibrahim S, Harris ND, Radatz M, Selmi F, Rajbhandari S, Brady L, Jakubowski J, Ward JD: A new minimally invasive technique to show nerve ischaemia in diabetic neuropathy. Diabetologia 1999, 42(6):737–742.
https://openalex.org/W4365141458	https://link.springer.com/content/pdf/10.1007/s40962-023-01020-x.pdf	English	null	Search for the Optimal Soaking Temperature for Hyperquenching of the Gx2crnimocun 25-6-3-3 Duplex Cast Steel	International journal of metalcasting	2,023	cc-by	6,471	Abstract hyperquenching guarantees the elimination of the brittle intermetallic phase in the steel microstructure. In addition, the most favorable strength-to-ductility ratio of the steel with an almost equal amount of austenite c and ferrite a in its microstructure was obtained using the soak temperature of 1080 C, which was considered optimal for the above- mentioned duplex cast steel. hyperquenching guarantees the elimination of the brittle intermetallic phase in the steel microstructure. In addition, the most favorable strength-to-ductility ratio of the steel with an almost equal amount of austenite c and ferrite a in its microstructure was obtained using the soak temperature of 1080 C, which was considered optimal for the above- mentioned duplex cast steel. The paper presents the research results on duplex corro- sion-resistant chromium–nickel–molybdenum cast steel of the GX2CrNiMoCuN 25-6-3-3 grade. The aim was to determine the effect of the soaking temperature for the hyperquenching process Tp, that is, 950 to 1200 C, on the microstructure and mechanical properties, that is UTS tensile strength, the yield strength, HB Brinell hardness, the elongation EL and impact strength KV of the steel, cast under industrial conditions with the minimum content of Cr, Ni, Mo and Cu according to the PN-EN 10283 stan- dard. On the basis of the results, it was conﬁrmed that the Keywords: cast steel, duplex, hyperquenching, mechanical properties, ferrite, austenite According to PN-EN 10283 standard, duplex cast steels contain C\0.08 wt% (usually for most grades even C\0.03 wt%), Cr = 21.0-27.0 wt%, Ni = 4.5-8.5 wt%, Mo = 0.1-5.0 wt%, N = 0.10-0.25 wt%, Mn\1.5 wt%, Si \ 1.0 wt%, S \ 0.025 wt%, P \ 0.035 wt% and possibly for some grades Cu = 1.0-3.5 wt%. Currently, considering the global economic conditions, the possibility of manu- facturing duplex cast steel at minimum concentrations as per the PN-EN 10283 standard is sought, which was pre- sented, among others, in the paper describing the example of the GX2CrNiMoCuN grade 25-6-3-3 (Table 1). In general, duplex cast steels are used in the chemical, petrochemical, energy, mining, marine, pulp and paper, food and medical industries.4,8 SEARCH FOR THE OPTIMAL SOAKING TEMPERATURE FOR HYPERQUENCHING OF THE GX2CrNiMoCuN 25-6-3-3 DUPLEX CAST STEEL Tomasz Wro´bel , Paweł Jurczyk , Czesław Baron and Jan Jezierski Department of Foundry Engineering, Silesian University of Technology, Towarowa, 7, 44-100 Gliwice, Poland Tomasz Wro´bel , Paweł Jurczyk , Czesław Baron and Jan Jezierski Department of Foundry Engineering, Silesian University of Technology, Towarowa, 7, 44-100 Gliwice, Poland Copyright 2023 The Author(s) https://doi.org/10.1007/s40962-023-01020-x Copyright 2023 The Author(s) https://doi.org/10.1007/s40962-023-01020-x Received: 30 December 2022 / Accepted: 07 March 2023 / Published online: 12 April 2023 This paper is an invited submission to IJMC selected from presen- tations at the 74th World Foundry Congress, held October 16 to 20, 2022, in Busan, Korea, and has been expanded from the original presentation. Introduction Among alloy cast steels, one of the most important groups is corrosion-resistant cast steels whose main alloying ele- ments are Cr, Ni, Mn and Mo. In this group, alloy cast steels with a ferritic–austenitic microstructure, commonly referred to as duplex steels, deserve special attention because of their properties. Compared to very popular austenitic cast steels, duplex cast steels contain a lower amount of scarce and expensive Ni and simultaneously have higher mechanical properties, particularly even two times higher YS yield strength while maintaining high corrosion resistance, also in environments containing chloride ions.1–7 The next advantage of duplex cast steels is high resistance to cracks at very low temperature.6 How- ever, it should be noted that the operating temperature of duplex cast steel should not exceed 300 C, as presented in papers.4,8–10 By analyzing the historical outline of duplex cast steel presented in,4 it can be considered the ‘‘youngest’’ in the group of corrosion-resistant alloy cast steels. Therefore, there is potential for research that applies mainly to heat treatment, such as hyperquenching, which is mandatory for duplex steel. According to PN-EN 10283, duplex alloy steel should be hyperquenched from the temperature ranging from 1120 to 1150 C to remove undesirable phases that occur in the microstructure in the as-cast state, including, in particular, the brittle intermetallic phase of This paper is an invited submission to IJMC selected from presen- tations at the 74th World Foundry Congress, held October 16 to 20, 2022, in Busan, Korea, and has been expanded from the original presentation. International Journal of Metalcasting/Volume 17, Issue 4, 2023 2524 the type r,4,6,8,11–13 the presence of which makes it impossible to meet the minimum impact strength of 50 J. Furthermore, the value of the soaking temperature used for the hyperquenching process affects the share of ferrite and austenite, which in turn translates into the mechanical properties. According to,4,13–15 along with the increase in the soaking temperature to the hyperquenching process in the range recommended by PN-EN 10283, the amount of ferrite increases at the cost of austenite and the strength of castings is higher. However, the use of a lower soaking temperature value for the hyperquenching process favors an increase in the amount of austenite at the cost of ferrite, which in turn boosts plastic properties of duplex cast steel. Introduction However, considering the economics of the duplex cast steel production process, there is a need for a related reduction in the consumption of expensive alloying addi- tives and electricity. Therefore, the aim of the investigation was to determine the effect of the normative and non- normative soaking temperature for the hyperquenching process Tp, that is, 950 to 1200 C, on the microstructure and mechanical properties of the cast steel GX2CrNiMo- CuN 25-6-3-3 grade, cast in industrial conditions with a minimum content of Cr, Ni, Mo and Cu as per the PN-EN 10283 standard. using the LECO GDS500A optical emission spectrometer. Based on the results of chemical composition analysis, the numerical simulation of predicting the presence of phases in the microstructure of studied duplex cast steel was performed in Thermocalc software. The samples of tested cast steel in the form of test castings with a standard shape of three-leaf clover (Figure 1) were made during industrial melt conducted in the medium frequency induction furnace Elzamet 750 with an approx- imate capacity of 700 kg. The pouring temperature was 1590 C. The test castings were obtained using a sand mold manufactured in Alphaset technology. The molding sand contains silica sand with the main fraction size of 0.2 mm, the alkaline-phenolic resin in the amount of 1.3% to sand mass and an ester curing agent in the amount of 18% to resin mass. Based on the results of numerical simulation made in Magmasoft, the values of temperature and time of knockout for the test castings were applied approx. 1000 C and approx. 1300 sec., respectively. Finally, after machining for each test, three samples for the tensile test and three for the impact test were prepared. The total amount of samples was 36 for both tensile and impact tests. As part of the tests for the duplex cast steel castings per- formed, heat treatment of the hyperquenching in 25 C water was carried out according to PN-EN 10283, as well as the procedure with a nonstandard soaking temperature Tp. In connection with the experiment, a Tp ranging from 1200 to 950 C was used in increments of 25 C. More- over, for each temperature from the range 950-1200 C, the constant soaking time dependent on the thickness of the Experimental Procedure The material for the tests was GX2CrNiMoCuN 25-6-3-3 alloy cast steel, manufactured under industrial conditions, that is, by GZUT S.A. foundry plant. Table 2 presents the chemical compositions of the tested cast steel determined Table 1. The Chemical Composition and Mechanical Properties of Duplex Cast Steel GX2CrNiMoCuN 25-6-3-3 Grade According to PN-EN 10283 Standard C Si Mn Cr Mo Ni Cu N P S Elements content, wt.% 24.50 2.50 5.00 2.75 0.12 B 0.03 B 1.00 B 1.50 – – – – – B 0.035 B 0.025 26.50 3.50 7.00 3.50 0.22 Mechanical properties UTS, MPa YS, MPa A, % KV, J HB C 650 C 480 C 22 C 50 Not required Table 1. The Chemical Composition and Mechanical Properties of Duplex Cast Steel GX2CrNi According to PN-EN 10283 Standard mposition and Mechanical Properties of Duplex Cast Steel GX2CrNiMoCuN 25-6-3-3 Grade According to PN-EN 10283 Standard Table 2. The Chemical Composition of Tested Duplex Cast Steel GX2CrNiMoCuN 25-6-3-3 Grade Elements content, wt% C Si Mn Cr Mo Ni Cu Nb N P S 0.026 0.40 0.55 24.60 2.61 5.15 2.79 0.05 0.12 0.015 0.006 International Journal of Metalcasting/Volume 17, Issue 4, 2023 2525 Table 2. The Chemical Composition of Tested Duplex Cast Steel GX2CrNiMoCuN 25-6-3-3 Grade Elements content, wt% C Si Mn Cr Mo Ni Cu Nb N P S 0.026 0.40 0.55 24.60 2.61 5.15 2.79 0.05 0.12 0.015 0.006 ble 2. The Chemical Composition of Tested Duplex Cast Steel GX2CrNiMoCuN 25-6-3-3 Grade International Journal of Metalcasting/Volume 17, Issue 4, 2023 2525 Figure 1. The scheme of test casting with location of the samples for tensile and impact tests. Figure 1. The scheme of test casting with location of the samples for tensile and impact tests. Figure 1. The scheme of test casting with location of the samples for tensile and impact tests. Figure 1. The scheme of test casting with location of the samples for tensile and impact tests. Figure 2. The scheme of hyperquenching type heat treatment used for tested duplex cast steel GX2CrNiMoCuN 25-6-3-3 grade. Figure 2. The scheme of hyperquenching type heat treatment used for tested duplex cast steel GX2CrNiMoCuN 25-6-3-3 grade. samples was used. Therefore, in the case of the samples to impact resistance the soaking time was 900 sec. while for the samples to tensile strength equaled 1800 sec. Figure 2 presents the scheme of heat treatment applied. Results and Discussion The phase composition consists of a ferrite, c austenite and r-type Fe-Cr intermetallic phase. It was found that in reality the amount of r phase is approx. 4% and is much lower than predicted numerically. The difference between the predicted and real value results from the applied accelerated knocking out of castings from the mold and their accelerated cooling in the air from temperature ranging from 1000 C, i.e., of r phase precipitation according to.4,11–13 Thus, the beneﬁcial effect of knocking out host castings from the mold on the microstructure of duplex cast steel was conﬁrmed. According to results obtained from the Thermocal analysis (Figure 3), it was concluded that the microstructure of the tested duplex cast steel with determined chemical compo- sition (Table 2) will contain 50% of ferrite a, 33% of austenite c, 16.4% of r-type Fe-Cr intermetallic phase and trace amounts, i.e., 0.6% of carbides M23C6. In reality, all the above-mentioned phases, without car- bides, are present in the microstructure of cast steel. Therefore, Figure 4 shows the microstructure of the GX2CrNiMoCuN 25-6-3-3 cast steel in the as-cast state. Based on the results of the EDS analysis (Figures. 4 and 5 and Table 3), it was found that the a phase is rich in ferrite- forming elements, i.e., Cr, Mo and Si in an average amount of 26 wt%, 4 wt% and 1 wt%, respectively. In addition, the presence of austenitizing elements, i.e., Ni, Mn and Cu, was observed in this phase, but in a smaller amount than in the c phase, i.e., Ni & 4.5 wt% in a and 5.5 wt% in c, Mn & 0.3 wt%. in a and 0.5 wt% in c, Cu & 1 wt% in a and 4.5 wt% in c. On the other hand, in the c phase, a lower concentration of ferrite-forming elements was observed than in the a phase, i.e., Cr & 22 wt%, Mo & 2 wt% and Si & 0.8 wt%. Both phases contain Fe in a comparable amount of about 63.5 wt%. The r phase is mainly located within the boundaries of austenite grains. On average, the r phase contains 61 wt% Fe, 30 wt% Cr, 3.5 wt% Ni and 1 wt% Mn, and is also enriched with Mo in an amount of 4 wt% and 1 wt% Si. Experimental Procedure The heat treatment of the tested samples was carried out using a Linn High Term VMK-1600-G electric chamber resistance furnace. The as-cast state and the hyperquenched state were met- allographically checked using a Nikon Eclipse LV150N light optical microscope (LOM) and a Phenom ProX scanning electron microscope (SEM) with an energy-dis- persive X-ray spectrometer (EDS). The metallographic samples were mechanically ground and polished and then electrolytically etched using LectroPol-5 Struers. In the presented paper, the etching reagent contained 3 g of ferric chloride, 10 cm3 of hydrochloric acid and 90 cm3 of ethanol (etching voltage 15 V at time 30 sec.). As part of the tests using LOM, a quantitative analysis of the share of ferrite and austenite and possibly other phases in the microstructure of duplex cast steel was carried out using the image analysis NIS-Elements v. F3.0 software. More- over, as part of the SEM studies, backscattered electron (BSE) imaging was used at an electron beam accelerating voltage of 10 and 15 kV and utilizing point microanalysis of the chemical composition using the energy-dispersive X-ray spectroscopy (EDS) method. Furthermore, the results of the studies were statistically elaborated using stepwise regression in the Statistica v.13.3 software. After completing the hyperquenching, the mechanical properties were tested, tensile strength UTS, yield strength YS and elongation EL were measured using the WPM Measure Machine, applying samples with a diameter of 14 mm and a gauge length of 70 mm. Hardness was measured using the Brinell Kabid Press B4CS using sin- tered carbide ball intender with a diameter of 10 mm loa- ded with a force of 29420 N. The impact energy KV was measured using the Charpy method, applying standard samples (10910955 mm) at an energy 300 J on the Losenhausenwerk hammer. All mechanical properties were determined at ambient temperature. International Journal of Metalcasting/Volume 17, Issue 4, 2023 2526 International Journal of Metalcasting/Volume 17, Issue 4, 2023 Results and Discussion The chemical composition determined is approximately consistent with the results presented in papers [4 and 16], and therefore, the correctness of phase identiﬁcation with respect to the intermetallic r phase should be conﬁrmed with full conviction. Figure 3. Phase content in the function of temperature for tested duplex cast steel GX2CrNiMoCuN 25-6-3-3 grade. According to data presented in papers,4,13,17 any presence of the r phase in the microstructure of ferritic–austenitic duplex alloy cast steel is undesirable, although papers 18,19 Figure 3. Phase content in the function of temperature for tested duplex cast steel GX2CrNiMoCuN 25-6-3-3 grade. Figure 4. Microstructure of tested GX2CrNiMoCuN 25-6-3-3 cast steel in as-cast state: (a) LOM, mag. 500x, (b) SEM, mag. 10,000x, a ferrite, c austenite and intermetallic r phase. Figure 4. Microstructure of tested GX2CrNiMoCuN 25-6-3-3 cast steel in as-cast state: (a) LOM, mag. 500x, (b) SEM, mag. 10,000x, a ferrite, c austenite and intermetallic r phase. 2527 International Journal of Metalcasting/Volume 17, Issue 4, 2023 Figure 5. The result of EDS analysis in: (a) point 1 from Figure 4b (b) point 2 from Figure 4b (c) point 3 from Figure 4b. ult of EDS analysis in: (a) point 1 from Figure 4b (b) point 2 from Figure 4b (c) point 3 fro Figure 5. The result of EDS analysis in: (a) point 1 from Figure 4b (b) point 2 from Figure 4b (c) point 3 from Figure 4b. Table 3. The Results of EDS Analysis in Points 1, 2 and 3 from Figure 4b Element Point no. 1 2 3 at.% Err. at.% wt% Err. wt% at.% Err. at.% wt% Err. wt% at.% Err. at.% wt% Err. Results and Discussion wt% Fe 63.3 ± 1.3 63.9 ± 1.3 63.7 ± 0.6 64.2 ± 0.6 60.1 ± 0.0 60.9 ± 0.0 Cr 26.6 ± 0.5 25.0 ± 0.5 23.8 ± 0.5 22.3 ± 0.4 30.8 ± 0.0 29.1 ± 0.0 Ni 4.4 ± 0.5 4.7 ± 0.6 5.3 ± 0.5 5.6 ± 0.6 3.3 ± 0.1 3.5 ± 0.1 Mo 2.2 ± 0.2 3.9 ± 0.3 1.3 ± 0.2 2.2 ± 0.3 2.4 ± 0.1 4.2 ± 0.1 Mn 0.3 ± 0.1 0.3 ± 0.1 0.5 ± 0.2 0.5 ± 0.2 1.1 ± 0.1 1.1 ± 0.1 Si 2.2 ± 0.3 1.1 ± 0.1 1.6 ± 0.2 0.8 ± 0.1 2.3 ± 0.1 1.2 ± 0.1 Cu 1.0 ± 0.5 1.1 ± 0.6 3.8 ± 0.6 4.4 ± 0.7 – – – – Table 3. The Results of EDS Analysis in Points 1, 2 and 3 from Figure 4b Table 3. The Results of EDS Analysis in Points 1, 2 and 3 from Figure 4b 45.9 J. In this state, the tested cast steel, due to its impact strength, does not meet the requirements speciﬁed for grade GX2CrNiMoCuN 25-6-3-3 according to the PN-EN 10283 standard (Table 1). present data proving the possibility of its use to increase the hardness and erosion resistance of this type of cast steel. However, the implementation of the objectives of the paper requires meeting the requirements of the PN-EN 10283 standard in terms of mechanical properties, which are adversely affected by the r phase. Furthermore, the nega- tive effect of the r phase on the corrosion resistance of duplex alloy cast steel is also signiﬁcant, as described in.4,20,21 To increase the mechanical properties and meet all the requirements of the PN-EN 10283 standard for the tested cast steel GX2CrNiMoCuN 25-6-3-3, the hyperquenching heat treatment was carried out. Results and Discussion By analyzing the TTT diagrams (temperature transformation time for continuous cooling) of duplex alloy cast steel, described in,4 the soaking temperature was selected with a value higher than that recommended in PN-EN 10283, that is 1200 and 1175 C, as recommended by the above-mentioned As a result, the microstructure obtained in the as-cast state determines the poor mechanical properties of the tested cast steel GX2CrNiMoCuN 25-6-3-3, i.e., UTS = 750.3 MPa; YS = 598.4 MPa; 255HB; EL = 16.8% and KV = 2528 International Journal of Metalcasting/Volume 17, Issue 4, 2023 standards, that is 1150 and 1125 C and with a value lower than the shown above, that is 1100, 1075, 1050, 1025, 1000, 975 and 950 C. The lower temperature range for hyperquenching approximately coincides with the upper value at the r phase separation starting temperature, which according to papers,2,4 is approx. 950 C. By determining the ﬁrst derivative for the correlations (1–5) and equating it to zero, the extremum of the poly- nomial function was sought. Due to the form of the determined polynomial functions (1-5), their extremum is equivalent to their maximum. Thus, the Tp(max) value was determined, for which the most favorable UTS, YS, HB, Table 4 presents the mechanical property test results of the GX2CrNiMoCuN 25-6-3-3 cast steel in the state after hyperquenching process ranging from 950 to 1200 C. Figure 6. Inﬂuence of soaking temperature Tp for hyper- quenching on average tensile strength UTS of tested duplex cast steel GX2CrNiMoCuN 25-6-3-3 grade. Statistical analysis was carried out for the obtained results, looking for the dependence of the average values of UTS, YS, HB, EL and KV as a function of soaking temperature Tp. Using the multiple regression method, the following sta- tistical relationships (1–5) with acceptable correlation coefﬁcients were determined: Figure 6. Inﬂuence of soaking temperature Tp for hyper- quenching on average tensile strength UTS of tested duplex cast steel GX2CrNiMoCuN 25-6-3-3 grade. national Journal of Metalcasting/Volume 17, Issue 4, 2023 Results and Discussion Inﬂuence of soaking temperature Tp for hyper- quenching on average elongation EL of tested duplex cast steel GX2CrNiMoCuN 25-6-3-3 grade. Summing up, the temperature of Tp(opt) = 1080 C was found to be the most favorable for the hyperquenching heat treatment of GX2CrNiMoCuN 25-6-3-3 cast steel, con- sidering all the mechanical properties tested, as it corre- sponds to the average value of Tp1 and Tp2. The performed quantitative analysis was used to determine the effect of soaking temperature in the hyperquenching process on the amount of ferrite a and austenite c. In the microstructure of the tested cast steel GX2CrNiMoCuN 25-6-3-3, it was found that with an increase in the Tp value, the amount of ferrite a increases, reaching 71% at Tp=1200 C, and simultaneously the amount of austenite c decreases, reaching the Tp value 29% (Figures 11 and 12a). However, for the value of Tp = 950 C the proportions are reversed and then the amount of austenite c is the largest and amounts to 64%, while the amount of ferrite a is the smallest and amounts to 36% (Figures 11 and 12b). The c/a ratio, which varies with the temperature Tp, directly affects the mechanical properties of the tested cast steel GX2CrNiMoCuN 25-6-3-3, that is at higher Tp, the higher strength was obtained on average (Figures 6, 7 and 8), and at lower Tp, the average ductility of the cast steel was higher (Figures 9 and 10). Furthermore, in none of the tested samples of GX2CrNiMoCuN 25-6-3-3 cast steel in the hyperquenched state, the presence of the brittle r phase or carbides Cr(Fe) was found, despite the fact that the start temperature of these phases precipitation is approx. 1000 C (Figure 13). However, it is not possible to achieve complete certainty in respect to this matter by using LOM and SEM research techniques, particularly when the decrease of impact resistance at a soaking temperature below 1000 C is noted. Figure 8. Inﬂuence of soaking temperature Tp for hyper- quenching on average hardness HB of tested duplex cast steel GX2CrNiMoCuN 25-6-3-3 grade. Figure 9. Inﬂuence of soaking temperature Tp for hyper- quenching on average elongation EL of tested duplex cast steel GX2CrNiMoCuN 25-6-3-3 grade. Figure 9. Inﬂuence of soaking temperature Tp for hyper- quenching on average elongation EL of tested duplex cast steel GX2CrNiMoCuN 25-6-3-3 grade. Figure 10. Results and Discussion UTS ¼ 34 104T2 p þ 7:73Tp 3602:1 at R2 ¼ 0:90 Eqn: 1 YS ¼ 2 103T2 p þ 4:70Tp 2141:6 at R2 ¼ 0:83 Eqn: 2 HB ¼ 16 104T2 p þ 3:61Tp 1804:5 at R2 ¼ 0:84 Eqn: 3 EL ¼ 3 104T2 p þ 0:58Tp 277:4 at R2 ¼ 0:55 Eqn: 4 KV ¼ 11 104T2 p þ 2:33Tp 1086:0 at R2 ¼ 0:65 Eqn: 5 EL and KV values of the tested GX2CrNiMoCuN 25-6-3-3 Figure 7. Inﬂuence of soaking temperature Tp for hyper- quenching on average yield strength YS of tested duplex cast steel GX2CrNiMoCuN 25-6-3-3 grade. Figure 7. Inﬂuence of soaking temperature Tp for hyper- quenching on average yield strength YS of tested duplex cast steel GX2CrNiMoCuN 25-6-3-3 grade. Graphical interpretation of Eqns. 1–5 is shown in Figures 6, 7, 8, 9 and 10. Graphical interpretation of Eqns. 1–5 is shown in Figures 6, 7, 8, 9 and 10. Table 4. Mechanical Properties of All Samples of Tested Duplex Cast Steel GX2CrNiMoCuN 25-6-3-3 Grade After Hyperquenching at Determined Soaking Temperature Tp Tp UTS DUTS YS DYS HB DHB EL DEL KV DKV C MPa MPa MPa MPa % % J J 1200 812.9 4.7 625.0 3.1 237 6.9 17.5 0.4 152.0 3.6 1175 851.3 3.7 668.3 4.5 241 0.0 27.6 0.6 155.9 4.5 1150 860.7 3.0 670.8 5.6 255 14.0 26.3 0.8 156.2 0.8 1125 859.7 0.4 665.3 3.6 255 14.0 27.2 0.5 161.2 0.8 1100 855.6 4.3 657.2 3.0 255 14.0 28.0 0.3 165.0 3.1 1075 806.5 3.0 618.5 2.6 242 68.8 27.3 1.1 178.0 3.5 1050 796.8 1.3 596.2 3.7 237 6.9 28.4 0.5 164.3 0.6 1025 777.0 1.5 589.7 1.8 210 53.3 26.2 1.8 157.9 3.0 1000 770.9 2.1 573.6 4.3 218 44.7 28.9 0.8 162.3 1.3 975 742.7 2.8 565.2 4.1 200 5.7 28.0 0.6 152.5 1.0 950 721.8 3.3 560.2 0.9 204 11.5 27.1 1.4 151.1 1.1 Table 4. Mechanical Properties of All Samples of Tested Duplex Cast Steel GX2CrNiMoCuN 25-6-3-3 Grade After Hyperquenching at Determined Soaking Temperature Tp International Journal of Metalcasting/Volume 17, Issue 4, 2023 2529 Figure 8. Inﬂuence of soaking temperature Tp for hyper- quenching on average hardness HB of tested duplex cast steel GX2CrNiMoCuN 25-6-3-3 grade. Figure 8. Inﬂuence of soaking temperature Tp for hyper- quenching on average hardness HB of tested duplex cast steel GX2CrNiMoCuN 25-6-3-3 grade. Figure 9. Results and Discussion 4.70 = 0 - 3210-4TP ? 3.61 = 0 Tp(max) = 1136 C Tp(max) = 1175 C Tp(max) = 1128 C Tp1 = 1146 C (4) Concerning EL (5) Concerning KV First derivative - 610-4TP ? 0.58 = 0 - 2210-4TP ? 2.33 = 0 Tp(max) = 967 C Tp (max) = 1059 C Tp2 = 1013 C Tp(opt) = 1080 C Figure 11. Inﬂuence of soaking temperature Tp for hyperquenching on average amount of a ferrite and c austenite in microstructure of tested duplex cast steel GX2CrNiMoCu25-6-3-3 grade. Table 5. The Method of Calculating the Optimal Soaking Temperature Tp(opt) for Hyperquenching of the Tested Duplex Cast Steel of the GX2CrNiMoCuN 25-6-3-3 Grade Equation no. (1) Concerning UTS (2) Concerning YS (3) Concerning HB First derivative - 6810-4TP ? 7.73 = 0 -410-3TP ? 4.70 = 0 - 3210-4TP ? 3.61 = 0 Tp(max) = 1136 C Tp(max) = 1175 C Tp(max) = 1128 C Tp1 = 1146 C (4) Concerning EL (5) Concerning KV First derivative - 610-4TP ? 0.58 = 0 - 2210-4TP ? 2.33 = 0 Tp(max) = 967 C Tp (max) = 1059 C Tp2 = 1013 C Tp(opt) = 1080 C Table 5. The Method of Calculating the Optimal Soaking Temperature Tp(opt) for Hyperquenching of the Tested Duplex Cast Steel of the GX2CrNiMoCuN 25-6-3-3 Grade Table 5. The Method of Calculating the Optimal Soaking Temperature Tp(opt) for Hyperquenching of the Tested Duplex Cast Steel of the GX2CrNiMoCuN 25-6-3-3 Grade Method of Calculating the Optimal Soaking Temperature Tp(opt) for Hyperquenching of the Teste Cast Steel of the GX2CrNiMoCuN 25-6-3-3 Grade Figure 11. Inﬂuence of soaking temperature Tp for hyperquenching on average amount of a ferrite and c austenite in microstructure of tested duplex cast steel GX2CrNiMoCu25-6-3-3 grade. Figure 11. Inﬂuence of soaking temperature Tp for hyperquenching on average amount of a ferrite and c austenite in microstructure of tested duplex cast steel GX2C NiM C 25 6 3 3 d Figure 11. Inﬂuence of soaking temperature Tp for hyperquenching on average amount of a ferrite and c austenite in microstructure of tested duplex cast steel GX2CrNiMoCu25-6-3-3 grade. Results and Discussion Inﬂuence of soaking temperature Tp for hyperquenching on average impact energy KV of tested duplex cast steel GX2CrNiMoCuN 25-6-3-3 grade. Figure 14 shows the microstructures of the tested cast steel GX2CrNiMoCuN 25-6-3-3 after hyperquenching from a temperature very close to the optimum, i.e., 1075 C. The microstructure of this cast steel is two-phase ferritic–aus- tenitic. Similarly to the as-cast state, after hyperquenching from the temperature of 1075 C, a high concentration of ferrite-forming elements is observed in ferrite, that is on average Cr & 25 wt%, Mo & 4 wt% and Si & 1 wt% and occasionally Nb up to 0.5 wt%. On the other hand, there are fewer austenitizing elements in the a phase than in the c phase, that is Ni & 3 wt%. in a and 6.5 wt% in c, Mn & 1 wt% in a and 1.5 wt% in c and Cu less than 0.1 wt% in a and 4 wt% in c. On the other hand, in the c phase, a lower concentration of ferrite-forming elements was found than in the a phase, i.e., Cr & 20.5 wt%, Mo & 2.5 wt%, and Si & 1 wt%. Also, as in the as-cast state, Fe is present in comparable amounts of about 64 wt% in both phases (Figures 14 and 15 and Table 6). Figure 10. Inﬂuence of soaking temperature Tp for hyperquenching on average impact energy KV of tested duplex cast steel GX2CrNiMoCuN 25-6-3-3 grade. cast steel were obtained. For mechanical properties, i.e., UTS, YS and HB were determined this way according to the value of the soaking temperature for the hyper- quenching Tp1, which is the most favorable considering the strength of the tested cast steel GX2CrNiMoCuN 25-6-3-3 (Table 5). The Tp1 value is the average of the Tp(max) values calculated for UTS, YS and HB, respectively. Similarly, for EL and KV, the temperature of soaking for the hyperquenching Tp2 was determined, which is the most favorable considering the ductility of the GX2CrNiMoCuN 25-6-3-3 tested cast steel (Table 5). 2530 International Journal of Metalcasting/Volume 17, Issue 4, 2023 Conclusions GX2CrNiMoCuN 25-6-3-3 grade, which is additionally characterized by reduced produc Table 5. The Method of Calculating the Optimal Soaking Temperature Tp(opt) for Hyperquenching of the Tested Duplex Cast Steel of the GX2CrNiMoCuN 25-6-3-3 Grade Equation no. (1) Concerning UTS (2) Concerning YS (3) Concerning HB First derivative - 6810-4TP ? 7.73 = 0 -410-3TP ? Conclusions GX2CrNiMoCuN 25-6-3-3 grade, which is additionally characterized by reduced produc- tion costs. GX2CrNiMoCuN 25-6-3-3 grade, which is additionally characterized by reduced produc- tion costs. Based on the research conducted, the following conclu- sions have been formulated: (2) 2) A variable factor in the process of manufactur- ing duplex alloy cast steel of GX2CrNiMoCuN 25-6-3-3 grade, which has the strongest inﬂu- ence on its mechanical properties, is the tem- perature of soaking for the hyperquenching process, the increase of which raises the share of ferrite a in the casting microstructure at the cost of austenite c and, as a result, increases the (1) It is possible to optimize the chemical compo- sition and soaking temperature for the hyper- quenching process, carried out, respectively, within the PN-EN 10283 standard and going beyond its recommendations, in order to obtain a fully technologically useful, considering mechanical properties, duplex alloy cast steel International Journal of Metalcasting/Volume 17, Issue 4, 2023 2531 Figure 12. Microstructure of tested GX2CrNiMoCuN 25-6-3-3 cast steel after hyperquenching from: (a) 1200 and (b) 950 C, LOM, mag. 100x, a ferrite and c austenite. Figure 12. Microstructure of tested GX2CrNiMoCuN 25-6-3-3 cast steel after hyperquenching from: (a) 1200 and (b) 950 C, LOM, mag. 100x, a ferrite and c austenite. strength properties, i.e., UTS, YS and HB while reducing ductility properties, i.e., EL and KV. strength properties, i.e., UTS, YS and HB while reducing ductility properties, i.e., EL and KV. (3) Due to the achievement of the most balanced strength and ductility properties with an equal proportion of ferrite to austenite, the most effective way to tailor the technological suit- ability of the castings made of duplex cast steel GX2CrNiMoCuN 25-6-3-3 grade is their hyper- quenching from the temperature of 1080 C. Figure 13. Microstructure of tested GX2CrNiMoCuN 25-6-3-3 cast steel after hyperquenching from 950 C, LOM, mag. 500x, a ferrite and c austenite. (3) (3) Due to the achievement of the most balanced strength and ductility properties with an equal proportion of ferrite to austenite, the most effective way to tailor the technological suit- ability of the castings made of duplex cast steel GX2CrNiMoCuN 25-6-3-3 grade is their hyper- quenching from the temperature of 1080 C. Conclusions (3) Due to the achievement of the most balanced strength and ductility properties with an equal proportion of ferrite to austenite, the most effective way to tailor the technological suit- ability of the castings made of duplex cast steel GX2CrNiMoCuN 25-6-3-3 grade is their hyper- quenching from the temperature of 1080 C. (4) q g p The suggested chemical composition, despite a signiﬁcant reduction in the concentration of such alloying elements as Ni, Mo, Cu and Cr, together with the use of the soaking temperature for the hyperquenching process lower by 40 C as compared to the minimum recommended by PN-EN 10283, allows us to obtain duplex alloy cast steel GX2CrNiMoCuN 25-6-3-3 grade with UTS = 806 MPa, YS = 618 MPa, EL = 27% and KV = 178 J, i.e., respectively, higher for UTS by 24%, for YS by 29%, and for EL by 24%, while in the case of KV by 256%, from the Figure 13. Microstructure of tested GX2CrNiMoCuN 25-6-3-3 cast steel after hyperquenching from 950 C, LOM, mag. 500x, a ferrite and c austenite. Figure 14. Microstructure of tested GX2CrNiMoCuN 25-6-3-3 cast steel after hyperquenching from 1075 C: (a) LOM, mag. 500x, (b) SEM, mag. 4000x, a ferrite and c austenite. Figure 14. Microstructure of tested GX2CrNiMoCuN 25-6-3-3 cast steel after hyperquenching from 1075 C: (a) LOM, mag. 500x, (b) SEM, mag. 4000x, a ferrite and c austenite. International Journal of Metalcasting/Volume 17, Issue 4, 2023 2532 Figure 15. The result of EDS analysis in: (a) point 1 from Figure 14b (b) point 2 from Figure 14b. Figure 15. The result of EDS analysis in: (a) point 1 from Figure 14b (b) point 2 from Figure 14b. Figure 15. The result of EDS analysis in: (a) point 1 from Figure 14b (b) point 2 from Figure 14b. Table 6. The Results of EDS Analysis in Points 1 and 2 from Figure 14b Table 6. The Results of EDS Analysis in Points 1 and 2 from Figure 14b Element Point no. 1 2 at.% Err. at.% wt% Err. wt% at.% Err. at.% wt% Err. REFERENCES 1. H. Eriksson, S. Bernhardsson, The applicability of duplex stainless steels in sour environments. Corros. Sci. 47(9), 719–725 (1991). https://doi.org/10.5006/1. Acknowledgements This paper is an invited submission to IJMC selected from presentations at the 74th World Foundry Con- gress, held October 16 to 20, 2022, in Busan, Korea, and has been expanded from the original presentation. Conclusions wt% Fe 64.6 ± 0.6 65.3 ± 0.6 63.1 ± 0.6 63.5 ± 0.6 Cr 26.6 ± 0.5 25.0 ± 0.5 22.2 ± 0.4 20.8 ± 0.4 Ni 2.9 ± 0.5 3.1 ± 0.5 6.2 ± 0.5 6.5 ± 0.6 Mo 2.3 ± 0.2 3.9 ± 0.3 1.3 ± 0.2 2.3 ± 0.3 Mn 1.1 ± 0.3 1.1 ± 0.3 1.5 ± 0.3 1.5 ± 0.3 Si 2.2 ± 0.2 1.1 ± 0.1 1.9 ± 0.3 1.0 ± 0.1 Cu – – – – 3.8 ± 0.6 4.4 ± 0.7 Nb 0.3 ± 0.1 0.5 ± 0.1 – – – – minimum requirements speciﬁed in the PN-EN 10283 standard for the mechanical properties of the above-mentioned cast steel grade. 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://creativecom mons.org/licenses/by/4.0/. 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 2. B. Voronenko, Austenitic-ferritic stainless steels: a state-of-the-art review. Met. Sci. Heat Treat. 39(10), 428–437 (1997) ( ) 3. M. Chen, W. Tsai, Stress corrosion cracking behaviour of 2205 duplex stainless steel in concentrated NaCl solution. Corros. Sci. 42(3), 545–559 (2000). https:// doi.org/10.1016/S0010-938X(99)00105-5 ( ) 3. M. Chen, W. Tsai, Stress corrosion cracking behaviour of 2205 duplex stainless steel in concentrated NaCl solution. Corros. Sci. 42(3), 545–559 (2000). https:// doi.org/10.1016/S0010-938X(99)00105-5 g 4. B. Kalandyk, Characteristics of microstructure and properties of castings made from ferritic-austenitic 2533 International Journal of Metalcasting/Volume 17, Issue 4, 2023 improving of casting quality from duplex cast steel. Arch. Foundry Eng. 7(3), 39–42 (2007) steel. (Archives of foundry engineering, Katowice– Gliwice, Poland, 2011) 5. E. Edwards, D. Hodgkinson, M. Maxﬁeld, Effect of chemistry and heat treatment on the pitting corrosion resistance of three duplex stainless steel alloys. Int. J. Metalcast. 10(1), 118–121 (2016). https://doi.org/10. 1007/s40962-015-0013-3 14. V. Kanˇa, V. Pernica, A. Zadera, V. Krutisˇ, Compar- ison of methods for determining the ferrite content in duplex cast steels. Arch. Foundry Eng. 19(2), 85–90 (2019). https://doi.org/10.24425/afe.2019.127121 15. P. Jurczyk, T. Wro´bel, C. Baron, The inﬂuence of hyperquenching temperature on microstructure and mechanical properties of alloy cast steel GX2CrNi- MoCuN 25-6-3-3. Arch. Metall. Mater. 66(1), 73–80 (2021). https://doi.org/10.24425/amm.2021.134761 6. N. Haghdadi, P. Cizek, P. Hodgson, H. Beladi, Microstructure dependence of impact toughness in duplex stainless steels. Mat. Sci. Eng. A. 745, 369–378 (2019). https://doi.org/10.1016/j.msea.2018.12.117 7. P. Nithin Raj, A. Sivan, K. Sekar, M. Joseph, Effect of austenite reformation on localized corrosion resistance of hyper-duplex stainless steel in hot chloride solution. Int. J. Metalcast. 14(1), 167–178 (2020). https://doi. org/10.1007/s40962-019-00348-7 16. M. Pohl, O. Storz, T. Głogowski, Effect of inter- metallic precipitation on the properties of duplex stainless steel. Mater. Charact. 58(1), 65–71 (2007). https://doi.org/10.1016/j.matchar.2006.03.015 17. R. Yamamoto, H. Yakuwa, M. Miyasaka, N. Hara, Effects of the a/c-phase ratio on the corrosion behavior of cast duplex stainless steel. International Journal of Metalcasting/Volume 17, Issue 4, 2023 ( ) ( org/10.1007/s40962-019-00348-7 Open Access Corrosion 76(9), 815–825 (2019). https://doi.org/10.5006/3464 8. J. Pearce, P. Bhandhubanyong, Factors inﬂuencing the microstructure and corrosion resistance of austenitic and duplex stainless steel castings. Int. Sci. J. Eng. Tech. 4(2), 41–51 (2020) 18. Z. Stradomski, A. Brodziak-Hyska, C. Kolan, Opti- mization of sigma phase precipitates with respect to the functional properties of duplex cast steel. Arch. Foundry Eng. 12(2), 75–78 (2012). https://doi.org/10. 2478/v10266-012-0040-y 9. T. Chen, K. Weng, J. Yang, The effect of high- temperature exposure on the microstructural stability and toughness property in a 2205 duplex stainless steels. Mater. Sci. Eng. A. 338(1–2), 259–270 (2002). https://doi.org/10.1016/S0921-5093(02)00093-X 19. Y. Wang, J. Han, H. Wu, B. Yang, X. Wang, Effect of sigma phase precipitation on the mechanical and wear properties of Z3CN20.09M cast duplex stainless steel. Nucl. Eng. Des 259, 1–7 (2013). https://doi.org/10. 1016/j.nucengdes.2013.02.037 10. Z. Li, Y. Hu, T. Chen, X. Wang, P. Liu, Y. Lu, Microstructural evolution and mechanical behavior of thermally aged cast duplex stainless steel. Materials 13(24), 5636–5649 (2020). https://doi.org/10.3390/ ma13245636 20. Z. Stradomski, A. Brodziak, The inﬂuence of sigma phase on erosion and corrosion properties of duplex steel. Arch. Foundry Eng. 10(3), 85–88 (2010) 11. T. Chen, J. Yang, Effects of solution treatment and continuous cooling on r-phase precipitation in a 2205 duplex stainless steel. Mater. Sci. Eng. A 311(1–2), 28–41 (2001). https://doi.org/10.1016/S0921- 5093(01)00911-X 21. P. Mu¨ller, V. Pernica, V. Kanˇa, Corrosion resistance of cast duplex steels. Arch Foundry Eng 22(3), 5–10 (2022). https://doi.org/10.24425/afe.2022.140230 12. V. Hosseini, L. Karlsson, S. Wessman, N. Fuertes, Effect of sigma phase morphology on the degradation of properties in a super duplex stainless steel. Materials 11(933), 1–20 (2018). https://doi.org/10. 3390/ma11060933 Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. 13. D. Dyja, Z. Stradomski, Optimization of heat treat- ment in aspect of production’s costs reducing and 13. D. Dyja, Z. Stradomski, Optimization of heat treat- ment in aspect of production’s costs reducing and 2534 International Journal of Metalcasting/Volume 17, Issue 4, 2023
https://openalex.org/W2112267796	https://pure.eur.nl/ws/files/47004678/20101214-16.pdf	English	null	Essential Role for the d-Asb11 cul5 Box Domain for Proper Notch Signaling and Neural Cell Fate Decisions In Vivo	PloS one	2,010	cc-by	6,691	Abstract ECS (Elongin BC-Cul2/Cul5-SOCS-box protein) ubiquitin ligases recruit substrates to E2 ubiquitin-conjugating enzymes through a SOCS-box protein substrate receptor, an Elongin BC adaptor and a cullin (Cul2 or Cul5) scaffold which interacts with the RING protein. In vitro studies have shown that the conserved amino acid sequence of the cullin box in SOCS-box proteins is required for complex formation and function. However, the in vivo importance of cullin boxes has not been addressed. To explore the biological functions of the cullin box domain of ankyrin repeat and SOCS-box containing protein 11 (d-Asb11), a key mediator of canonical Delta-Notch signaling, we isolated a zebrafish mutant lacking the Cul5 box (Asb11Cul). We found that homozygous zebrafish mutants for this allele were defective in Notch signaling as indicated by the impaired expression of Notch target genes. Importantly, asb11Cul fish were not capable to degrade the Notch ligand DeltaA during embryogenesis, a process essential for the initiation of Notch signaling during neurogenesis. Accordingly, proper cell fate specification within the neurogenic regions of the zebrafish embryo was impaired. In addition, Asb11Cul mRNA was defective in the ability to transactivate a her4::gfp reporter DNA when injected in embryos. Thus, our study reporting the generation and the characterization of a metazoan organism mutant in the conserved cullin binding domain of the SOCS-box demonstrates a hitherto unrecognized importance of the SOCS-box domain for the function of this class of cullin-RING ubiquitin ligases and establishes that the d-Asb11 cullin box is required for both canonical Notch signaling and proper neurogenesis. Editor: Marc Vooijs, University Medical Center Maastricht, Netherlands Received April 8, 2010; Accepted October 24, 2010; Published November 19, 2010 Copyright: 2010 Sartori da Silva 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: MASdS and JMT are paid by Algemene Levens Wetenschappen Grant #817.02.002 and #81502006 respectively (www.nwo.nl) and SHD and MPP receive financial support from Top Institute Pharma grant T1-215 and T3-103 (http://www.tipharma.com/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: M.Peppelenbosch@erasmusmc.nl substrate recognition subunit [3]. Abstract Among the cullin-RING E3s, the group collectively denominated as ECS (Elongin BC-Cul2/ Cul5-SOCS-box protein) ubiquitin ligases has recently attracted special attention [4]. This group of E3 ligases has been implicated in transduction of extracellular cues to altered gene transcription. Many details of its modus operandi remain, however, obscure. Specifically, there is remarkably little insight into the in vivo relevance of the different components of ECS ubiquitin ligases. In vitro studies have shown that in ECS ubiquitin ligases the SOCS- box protein works as the substrate recognition subunit. SOCS-box proteins are composed of two distinct protein-protein interaction domains, a substrate binding domain and a SOCS-box domain. The SOCS-box motif is found at the C-terminus of over 70 human proteins in nine different families. In vitro studies show that SOCS boxes act as substrate recognition modules of the ECS type E3 ubiquitin ligase complex (Fig. 1A) [2]. The SOCS-box domain is divided into two sub-domains: the BC box, which links SOCS- box proteins to the cullin-Rbx module and a motif termed cullin box, located immediately downstream of the BC box. The cullin box is proposed to determine whether a given SOCS-box protein Essential Role for the d-Asb11 cul5 Box Domain for Proper Notch Signaling and Neural Cell Fate Decisions In Vivo Maria A. Sartori da Silva1, Jin-Ming Tee1, Judith Paridaen1, Anke Brouwers1, Vincent Runtuwene1, Danica Zivkovic1, Sander H. Diks2, Daniele Guardavaccaro1, Maikel P. Peppelenbosch3* 1 Hubrecht Institute-KNAW and University Medical Center Utrecht, Utrecht, The Netherlands, 2 Department of Cell Biology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands, 3 Department of Gastroenterology and Hepatology, Erasmus MC-University Medical Center Rotterdam, Rotterdam, The Netherlands 1 Hubrecht Institute-KNAW and University Medical Center Utrecht, Utrecht, The Netherlands, 2 Department of Cell Biology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands, 3 Department of Gastroenterology and Hepatology, Erasmus MC-University Medical Center Rotterdam, Rotterdam, The Netherlands November 2010 \| Volume 5 \| Issue 11 \| e14023 ation: Sartori da Silva MA, Tee J-M, Paridaen J, Brouwers A, Runtuwene V, et al. (2010) Essential Role for the d-Asb11 cul5 Box Do naling and Neural Cell Fate Decisions In Vivo. PLoS ONE 5(11): e14023. doi:10.1371/journal.pone.0014023 Generation and characterization of d-asb11 mutants The consensus sequence wXXLPwPXXwXX(Y/F) corresponds to the Cul5-box in the C-terminal portion of the canonical SOCS- box proteins, and is highly conserved in vertebrates [5,9] (Fig. 1B). We performed a TILLING screen on an F1 N-ethyl-N-nitrosurea (ENU)-mutagenized zebrafish library for d-asb11 mutations mapping to the putative consensus sequence [18]. A premature stop codon corresponding to amino acid 281 in the conserved LPwP sequence of the d-Asb11 was identified (Fig. 1C), and the homozygous allele was designated asb11cul. To our knowledge, this is the first report of a metazoan mutant presenting a mutation in the consensus sequence of any SOCS-box protein, allowing for the first assessment of the in vivo function of the cullin box. l Ankyrin repeat and SOCS-box containing proteins (ASB) constitute the largest subclass of the SOCS-box protein family. ASB members (ASB1-ASB18) are structurally characterized by a variable number of N-terminal ankyrin repeats, which mediate the association with the substrate [9]. ASB proteins in general participate in various important biological processes [10–17], but like the superfamily of SOCS-box proteins in toto, their role in vivo remains largely unknown. We have recently showed that Danio rerio Asb11 (d-Asb11) regulates compartment size in the endodermal and neuronal lineages [10] via ubiquitylation of DeltaA, leading to the activation of the canonical Notch pathway [11]. Thus, d-Asb11 is an attractive protein to assess the elusive functions of the cullin box motif in the SOCS-box holodomain. All ASB proteins share, with slight divergences, the consensus sequences of BC box and Cul5 box in their C-terminal (Fig. 1B)[5,6,8]. Thus elucidation of the in vivo mode of action of d-Asb11 should also provide important clues for this family in its entirety. Together, these considerations prompted us to explore the function of Asb11 cullin box in vivo. Morphological analysis of asb11cul revealed a slight hyperper- icardium at 48 and 72 hours post-fertilization (hpf) (Fig. 2A). This corresponds to the Asb11 knockdown morphant phenotype we described previously, although with less severity [10]. Next, to further identify the functional consequences of the mutated allele, we performed whole-mount in situ hybridization (WISH) with d-asb11 probe on 10 hpf embryos. Strikingly, d-asb11 transcripts were enhanced in asb11cul mutants compared to wild type, showing expanded expression in the polster, a U-shaped structure surrounds the head [19], and along the margins of the neural plate (Fig. 2B). Quantitative RT-PCR (qPCR), confirmed the increase of mRNA transcripts in asb11cul. Introduction The ubiquitin-proteasome system plays a fundamental role in the control of numerous cellular processes, including cell cycle progression, gene transcription, signal transduction, proliferation and differentiation [1]. In this system, ubiquitin is first activated by an E1 ubiquitin-activating enzyme. Activated ubiquitin is then transferred to the active-site cysteine of an E2 ubiquitin- conjugating enzyme. Subsequently, an E3 ubiquitin ligase mediates the transfer of ubiquitin from E2 to a lysine residue on the protein substrate. Multiple rounds of these reactions lead to the formation of polyubiquitylated substrates that are targeted to the 26S proteasome [2]. There are two major classes of E3 ubiquitin ligases, proteins with a HECT (homologous to E6-AP carboxyl terminus) domain and proteins with a RING (Really Interesting New Gene)–like motif. Within this class, cullin-RING E3s are multisubunit ubiquitin ligases composed of a scaffold protein known as cullin, a RING finger protein, which mediates the interaction with the E2, a variable substrate-recognition subunit and an adaptor that links the cullin-RING complex to the November 2010 \| Volume 5 \| Issue 11 \| e14023 1 PLoS ONE \| www.plosone.org Role of the d-Asb11 cul5 Box Figure 1. Schematic representation of Asb11 proteins. (A), Asb11 functions as a substrate recognition module in a putative Elongin BC-Cullin- SOCS-box (ECS) type E3 ubiquitin ligase complex. (B), Sequence alignment of conserved Asb11 SOCS-box domain in different species. The cul5-box consensus sequence is indicated below the alignment. Identical amino acids are highlighted in red and similar ones in yellow. Dr: Danio rerio; Mm: Mus musculus; Hs: Homo sapiens. (C), (left) Illustration of the wild type and mutant d-asb11 gene products. Mutated protein is represented as Asb11cul showing the predicted residual fragment and the position of the identified mutation. The different domains are indicated. (right) The TRA mutation changes a leucine into a stop codon. doi:10.1371/journal.pone.0014023.g001 Figure 1. Schematic representation of Asb11 proteins. (A), Asb11 functions as a substrate recognition module in a putative Elongin BC-Cullin- SOCS-box (ECS) type E3 ubiquitin ligase complex. (B), Sequence alignment of conserved Asb11 SOCS-box domain in different species. The cul5-box consensus sequence is indicated below the alignment. Identical amino acids are highlighted in red and similar ones in yellow. Dr: Danio rerio; Mm: Mus musculus; Hs: Homo sapiens. (C), (left) Illustration of the wild type and mutant d-asb11 gene products. Mutated protein is represented as Asb11cul showing the predicted residual fragment and the position of the identified mutation. Introduction The different domains are indicated. (right) The TRA mutation changes a leucine into a stop codon. doi:10.1371/journal.pone.0014023.g001 PLoS ONE \| www.plosone.org Results and Discussion assembles into either a Cul2-Rbx1 or a Cul5-Rbx2 module to recruit and activate the E2 ubiquitin-conjugating enzymes for substrates ubiquitylation [5–8]. In vivo evidence that the cullin box is involved in mediating the biological action of any SOCS-box protein has not been provided hitherto. Generation and characterization of d-asb11 mutants Generation and characterization of d-asb11 mutants The other Notch genes have not been reported to change expression of her5 at this stage of zebrafish embryogenesis, tion of notch3 (Fig. 4D), which has been shown to repress hes5, a mammalian homologue of zebrafish her5 [24] (although Notch inhibition does not expand the her5 expression domain per se [25], and thus the exact status of her5 as a Notch target gene remains uncertain). The other Notch genes have not been reported to change expression of her5 at this stage of zebrafish embryogenesis, can exclude that this works through reduced Notch signaling as DAPT treatment reduces Asb-11 and forced Notch signaling increases Asb-11 expression [11]), implying that the cullin box mutation has consequences for d-Asb11 function. Generation and characterization of d-asb11 mutants Accordingly, higher protein expression levels were detected by immunoblotting on 12 hpf lysates from asb11cul embryos (Fig. 2C). No significant quantitative differences between wild type and heterozygous embryos confirmed the recessive nature of the mutation. The higher mRNA transcripts and protein levels suggest a compensa- tory effect of a hypomorphic mutation in the asb11cul embryo (we Here, we describe the isolation of a zebrafish carrying a mutant allele in the conserved LPwP sequence of the d-Asb11 cullin box. This mutant represents the first metazoan harboring a mutated cullin box. asb11Cul fish are defective in Notch signalling and have severely affected cell fate specification within the neurogenic regions of zebrafish embryos. Thus, our results establish a previously unrecognised in vivo importance of the cullin box for SOCS-box proteins in general and for Asb11 SOCS-box protein function in particular. November 2010 \| Volume 5 \| Issue 11 \| e14023 2 PLoS ONE \| www.plosone.org Role of the d-Asb11 cul5 Box Figure 2. Phenotypic assays on wild type and asb11cul embryos. (A), Morphological analysis of wild type and mutant embryos at 48 and 72hpf. (B), (left) Anterior view of wild type and mutant embryos at 10hpf after whole mount in situ hybridization, WISH, using probe against d-asb11. (right) Graph shows the quantification of the respective expressions using qPCR. (C), (left) Endogenous d-Asb11 in wild type (WT), heterozygous (asb11+/2) and mutant (asb11cul) embryos at 12 hpf was detected by immunoblotting using anti-d-Asb11 antibody. (right) Graph quantifies 3 individual experiments, with 30 embryos/genotype/experiment. doi:10.1371/journal.pone.0014023.g002 Figure 2. Phenotypic assays on wild type and asb11cul embryos. (A), Morphological analysis of wild type and mutant embryos at 48 and 72hpf. (B), (left) Anterior view of wild type and mutant embryos at 10hpf after whole mount in situ hybridization, WISH, using probe against d-asb11. (right) Graph shows the quantification of the respective expressions using qPCR. (C), (left) Endogenous d-Asb11 in wild type (WT), heterozygous (asb11+/2) and mutant (asb11cul) embryos at 12 hpf was detected by immunoblotting using anti-d-Asb11 antibody. (right) Graph quantifies 3 individual experiments, with 30 embryos/genotype/experiment. doi:10.1371/journal.pone.0014023.g002 tion of notch3 (Fig. 4D), which has been shown to repress hes5, a mammalian homologue of zebrafish her5 [24] (although Notch inhibition does not expand the her5 expression domain per se [25], and thus the exact status of her5 as a Notch target gene remains uncertain). PLoS ONE \| www.plosone.org Cullin box is required for correct expression of Notch target genes Morpholino-mediated knockdown of d-asb11 causes repression of specific Delta-Notch elements and their transcriptional targets, whereas misexpression of d-asb11 induces Delta-Notch activity [11]. To test whether the cullin box mutation has comparable consequences for d-Asb11 function in regulating Delta-Notch signaling pathway, we first explored the capacity of the cullin box- deleted protein to activate, upon its overexpression, Notch- dependent transcription in vitro. We observed that overexpression of wild type d-Asb11 in human neuronal precursor cell line, NTera2 [20] led to a strong activation of the Notch target gene hes1, however, overexpression of the mutant protein was not capable of doing so (Fig. 3). Because Notch signaling induces activation of hes1 gene through the CSL transcriptional complex [21], we used a hes1 reporter lacking the conserved CSL-binding site (hes1-RBP) to confirm Notch-specificity for this transactivation. However, neither d-Asb11 nor Asb11Cul were capable of transactivating hes1-RBP. These results showed that the Cul5 box of d-Asb11 is essential for its function to activate the Notch target gene hes1 through Notch pathway. Figure 3. Cullin box domain promotes induction of hes1 gene in vitro. nTera-d1 cells were co-transfected with hes1-luciferase (hes1) or hes1-luciferase lacking the conserved CSL-binding site (hes1-RBPdel) and myc-tag (MT) as a control, or myc-tagged d-asb11 full length (MT-Asb11) or myc-tagged asb11cul (MT-Asb11cul) cDNA. Hes1-dependent Notch activity was analyzed by luciferase measurement. doi:10.1371/journal.pone.0014023.g003 Next, we investigated the expression of Notch target genes in vivo by performing WISH for the Hairy/E(spl)-related transcription factors, her1, her4 and her5 on 12 hpf asb11cul and wild type embryos. At this time point, the expression of her1 and her4 was considerably reduced in asb11cul embryos (Fig. 4A–B). As her1 and her4 are known to be activated by the Notch signaling [22], this result suggests that the Notch signaling pathway is disrupted in embryos lacking the cullin box domain of Asb11. In contrast, asb11cul showed a significant increase in the expression of her5 (Fig. 4C), which is known to be downregulated by the Notch1A- intracellular domain [23]. Consistently, we observed downregula- Figure 3. Cullin box domain promotes induction of hes1 gene in vitro. nTera-d1 cells were co-transfected with hes1-luciferase (hes1) or hes1-luciferase lacking the conserved CSL-binding site (hes1-RBPdel) and myc-tag (MT) as a control, or myc-tagged d-asb11 full length (MT-Asb11) or myc-tagged asb11cul (MT-Asb11cul) cDNA. Hes1-dependent Notch activity was analyzed by luciferase measurement. November 2010 \| Volume 5 \| Issue 11 \| e14023 Cullin box is required for correct expression of Notch target genes doi:10.1371/journal.pone.0014023.g003 PLoS ONE \| www.plosone.org November 2010 \| Volume 5 \| Issue 11 \| e14023 3 Role of the d-Asb11 cul5 Box PLoS ONE \| www.plosone.org 4 November 2010 \| Volume 5 \| Issue 11 \| e14023 November 2010 \| Volume 5 \| Issue 11 \| e14023 PLoS ONE \| www.plosone.org PLoS ONE \| www.plosone.org Role of the d-Asb11 cul5 Box Role of the d-Asb11 cul5 Box Figure 4. asb11cul presented altered expression of Delta-Notch pathway components. Wild type (left panel) and mutant (middle panel) embryos at 12 hpf were analyzed for WISH using probes against her1, A; her4, B; her5, C; notch3, D; deltaD, E; and deltaA, F. (G), Higher magnification shows detailed analysis of deltaA expression. (left) Graphs quantify the mRNA expression levels. doi:10.1371/journal.pone.0014023.g004 We performed WISH to investigate the expression of ngn1, a bHLH transcription factor, which is expressed in neuronal precursors and differentiated neural cells [30] and is negatively regulated by Notch signaling [31]. As expected, wild type embryos at 12 hpf displayed the typical clustered expression of ngn1 (Fig. 5B). However, asb11cul embryos expressed ngn1 at a uniform high level with less evidence of clustering. The increase in ngn1 mRNA expression was confirmed by qPCR. d-Asb11 morphants showed a similar phenotype [10], confirming that the higher expression of ngn1 is caused by loss of d-Asb11 function in the mutant. thus we did not attempt to assess their expression levels in the context of the analysis of her5 expression patterns. Next, we analyzed expression of the Notch ligands DeltaA and DeltaD in asb11cul embryos. deltaA transcripts showed increased expression in asb11cul embryos (Fig. 4F), whereas deltaD remained unaffected (Fig. 4E). Detailed examination of the WISH expression patterns of deltaA revealed a change in distribution of mRNA in the neural plate (Fig. 4G). Wild type embryos exhibit a distinct ‘‘salt and pepper’’ aspect of deltaA mRNA distribution whereby some cells have stronger expression than their neighbors, consistent with the notion of Delta-Notch lateral signaling [26]. In contrast, asb11cul embryos showed a smear of deltaA mRNA transcript across the neural plate, indicating an impaired Notch-mediated lateral inhibition. Thus, the mutation in the d-asb11 cullin box results in the disruption of canonical Delta-Notch signaling. Some studies have shown that her4 is also expressed in Islet1/2- positive sensory neurons and its expression is not involved in canonical Notch signaling [29]. The cullin box domain of Asb11 is a bona-fide promoter of Notch-mediated her4 induction expression All together our data suggest that the cullin box domain of d- Asb11 is essential to regulate Notch targets genes although d- Asb11 lacking the cullin box may yet affect protein expression independently of Notch, via proneural genes. p It was reported that Hairy/E(Spl) expression and activity can be independent of Notch signaling in vivo [27]. Hence, to determine whether the altered regulation of Hairy/E(spl)-related transcrip- tion factors in asb11cul embryos was mediated by Notch activity, we co-injected her4::gfp reporter DNA with d-asb11 or asb11cul mRNA in zebrafish embryos, which were then treated with DAPT, a c- secretase inhibitor that blocks Notch signaling [28]. her4 transac- tivation was determined as a summation of all green fluorescent protein (GFP) present in the embryo. Confocal microscopy was used to trinomially classify transactivation of the her4 promoter as weak, medium or strong (Fig. 5A). When her4::gfp was injected with myc tag (MT) mRNA as a control, embryos presented 80%, 12% and 8% of weak, medium and strong GFP signals, respectively. Upon DAPT treatment, the number of medium and strong signal expressing embryos decreased to 8% and 4%, respectively, showing that Notch signaling was disrupted in response to DAPT treatment. Misexpression of MT-d-asb11 mRNA resulted in an increase in embryos expressing medium GFP signals (52%, c.f. 12% in MT-injected embryos; p,0.05), and strong GFP signals (24% c.f. 8% in MT-injected embryos; p,0.05). In agreement with previous data, MT-dAsb11 was unable to induce her4:gfp upon exposure of DAPT [11], showing the hierarchical upstream function of d-Asb11 in canonical Notch activation. Cullin box is required for correct expression of Notch target genes Consistently, islet1, detected by WISH, was also increased in zebrafish mutants at 16 hpf. Interestingly, islet1 expression was higher in the polster region where asb11cul were significantly increased in mutants (Fig. 5C). The cullin box is essential for DeltaA degradation and regulation of neural committed cells We have previously shown that d-Asb11 affects Delta-Notch signaling by targeting DeltaA for ubiquitylation and subsequent degradation. This effect, strictly dependent on the presence of the SOCS-box [11], establishes the lateral inhibition gradients between DeltaA and Notch facilitating canonical Notch signaling. To study the role of the cullin box domain in d-Asb11-mediated degradation of DeltaA, we injected zebrafish embryos with Myc- tagged deltaA (MT-dlA) and d-asb11 or asb11cul mRNA at one-cell stage. Embryos were analyzed for the presence of MT-DeltaA protein at 12 hpf. Wild type embryos injected with full-length d- asb11 displayed substantial DeltaA degradation. In contrast, injected asb11cul was not capable of degrading DeltaA when compared to control (Fig. 6A; p,0.05). Thus, we show that the cullin box domain of d-Asb11 is essential for degradation of Notch ligand DeltaA in zebrafish embryos, providing the first in vivo (but not in vitro, e.g. [32]]) evidence that absence of a cullin box interferes with a protein degradation function of a SOCS-box- protein. Moreover, the expression of deltaA in the three longitudinal domains of zebrafish neural plate corresponds to regions that express elevated levels of ngn1 and in which the earliest neurons are born [33]. As Asb11cul was unable to degrade DeltaA and acted as a dominant negative increasing the quantity of DeltaA protein in mutant embryos, we propose that the premature neuronal commitment in asb11cul embryos, assessed by the increased expression of the proneural gene ngn1, is a consequence of DeltaA accumulation in the neural plate. Interestingly, injection of MT-Asb11cul mRNA caused an increase in the number of embryos expressing medium signals, whereas the number of embryos with strong her4::gfp expression was slightly increased compared with control MT-injected embryos. However this effect was observed in both DAPT treated and untreated embryos (24% and 28%, respectively), suggesting that d-Asb11 lacking the cullin box domain (Asb11cul) is much less efficient in inducing the her4 reporter than wild type d-Asb11 and its function is independent of Notch signaling. These data are consistent with studies showing that her4 may be expressed in a Notch-independent manner in specific regions of the nervous system [27]. Although during early neurogenesis her4 expression requires Notch activation, during late neuronal development the her4 induction in sensory neurons is independent of Notch signaling and dependent on proneural genes, as neurogenin1 (ngn1) and zath3 [29]. Absence of the cullin box alters proliferation patterns p p As Notch signaling drives (or maintain) precursor cell proliferation within the neurogenic regions of the embryo, a prediction from our findings would be that the loss of d-Asb11 cullin box would impair such proliferation. Indeed, fluorescent whole-mount antibody labeling with the mitotic marker anti- November 2010 \| Volume 5 \| Issue 11 \| e14023 PLoS ONE \| www.plosone.org 5 Role of the d-Asb11 cul5 Box Figure 5. her4::gfp transactivation and premature differentiation of neural cells in asb11cul. (A), the her4::gfp reporter was co-injected with myc-tag (MT) mRNA as a control, myc-tagged d-asb11 full length (MT-Asb11) or myc-tagged asb11cul (MT-Asb11cul) mRNA in zebrafish embryos. Injected embryos were treated with (+) (n = 25) or without (2) (n = 25) DAPT, from 1.5 hpf. At 14 hpf, embryos were analyzed for her4 transactivation based on the intensity of the GFP signal. Positive embryos were counted and percentages of embryos presenting weak (blue), medium (green) or strong (red) signal were given. (B), Wild type (left panel) and mutant (middle panel) embryos at 12 hpf were analyzed for WISH using probe against ngn1. (right) Graph quantifies expression of ngn1 using qPCR. (C) Wild type (left panel) and mutant (right panel) polster of embryos at 16 hpf were analyzed for WISH using probe against islet1. doi:10 1371/journal pone 0014023 g005 phosphohistone 3 (PH 3) antibody showed a significant decrease In summary he Figure 5. her4::gfp transactivation and premature differentiation of neural cells in asb1 myc-tag (MT) mRNA as a control, myc-tagged d-asb11 full length (MT-Asb11) or myc-tagged a Injected embryos were treated with (+) (n = 25) or without (2) (n = 25) DAPT, from 1.5 hpf. At 14 based on the intensity of the GFP signal. Positive embryos were counted and percentages of e strong (red) signal were given. (B), Wild type (left panel) and mutant (middle panel) embryos at ngn1. (right) Graph quantifies expression of ngn1 using qPCR. (C) Wild type (left panel) and mut analyzed for WISH using probe against islet1. doi:10.1371/journal.pone.0014023.g005 Figure 5. her4::gfp transactivation and premature differentiation of neural cells in asb11cul. (A), the her4::gfp reporter was co-injected with myc-tag (MT) mRNA as a control, myc-tagged d-asb11 full length (MT-Asb11) or myc-tagged asb11cul (MT-Asb11cul) mRNA in zebrafish embryos. Injected embryos were treated with (+) (n = 25) or without (2) (n = 25) DAPT, from 1.5 hpf. RNA isolation and qRT-PCR Total RNA was extracted from whole wild type and mutant embryos at 10 or 12 hpf. Total RNA extraction, cDNA synthesis and qPCR quantification were performed according to previously described methods [38]. DAPT treatment Half of each injected group (n=50) (Fig. 5A) was incubated in 100 mM DAPT diluted in 1% DMSO in embryo-medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4, 0.00005% Meth Blue). The other half was incubated in 1% DMSO in embryo- medium. The embryos were incubated from 1.5hpf till 14hpf, fixed with 4% PFA overnight at 4uC and analyzed for GFP expression. Immunoblotting Whole mount in situ hybridizations were performed according to methods previously described [36]. At 12hfp, chorion and yolk were removed. Embryos were lysed in cell lyses buffer (50 mM Tris-Cl pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.1% Na-deoxycholate, 1% NP-40, 10 u, 1% protease inhibitor (ROCHE), 2 ml/embryo. Primary antibodies were diluted in PBS containing 5% milk (fig. 2: rabbit anti-asb11 1:100, fig. 5: rabbit anti-MT 1:1000, Bioke) and used for immunoblotting as previously described [37]. As loading control an anti-actin body was used in addition to coomassie staining of the membrane. For densitometric analysis all bands were measured with a GS-800 Densitometer (Biorad), and total area counts (OD x mm2) were corrected for back ground (equivalent area on a non-relevant place on the blot). Subsequently samples were corrected for loading using the control band and finally values were expressed relative, defining the intensity of the wild type sample as 1. Figure 6. Cullin box is essential for DeltaA degradation and for maintaining a cell proliferating state in vivo. (A) Zebrafish embryos were injected with Myc-tagged deltaA (MT-DeltaA) and d- asb11 (Asb11) or asb11cul (Asb11cul) mRNA at one-cell stage. (lower panel) Lysates of 12 hpf embryos were analyzed by immunoblotting for the presence of DeltaA. (higher panel) Graph quantifies 2 individual experiments, each with 30 injected embryos/group. (B), Fluorescent whole-mount antibody labeling of wild type (WT) and asb11cul embryos at 24 hpf for the mitotic marker anti-phosphohistone-3 (PH 3) antibody (green) and the neuronal marker Hu(C). Graph shows the number of positive cells per area (5 somites from beginning of yolk extension) of 5 embryos for each genotype. doi:10 1371/journal pone 0014023 g006 mRNA synthesis, mRNA and DNA microinjections mRNA synthesis, mRNA and DNA microinjections Capped mRNAs were synthesized using the mMESSAGE mMACHINE kit (Ambion). Fig. 6A, embryos were injected with 600 pg MT-deltaA and 350 pg d-asb11 or 350 pg asb11cul mRNAs. Fig. 5A, embryos were injected with 5 pg her4::gfp DNA or 5 pg her4::gfp +300 pg d-asb11 or asb11cul mRNA. Total volume of the injection was set at 1 nL. Capped mRNAs were synthesized using the mMESSAGE mMACHINE kit (Ambion). Fig. 6A, embryos were injected with 600 pg MT-deltaA and 350 pg d-asb11 or 350 pg asb11cul mRNAs. Fig. 5A, embryos were injected with 5 pg her4::gfp DNA or 5 pg her4::gfp +300 pg d-asb11 or asb11cul mRNA. Total volume of the injection was set at 1 nL. In situ hybridization Whole mount in situ hybridizations were performed according to methods previously described [36]. All probes used in this study are previously described [10,11]. Fish and embryos Figure 6. Cullin box is essential for DeltaA degradation and for maintaining a cell proliferating state in vivo. (A) Zebrafish embryos were injected with Myc-tagged deltaA (MT-DeltaA) and d- asb11 (Asb11) or asb11cul (Asb11cul) mRNA at one-cell stage. (lower panel) Lysates of 12 hpf embryos were analyzed by immunoblotting for the presence of DeltaA. (higher panel) Graph quantifies 2 individual experiments, each with 30 injected embryos/group. (B), Fluorescent whole-mount antibody labeling of wild type (WT) and asb11cul embryos at 24 hpf for the mitotic marker anti-phosphohistone-3 (PH 3) antibody (green) and the neuronal marker Hu(C). Graph shows the number of positive cells per area (5 somites from beginning of yolk extension) of 5 embryos for each genotype. doi 10 1371/journal pone 0014023 g006 Zebrafish were kept at 27.5uC. Embryos were obtained by natural matings, cultured in embryo medium and staged according to methods previously described [34]. Absence of the cullin box alters proliferation patterns At 14 hpf, embryos were analyzed for her4 transactivation based on the intensity of the GFP signal. Positive embryos were counted and percentages of embryos presenting weak (blue), medium (green) or strong (red) signal were given. (B), Wild type (left panel) and mutant (middle panel) embryos at 12 hpf were analyzed for WISH using probe against ngn1. (right) Graph quantifies expression of ngn1 using qPCR. (C) Wild type (left panel) and mutant (right panel) polster of embryos at 16 hpf were analyzed for WISH using probe against islet1. doi:10.1371/journal.pone.0014023.g005 In summary, here we show that the Cul5 domain of d-Asb11 is necessary for proper Notch signaling in vitro and in vivo. Zebrafish embryos lacking the cullin box of d-Asb11 displayed alterations in the expression of Notch pathway components and defective neurogenesis. Thus, our in vivo study reveals a novel role of cullin boxes previously unrecognized in in vitro experiments. phosphohistone-3 (PH 3) antibody showed a significant decrease in the rate of cellular proliferation of asb11cul embryos at 24 hpf (Fig. 6B, green label), indicating that the d-Asb11 cullin box is necessary for proper cell proliferation. Alternatively, the prema- ture differentiation of precursor cells in d-asb11 mutants led to diminished number of proliferating cells. PLoS ONE \| www.plosone.org November 2010 \| Volume 5 \| Issue 11 \| e14023 November 2010 \| Volume 5 \| Issue 11 \| e14023 6 Role of the d-Asb11 cul5 Box Plasmid construction Plasmids were constructed and/or provided as previously described [10,11]. The pCS2+MT-DeltaA construct was provided by B. Appel (Vanderbilt University, Nashville TN) [35]. The her4::gfp reporter was provided by S. Yeo (Kyungpook National University, Korea) [2]. For asb11cul, mutant zebrafish cDNA was isolated and cloned into the EcoRI and XhoI sites of pCS2+MT and pCS2+. References Notch-dependent HES1, HES5, HEY1, HEY2, HEYL transcription in fetal tissues, adult tissues, or cancer. Int J Oncol 31(2): 461–6. 1. Gao M, Karin M (2005) Regulating the regulators: control of protein ubiquitination and ubiquitin-like modifications by extracellular stimuli. Mol Cell 19: 581–931. J ( ) 22. Takke C, Campos-Ortega JA (1999) her1, a zebrafish pair-rule like gene, acts 22. Takke C, Campos-Ortega JA (1999) her1, a zebrafish pair-rule like gene, acts downstream of notch signalling to control somite development. Development 126: 3005–14. , p g J ( ) , p g , downstream of notch signalling to control somite development. Development 126: 3005–14. 2. Kile BT, Schulman BA, Alexander WS, Nicola NA, Martin HM, et al. (2002) The SOCS box: a tale of destruction and degradation. Trends Biochem Sci 27: 235–241. 23. Hans S, Scheer N, Riedl I, v Weizsacker E, Blader P, et al. (2004) her3, a zebrafish member of the hairy-E(spl) family, is repressed by Notch signalling. Development 131: 2957–2969. 3. Deshaies RJ, Joazeiro CA (2009) RING domain E3 ubiquitin ligases. Annu Rev Biochem 78: 399–434. 4. Piessevaux J, Lavens D, Peelman F, Tavernier J (2008) The many faces of the SOCS box. Cytokine Growth Factor Rev 19: 371–81. 24. Beatus P, Lundkvist J, Oberg C, Lendahl U (1999) The notch 3 intracellular domain represses notch 1-mediated activation through Hairy/Enhancer of split (HES) promoters. Development 126: 3925–3935. y 5. Kamura T, Maenaka K, Kotoshiba S, Matsumoto M, Kohda D, et al. (2004) VHL-box and SOCS-box domains determine binding specificity for Cul2-Rbx1 5. Kamura T, Maenaka K, Kotoshiba S, Matsumoto M, Kohda D, et al. (2004) VHL-box and SOCS-box domains determine binding specificity for Cul2-Rbx1 and Cul5-Rbx2 modules of ubiquitin ligases. Genes Dev 18: 3055–3065. 25. Geling A, Plessy C, Rastegar S, Stra¨hle U, Bally-Cuif L (2004) Her5 acts as a prepattern factor that blocks neurogenin1 and coe2 expression upstream of Notch to inhibit neurogenesis at the midbrain-hindbrain boundary. Develop- ment 31: 1993–2006. and Cul5-Rbx2 modules of ubiquitin ligases. Genes Dev 18: 305 6. Kohroki J, Nishiyama T, Nakamura T, Masuho Y (2005) ASB proteins interact with Cullin5 and Rbx2 to form E3 ubiquitin ligase complexes. FEBS Lett 579: 6796–6802. 26. Skeath JB, Thor S (2003) Genetic control of Drosophila nerve cord development. Curr Opin Neurobiol 13: 8–15. 7. Krebs DL, Hilton DJ (2000) SOCS: physiological suppressors of cytokine signaling. J Cell Sci 113: 2813–2819. 27. References Yeo SY, Kim M, Kim HS, Huh TL, Chitnis AB (2007) Fluorescent protein expression driven by her4 regulatory elements reveals the spatiotemporal pattern of Notch signaling in the nervous system of zebrafish embryos. Dev Biol 301: 555–567. 8. Mahrour N, Redwine WB, Florens L, Swanson SK, Martin-Brown S, et al. (2008) Characterization of Cullin-box sequences that direct recruitment of Cul2- Rbx1 and Cul5-Rbx2 modules to Elongin BC-based ubiquitin ligases. J Biol Chem 283: 8005–8013. 28. Geling A, Steiner H, Willem M, Bally-Cuif L, Haass C (2002) A gamma- secretase inhibitor blocks Notch signaling in vivo and causes a severe neurogenic phenotype in zebrafish. EMBO Rep 3: 688–694. 9. Hilton DJ, Richardson RT, Alexander WS, Viney EM, Willson TA, et al. (1998) Twenty proteins containing a C-terminal SOCS box form five structural classes. Proc Natl Acad Sci U S A 95: 114–119. 29. So JH, Chun HS, Bae YK, Kim HS, Park YM, et al. (2008) Her4 is necessary for establishing peripheral projections of the trigeminal ganglia in zebrafish. Biochem Biophys Res Commun 379: 22–26. 10. Diks SH, Bink RJ, van de Water S, Joore J, van Rooijen C, et al. (2006) The novel gene asb11: a regulator of the size of the neural progenitor compartment. J Cell Biol 174: 581–592. 30. Ma Q, Chen Z, del Barco Barrantes I, de la Pompa JL, Anderson DJ (1998) neurogenin1 is essential for the determination of neuronal precursors for proximal cranial sensory ganglia. Neuron 20: 469–482. J 11. Diks SH, Sartori da Silva MA, Hillebrands JL, Bink RJ, Versteeg HH, et al. (2008) d-Asb11 is an essential mediator of canonical Delta-Notch signalling. Nat Cell Biol 10: 1190–1198. 12. Boengler K, Pipp F, Fernandez B, Richter A, Schaper W, et al. (2003) The ankyrin repeat containing SOCS box protein 5: a novel protein associated with arteriogenesis. Biochem Biophys Res Commun 302: 17–22. 31. Blader P, Fischer N, Gradwohl G, Guillemot F, Strahle U (1997) The activity of neurogenin1 is controlled by local cues in the zebrafish embryo. Development 124(22): 4557–4569. 13. Ferguson JE, 3rd, Wu Y, Smith K, Charles P, Powers K, et al. (2007) ASB4 is a hydroxylation substrate of FIH and promotes vascular differentiation via an oxygen-dependent mechanism. Mol Cell Biol 27: 6407–6419. 32. Chung AS, Guan YJ, Yuan ZL, Albina JE, Chin YE (2005) Ankyrin repeat and SOCS box 3 (ASB3) mediates ubiquitination and degradation of tumor necrosis factor receptor II. References Mol Cell Biol 25: 4716–4726. 14. Kile BT, Metcalf D, Mifsud S, DiRago L, Nicola NA, et al. (2001) Functional analysis of Asb-1 using genetic modification in mice. Mol Cell Biol 21: 6189–6197. 33. Appel B, Eisen JS (1998) Regulation of neuronal specification in the zebrafish spinal cord by Delta function. Development 125: 371–380. 33. Appel B, Eisen JS (1998) Regulation of neuronal specificati spinal cord by Delta function. Development 125: 371–380. 34. Kimmel CB, Ballard WW, Kimmel SR, Ullmann B, Schilling TF (1995) Stages of embryonic development of the zebrafish. Dev Dyn 203(3): 253–310. 15. Kim KS, Kim MS, Kim SK, Baek KH (2004) Murine Asb-17 expression during mouse testis development and spermatogenesis. Zygote 12(2): 151–15. of embryonic development of the zebrafish. Dev Dyn 203(3): 2 35. Appel B, Fritz A, Westerfield M, Grunwald DJ, Eisen JS, et al. (1999) Delta- mediated specification of midline cell fates in zebrafish embryos. Curr Biol 11: 247–56. 16. McDaneld TG, Hannon K, Moody DE (2006) Ankyrin repeat and SOCS box protein 15 regulates protein synthesis in skeletal muscle. Am J Physiol Regul Integr Comp Physiol 290: R1672–1682. 36. Oxtoby E, Jowett T (1993) Cloning of the zebrafish krox-20 gene (krx-20) and its expression during hindbrain development. Nucleic Acids Res 21: 1087–109. g p y 17. McDaneld TG, Spurlock DM (2008) Ankyrin repeat and suppressor of cytokine signaling (SOCS) box-containing protein (ASB) 15 alters differentiation of mouse C2C12 myoblasts and phosphorylation of mitogen-activated protein kinase and Akt. J Anim Sci 86: 2897–290. 37. Versteeg HH, Sørensen BB, Slofstra SH, Van den Brande JH, Stam JC, et al. (2002) VIIa/tissue factor interaction results in a tissue factor cytoplasmic domain-independent activation of protein synthesis, p70, and p90 S6 kinase phosphorylation. J Biol Chem 277: 27065–72. J 18. Wienholds E, van Eeden F, Kosters M, Mudde J, Plasterk RH, et al. (2003) Efficient target-selected mutagenesis in zebrafish. Genome Res 13: 2700–2707. 38. Braat H, Stokkers P, Hommes T, Cohn D, Vogels E, et al. (2005) Consequence of functional Nod2 and Tlr4 mutations on gene transcription in Crohn’s disease patients. J Mol Med 83: 601–9. 19. Kimmel CB, Ballard WW, Kimmel SR, Ullmann B, Schilling TF (1995) Stages of embryonic development of the zebrafish. Dev Dyn 203(3): 253–310. 20. Pleasure SJ, Lee VM (1993) NTera 2 cells: a human cell line which displays characteristics expected of a human committed neuronal progenitor cell. J Neurosci Res 35: 585–602. We thank Dr. Paula van Tijn for helpful discussions. We thank Dr. Paula van Tijn for helpful discussions. Whole mount immunolabelling, microscopy and image quantification Whole-mount immunohistochemistry and picture capture and analysis was performed as described [13,39]. For figure 6B anti- November 2010 \| Volume 5 \| Issue 11 \| e14023 7 PLoS ONE \| www.plosone.org Role of the d-Asb11 cul5 Box HuC (red) and anti-PH 3 (green) antibodies (Upstate Biotechnol- ogy) were used. For the analysis of fluorescent stainings, Leica Confocal TCS SPE was used. To quantify the intensity of signal, a z-stack (z-slices of 7 mM) was made, scanning the whole embryo. Leica software (Application Suite 1.8.0) was used to create a maximum projection of the z-stack. Author Contributions Conceived and designed the experiments: MSdS JMT DZ MP. Performed the experiments: MSdS JMT JP AB SHD. Analyzed the data: MSdS JMT VR DZ MP. Contributed reagents/materials/analysis tools: DG. Wrote the paper: MSdS JMT DG MP. VR DZ MP. Contributed reagents/materials/analysis tools: DG. Wrote the paper: MSdS JMT DG MP. Statistical testing Each value with a standard deviation is the average of at least two independent experiments performed in triplicate. Statistical tests were performed using two-tailed t-test. All bars in graphs depict mean values with error bars depicting standard deviations. Statistical x2-test was performed for Fig. 5A. Acknowledgments nTera2/d1 cells were maintained in DMEM containing 10% FCS. The culture media were supplemented with 5 mM glutamine and antibiotics/antimycotics. Cells were incubated at 5% CO2 in a humidified incubator at 37uC. NTera2/d1 cells were seeded in a 96-well plate and transfected using IBAfect and MA- enhancer (IBA Biosciences, GmbH) using the suppliers protocol. Luciferase was measured on a Packard TOPCOUNT Microplate Scintillation Counter (Packard). The experiments were performed two times in triplicate. Values were normalised with TAL-luc [11]. nTera2/d1 cells were maintained in DMEM containing 10% FCS. The culture media were supplemented with 5 mM glutamine and antibiotics/antimycotics. Cells were incubated at 5% CO2 in a humidified incubator at 37uC. NTera2/d1 cells were seeded in a 96-well plate and transfected using IBAfect and MA- enhancer (IBA Biosciences, GmbH) using the suppliers protocol. Luciferase was measured on a Packard TOPCOUNT Microplate Scintillation Counter (Packard). The experiments were performed two times in triplicate. Values were normalised with TAL-luc [11]. We thank Dr. Paula van Tijn for helpful discussions. References 39. Peppelenbosch M, Boone E, Jones GE, van Deventer SJ, Haegeman G, et al. (1999) Multiple signal transduction pathways regulate TNF-induced actin reorganization in macrophages: inhibition of Cdc42-mediated filopodium formation by TNF. J Immunol 162: 837–45. 21. Katoh M, Katoh M (2007) Integrative genomic analyses on HES/HEY family: Notch-independent HES1, HES3 transcription in undifferentiated ES cells, and PLoS ONE \| www.plosone.org November 2010 \| Volume 5 \| Issue 11 \| e14023 8
https://openalex.org/W4393014612	https://www.frontiersin.org/articles/10.3389/fsoc.2024.1374488/pdf?isPublishedV2=False	English	null	Between nature and culture – Interpreting students’ sexuality in physical education	Frontiers in sociology	2,024	cc-by	10,604	COPYRIGHT COPYRIGHT © 2024 Böhlke, Zander and Rode. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Results: Our discourse analysis reveals that the multifaceted and often controversial online discussions are structured by two dominant schemes of interpreting students’ sexuality in PE, both of which are differentiated in complex ways: The online communication draws on – and by that reproduces – a nature and a culture perspective on constellations of body, sex, gender, and sexuality. We detail how from each perspective, different knowledge about these constellations, different everyday phenomena and problems in PE, and different norms for dealing with these phenomena and problems become important. – a nature and a culture perspective on constellations of body, sex, gender, and sexuality. We detail how from each perspective, different knowledge about these constellations, different everyday phenomena and problems in PE, and different norms for dealing with these phenomena and problems become important. Discussion: Discussing these results in the context of previous literature, we argue that it is important to address sexuality in a subject-specific approach and take the discursive knowledge and fundamental schemes of interpretation into account that shape the (im-)possibilities of addressing sexuality in PE. Sexuality, physical education, online research, discourse analysis, interpretive schemes Sexuality, physical education, online research, discourse analysis, interpretive schemes TYPE Original Research PUBLISHED 20 March 2024 DOI 10.3389/fsoc.2024.1374488 TYPE Original Research PUBLISHED 20 March 2024 DOI 10.3389/fsoc.2024.1374488 TYPE Original Research PUBLISHED 20 March 2024 DOI 10.3389/fsoc.2024.1374488 frontiersin.org OPEN ACCESS OPEN ACCESS EDITED BY Sónia Vladimira Correia, Lusofona University, Portugal REVIEWED BY Jonathan Glazzard, University of Hull, United Kingdom Chelsea Litchfield, Charles Sturt University, Australia CORRESPONDENCE Nicola Böhlke n.boehlke@tu-braunschweig.de RECEIVED 24 January 2024 ACCEPTED 04 March 2024 PUBLISHED 20 March 2024 CITATION Böhlke N, Zander B and Rode D (2024) Between nature and culture – Interpreting students’ sexuality in physical education. Front. Sociol. 9:1374488. doi: 10.3389/fsoc.2024.1374488 EDITED BY Sónia Vladimira Correia, Lusofona University, Portugal Nicola Böhlke 1, Benjamin Zander 2 and Daniel Rode 3 1 Institute of Sport Science and Movement Pedagogic, Technische Universität Braunschweig, Braunschweig, Germany, 2 Institute of Sport Science, University of Göttingen, Göttingen, Germany, 3 Department of Sport and Exercise Science, Paris Lodron University Salzburg, Salzburg, Austria Nicola Böhlke 1, Benjamin Zander 2 and Daniel Rode 3 1 Institute of Sport Science and Movement Pedagogic, Technische Universität Braunschweig, Nicola Böhlke 1, Benjamin Zander 2 and Daniel Rode 3 1 Institute of Sport Science and Movement Pedagogic, Technische Universität Braunschweig, Braunschweig, Germany, 2 Institute of Sport Science, University of Göttingen, Göttingen, Germany, 3 Department of Sport and Exercise Science, Paris Lodron University Salzburg, Salzburg, Austria Introduction: As sexuality in physical education (PE) is often treated as a taboo subject, social media platforms, online chats, and internet forums are emerging as spaces where it is negotiated more openly and broadly by current and former actors of the field. This paper contributes to a better understanding of the discursive construction of sexuality in PE in such online communication. Böhlke N, Zander B and Rode D (2024) Between nature and culture – Interpreting students’ sexuality in physical education. Front. Sociol. 9:1374488. doi: 10.3389/fsoc.2024.1374488 Methods: In line with The Sociology of Knowledge Approach to Discourse (SKAD) we investigate basic schemes of interpretation of sexuality with a heterogeneous sample of threads (17 threads from seven different online forums) on different PE situations in Germany. The threads are analyzed using grounded theory coding procedures. 1 Introduction Studies are especially concerned with analyzing the significance of (hetero-)sexuality in the construction of gender dichotomies and hierarchical gender relations in PE. They show that LGBTIQ students often face problems of being excluded, insulted, or attacked (e.g. Pérez-Samaniego et al., 2016; Müller and Böhlke, 2021) in PE, which is characterized by “narrow and defined norms of gender [and] sexuality and the body”1 (Piedra et al., 2016, p. 117). In this regard, LGBTIQ individuals or queer bodies in PE are often theorized as abject or oppressed (Pérez-Samaniego et al., 2016). Many queer educators and students still feel uncomfortable and limited in schools, despite progress in the acceptance of queerness in Western culture slowly making its way into schools (e.g., Ferfolja and Ullman, 2020). Particularly problematic practices in PE include dancing, changing rooms and restrooms as well as swimming (Landi, 2019a; Kosciw et al., 2020). Recent studies (Devís-Devís et al., 2018; Berg and Kokkonen, 2022) support the notion that traditional PE settings often support heteronormativity. Yet, they also show that PE can offer spaces to potentially challenge this and thereby provide positive experiences for queer individuals. matters of body and gender, have a notable impact on teachers’ and students’ experiences of PE. While this is often treated as a taboo subject, social media platforms, online chats, and internet forums are emerging as spaces where current and former actors of PE articulate and discuss a broad range of aspects and topics related to sexuality in PE more openly (Böhlke and Zander, 2022). Building on these insights, existing scholarship emphasizes the need of critically interrogating PE as a field for either reproducing or surfacing, challenging, and transforming the “truths,” norms, and power relations that are tied to students’ sexuality in this field. i In this paper, we argue that gaining a better understanding of how students’ sexuality is interpreted in PE-related discourses is crucial for this task. Following a discourse analytical perspective, we regard PE as a field that is permeated by various discourses. As practices that systematically produce the objects of which they speak (Foucault, 1983), these discourses provide specific knowledge and “truths” about students’ sexuality that help shape realities and experiences of PE. Students and teachers are, however, not passively at the mercy of such discourses. Rather, they (re-) produce, contribute to, and negotiate them actively – for instance in emerging internet spaces. 1 Introduction According to a sociology of knowledge approach to discourse, these negotiations are structured by fundamental schemes of interpretation (Keller, 2013, 2018). This means that the diverse discussions in internet forums draw on underlying patterns of how the sexuality of students can be contextualized, referred to, valued, and interpreted so that students, their bodies, and their behavior can become intelligible within PE. Beyond research on these aspects, there is a small number of studies that focuses on desire in PE. In her research on experiences of PE teachers, Sykes (2003) explores topics such as homoerotic desire between lesbian teachers and students. She describes the active suppression and concealment of desire by the individuals themselves, with the goal of fitting into the heteronormative system of PE. An example of this is pretending to be heterosexual, which is discussed by Landi (2019a,b) in relation to the central role of the body in physical education. Focusing on the experiences of queer male students, he emphasizes the active role of the field of PE in the production (not just suppression) of queer desire. PE materially produces and shapes such desires. Thus, there is a mutual influence between queer desire and practices within the structures of this field. This includes discussions about changing rooms as ambivalent homoerotic spaces that evoke both discomfort and erotic desire in queer students, as they involuntarily must deal with phenomena of physiological arousal such as erections (Landi, 2019a). Our paper draws on a qualitative discourse analysis of 17 threads from 7 different internet forums in which the sexuality of students in PE is discussed by users that, in their communication, identify as actors related to PE. Our purpose is to answer the following research question: Which schemes of interpretation structure the discussions of students’ sexuality in PE in these internet threads? Answering this question, our study provides a better understanding of the discursive production of the norms, subjectivities, and power relations that are tied to the phenomenon of students’ sexuality in PE. 2 Materials and methods Building on existing research, critical pedagogical scholarship discusses PE as a learning space in which students of all sexualities can be sensitized and empowered in a unique way. Fitzpatrick and McGlashan (2016) criticize the dominance of a “straight pedagogy,” where heterosexuality is assumed, and they call for a radical rethinking of PE teacher education. Other scholars also propose pedagogical implications aimed at disrupting the field, with the overarching goal of challenging narrow and restrictive norms of gender, sexuality, and the body by conveying critical perspectives on health and physical activity (Larsson et al., 2014; Landi, 2018). Aspects such as the role of the teacher and the inclusion of or targeted focus on LGBTIQ students are discussed as particularly relevant in this context. Frontiers in Sociology 1 All translations from German or other languages by the authors. 1 Introduction Physical education (PE) has long been discussed as a school subject that is inseparably linked to issues of the body such as health, performance, gender, or dis−/ability (Kirk et al., 2006). In this context, sexuality is a phenomenon that has often been neglected (Clarke, 2006). Yet, there is a growing body of scholarship that has been establishing sexuality in PE as a field of empirical research and pedagogical consideration (Landi, 2019a; Van der Steeg et al., 2021; Varea and Öhman, 2022), with current studies emphasizing specific focuses, such as queerness or sexuality education, but devoting less consideration to the phenomenon of sexuality in PE in general. The previous scholarship demonstrates that aspects of sexuality, including sexualized boundary violations, physical contact, queerness, desire, and their association with Frontiers in Sociology 01 frontiersin.org Böhlke et al. 10.3389/fsoc.2024.1374488 sexual integrity (van der Steeg et al., 2021). LGBTIQ research explores the experiences of LGBTIQ individuals (e.g., Landi, 2019b; Müller and Böhlke, 2021) or focuses on pedagogical practices and structures of PE (Sykes, 2011; Landi, 2019a). Studies are especially concerned with analyzing the significance of (hetero-)sexuality in the construction of gender dichotomies and hierarchical gender relations in PE. They show that LGBTIQ students often face problems of being excluded, insulted, or attacked (e.g. Pérez-Samaniego et al., 2016; Müller and Böhlke, 2021) in PE, which is characterized by “narrow and defined norms of gender [and] sexuality and the body”1 (Piedra et al., 2016, p. 117). In this regard, LGBTIQ individuals or queer bodies in PE are often theorized as abject or oppressed (Pérez-Samaniego et al., 2016). Many queer educators and students still feel uncomfortable and limited in schools, despite progress in the acceptance of queerness in Western culture slowly making its way into schools (e.g., Ferfolja and Ullman, 2020). Particularly problematic practices in PE include dancing, changing rooms and restrooms as well as swimming (Landi, 2019a; Kosciw et al., 2020). Recent studies (Devís-Devís et al., 2018; Berg and Kokkonen, 2022) support the notion that traditional PE settings often support heteronormativity. Yet, they also show that PE can offer spaces to potentially challenge this and thereby provide positive experiences for queer individuals. sexual integrity (van der Steeg et al., 2021). LGBTIQ research explores the experiences of LGBTIQ individuals (e.g., Landi, 2019b; Müller and Böhlke, 2021) or focuses on pedagogical practices and structures of PE (Sykes, 2011; Landi, 2019a). 2.1 State of research: sexuality in PE In PE research, sexuality presents a marginal research topic that has mostly been investigated in the context of research on bodies, gender, and heteronormativity in PE, and specifically in LGBTIQ research. In these lines of research, gender is emphasized as a relevant category of difference in PE (e.g., Penney, 2002) with studies showing that the actualization of gender takes place in the context of peer affiliations, body-related practices or physical staging practices (e.g., Gorely et al., 2003). Sexuality appears to be closely interwoven with gender constructions (e.g., Clarke, 2006) and can function as a means of exercising violence or as a means of power (Fitzpatrick and Enright, 2016), e.g., in the context of the teacher-student relationship (e.g., Böhlke and Zander, 2022). Existing research on sexuality focusses on specific aspects of sexuality in PE, providing important insights into these aspects while leaving much room for further exploration. Previous work deals with sexualized boundary violations (Gråstén and Kokkonen, 2022; Wagner and Knoke, 2022), interpersonal body contact (Varea and Öhman, 2022) and ethical concepts of positive In sum, existing scholarship provides important empirical insights into the social construction of sexuality in PE while being mainly concerned with heteronormativity and LGBTIQ issues, yielding approaches for disrupting heteronormative structures within critical frontiersin.org frontiersin.org 02 Böhlke et al. 10.3389/fsoc.2024.1374488 10.3389/fsoc.2024.1374488 analysis focuses on the interpretative schemes that structure online communication on students’ sexuality in PE. Following the sociology of knowledge approach to discourse according to Keller (2013; Keller, 2018), the concept of interpretive schemes (in German: Deutungsmuster) refers to “social/collective meaning and action- organizing schemata, which are combined in and circulated through discourses” (Keller, 2018, p. 32). Interpretative schemes describe how people refer to discourses by interpreting specific everyday situations, actions, or phenomena in particular ways: “This concept has a particular importance for the relation between discourses and our everyday practices and self-understanding” (Keller, 2018, p. 33). Our research interest is to reconstruct which general patterns of interpretation – i.e., interpretative schemes – underly and structure online discussions about students’ sexuality in PE. Based on Keller (2013), our analysis was guided by the following sub-questions: pedagogies. Studies about sexuality in general are lacking. 2.1 State of research: sexuality in PE In a previous study by two of the authors of this paper (Böhlke and Zander, 2022), emerging online spaces could be identified as arenas where current and former actors negotiate sexuality in PE more broadly, thus providing a promising research field to explore the phenomenon of sexuality in PE more generally. Focusing on students as central actors of PE and following a discourse theoretical perspective, our study is thus concerned with exploring the underlying patterns that structure the discursive construction of the phenomenon of students’ sexuality in online forums. Frontiers in Sociology 2.2 Theoretical background: sexuality as a broad field of research Sexuality can be defined as a bio-psycho-social field of research (Dekker, 2013). There is a multitude of scientific perspectives (e.g., medical, biological, psychoanalytical, evolutionary psychological, social science) that draw on divers conceptual approaches and theoretical traditions to investigate sexuality not only as the reproduction of living beings but also with regard to aspects such as expressions of desire, relationship practices or forms of staging gendered bodies (Löw, 2008). In sometimes controversial debates, various explanatory approaches face each other, with attempts being made to find an approach that unites the disciplinary perspectives (Dannecker, 2017). In an interdisciplinary dialog, it is argued that physicality and sociality should no longer be analytically separated in terms of their influence on sexuality but should be more consistently related to each other (Dekker, 2013). A discourse-analytical approach can reveal which interpretations, perspectives and positions regarding the phenomenon of sexuality are taken up in what manner in a specific discourse field or discourse space. • Fundamental idea of sexuality: Which reference topics and fundamental ideas of interpreting students’ sexuality in PE are articulated in internet forums? • Manifestations of students’ sexuality in PE: How are reference topics and fundamental ideas of interpreting students’ sexuality linked to (which?) everyday phenomena and problems in PE? • Dealing with students’ sexuality in PE: Which ways, values, and norms of dealing with everyday phenomena and problems of students’ sexuality in PE are articulated? • Social differentiation of students: Which social differentiations of students (e.g., in terms of gender or body) are made within articulations of ways, norms, and values of dealing with sexuality in PE? 2.2.1 The discursive construction of sexuality Rather than following essentialist notions, a discourse analytical perspective considers sexuality, sexual bodies, feelings, behaviors, and identities as being continuously constructed in socio-cultural practices and processes. In these socio-cultural practices and processes, discursively disseminated knowledge and offers of interpretation are drawn upon. Following Foucault (1983), sexuality can be understood as a phenomenon that emerges in discourses in a historically and socio-culturally specific form. This form makes it possible to “combine anatomical elements, biological functions, behaviors, sensations, and pleasures into an artificial unity and to allow this fictitious unity to function as a causal principle, as an omnipresent meaning and mystery to be decoded everywhere” (Foucault, 1983, pp. 148–149). Discourses thus produce ideas about multifaceted aspects such as anatomy, behavior, feelings or desires as something that belongs together. They relate these ideas to concepts of normality and morality (e.g., of certain sexual practices or orientations) as well as concepts of the functions of sexual desire (e.g., reproduction or relationship building). These ideas, concepts, and their relations are discursive knowledge constructions that function as generators and stabilizers of everyday actions and ways of interpreting oneself and others. Internet forums are online spaces for asynchronous (and mostly text-based) communication. Communication is initiated in a post about a certain topic and that post is then followed chronologically by responding posts, which often refer to and/or build on each other. This results in thematic sequences of communication, which are called threads. The users participating in this communication can choose their username and the personal information they want to convey, thus giving them the opportunity to also post anonymously. Because of that, internet forums can provide low-threshold spaces to connect with peers but also gain access to expert knowledge (Döring, 2013). Notably, expertise, just like other personal information and characteristics, is defined according to the information the users themselves share, which is rarely verified independently. Previous research has found that sexuality in PE is discussed openly and broadly in internet forums (Böhlke and Zander, 2022). From our discourse theoretical perspective, internet forums thus present a field of research that is suited for exploring the collectively shared schemes of interpretation that constitute the sexuality of students in PE as a discourse phenomenon. We consider internet forums to be a discourse space in which users through their posts engage in social processes of interpretative meaning-making that discursively construct students’ sexuality in PE according to such schemes of interpretation. frontiersin.org 3.2 Data collection excluded. Our sampling process led us to identify six topics that most online discussions about students’ sexuality in PE revolved around (Table 1): desiring the PE teacher; erotic peer relationships; erotically connoted behavior of the teacher; nudity in changing room and showers; skimpy clothing; visible arousal. We looked to include threats in which these topics were discussed from different perspectives, that is, from the point of view of students, teachers, and parents (according to the users’ self-presentation), ceasing data collection when the variation of these perspectives and the criteria mentioned before was saturated. We pasted the threads into Word documents, saved screenshots to capture the visual elements of the websites, and wrote memos about our initial observations. Our study draws on a sample of 17 threads from the period 2015– 2022 about students’ sexuality in PE from seven different German- speaking internet forums (Table 1). For generating this sample, we systematically considered relevant methodological literature (Holtz et al., 2012; Smedley and Coulson, 2021), carefully documenting and continuously reflecting our own strategies (e.g., with regard to relevant selection criteria of forums, threads and posts). Our data collection followed a phenomenon-oriented theoretical sampling strategy, which is appropriate for exploratory research aimed at developing a local theory about a specific phenomenon directly from the data. Theoretical sampling methods are originally situated in Grounded Theory Methodology (Glaser and Strauss, 1998, p. 53) but have since been adapted in other qualitative research approaches, including the sociology of knowledge approach to discourse (Keller, 2013) that our study draws on. Following this method, data collection was part of an ongoing iterative process in which the analysis of available data informed the collection of new data, the comparative analysis of which led to a richer and deeper understanding of the phenomenon. Specifically, our aim was to compile a sample whose threats and posts covered a maximum of different topics, aspects, and facets of the phenomenon of students’ sexuality in PE. Drawing on the existing research outlined above and particularly on a previous study on sexuality in internet forums (Böhlke and Zander, 2022), we searched various internet forums with search strings that combined PE-related keywords (e.g., physical education, sports, school, teacher, student) with keywords related to aspects of sexuality (e.g., touching, relationship, boobs, erection, aroused, horny). The keywords and search strings evolved during the iterative research process. 3.3 Data analysis Our analysis was concerned with reconstructing the collectively shared interpretative schemes that underly the online communication about central topics of students’ sexuality in PE. Following the sociology of knowledge approach to discourse analysis according to Keller (2013, 2018), we analyzed the data material using two procedures. First, we conducted open coding of all data material. Second, we conducted sequential analyses of passages that we identified as particularly rich and relevant. Both procedures were guided by the sub-questions for reconstructing interpretative schemes that we presented in the theory section above. 3.2 Data collection We included all threats/posts that dealt with aspects related to students’ sexuality in PE and that met our ethical considerations (see below). All other threats/posts that did not meet these criteria were 2.2.2 Interpretative schemes To investigate this relation between discourses and everyday ways of interpreting certain actions, phenomena, oneself, and others, our 03 frontiersin.org 10.3389/fsoc.2024.1374488 Böhlke et al. TABLE 1 Data corpus. TABLE 1 Data corpus. Topics Thread Contributions Period (year) Forum/ Website Desiring the PE teacher 1 In love with my (almost) former teacher 277 2022 Website A 2 In love with hot PE teacher 10 2016–2020 Website B 3 How to attract glances from my PE teacher? 12 2015–2016 Website B Erotic peer relationships 4 Touch BFF in locker room ok? 31 2020–2022 Website C 5 Getting boys hot in gym class? 6 2017 Website B 6 Gay when I look at classmates’ underpants? 10 2017 Website B Erotically connoted behavior of the teacher 7 Where is a PE teacher allowed to touch a female student? 14 2018 Website B 8 Sexual harassment of teacher? 16 2017–2019 Website B 9 My teacher is grabbing me! 12 2013–2016 Website B Nudity in changing room and showers 10 In locker room pants down 44 2015 Website D 11 Showering after PE and changing clothes 36 2010 Website E 12 Swimming lessons and supervision in the locker room 58 2022 Website F 13 Showering and changing with classmates 11 2018–2020 Website G Skimpy clothing 14 Short tight sports pants too cheap? 13 2016–2022 Website B 15 Tight leggings and a belly in gym class? 9 2017–2018 Website B Visible arousal 16 Boner in PE, what to do? 8 2020 Website B 17 How do you feel about seeing a boy in gym class with a stiffy? 23 2011–2019 Website B All thread titles were translated from German by the authors and modified for the purpose of anonymization. Frontiers in Sociology frontiersin.org 3.4 Ethics statement Based on recent discussions (Eysenbach and Till, 2001; Smithson, 2015; Schmidt-Lux and Wohlrab-Sahr, 2020) and in adherence to current guidelines (Franzke et al., 2020) on ethics in online research, 04 frontiersin.org Böhlke et al. 10.3389/fsoc.2024.1374488 dealing with the everyday phenomena and problems of this manifestation? How are students socially differentiated in this context? our study followed a dynamic and situational approach to research ethics. In line with our sampling strategy presented above, this approach emphasizes an openly and reflexively designed research process in which ethical assessments and decisions must continuously take place as researchers gradually develop a deeper understanding of the characteristics of their online research field. Our study exclusively relies on internet forums that require no registration and are publicly accessible, justifying their consideration as “public behavior” (Holtz et al., 2012). This behavior happened without our interference as a form of ‘natural’ communication. Continuously deliberating this research field and the potential data material in our research team, we concluded that users in the online threads we included understand the public nature of their communication. They have moderate to low expectations of privacy, and they control which information to disclose, for example, through their chosen usernames or in their posts. Following the model for supporting ethical decision in (online) fieldwork by Heibges et al. (2019), we determined that, given these privacy expectations and characteristics of our research field, obtaining individual informed consent was not necessary. This decision is in line with recent discussions and guidelines on online research (e.g., Eysenbach and Till, 2001; Smithson, 2015; Franzke et al., 2020) as well as with existing studies (e.g., Lauritsalo et al., 2012; Sciberras and Tanner, 2023) that stress that informed consent can be waived if online communication is deemed public, the identity of the users can be protected and the potential for harm can be ruled out. To ensure the non-identifiability of individual users, safeguard personal rights, and prevent harm, we anonymized the data as required (e.g., removal of demographic or potentially identifying user data; omission of forum names in this publication) and maintained this anonymity throughout the research process. Moreover, we do not focus on individual persons and their personal experiences, as our discourse analytic research perspective is centered on collectively shared interpretative frameworks. 4.1 Students’ sexuality in PE as a phenomenon of biological development In this discursive scheme of interpretation, sexuality is understood as a biologically determined developmental phenomenon. The posts that draw on this scheme tend to focus on bodily sensations and expressions of pleasure, desire, or arousal. They view these as biological facts, constructing students’ sexuality in terms of a human nature that must be understood and controlled. Frontiers in Sociology 4.1.1 Basic idea of sexuality Bodily sensations are the central reference theme of this interpretive scheme. They are interpreted as expressions of a sexuality that is located in the body. Viewed as a desire that manifests itself physically, these expressions are described in the posts as biologically regulated bodily processes. The body is thereby constructed as an anthropological fact imposed on all human beings, subject to age and gender. Underlying this notion is an assumption of a human being whose sexual acts serve a natural drive to procreate (“That man and woman are interested in each other was already the case with Adam and Eve!”). Further, this interpretive scheme contains the notion of puberty as a peak phase of sexual development in which the superiority of the body over the mind intensifies (“if something moves in the pants of boys in puberty, then it is simply natural”). Moreover, sexuality is not only interpreted as a phenomenon expressed through bodily sensations but also as exerting an influence on the body. This shows in posts that characterize puberty as a phase in which the development of sexuality leads students to examine their own bodies and the bodies of others anew. Additionally, posts that draw on this interpretive scheme refer to quasi-objective (technical) knowledge about human biology regarding the body and human sexual drive (“reason for spontaneous erections are hormonal changes”). 4 Results Our analysis shows that students’ sexuality in PE is an ambiguous discourse phenomenon that is constructed in internet forums in a multifaceted way and leads to controversial discussions. However, we were able to reconstruct that this multifaceted online communication is grounded in and structured by two dominant interpretative schemes: The sexuality of students in PE is mostly interpreted as a phenomenon of biological development and as cultural phenomenon that is part of students’ identity. Notably, these two interpretative schemes offer different views on students’ sexuality in PE. Their common position is that both interpretative schemes recognize (from their respective point of view) the existence and the relevance of students’ sexuality in the PE classroom. Further, both view students’ sexuality as something that is subject to restrictive norms according to which it should be controlled or disciplined. frontiersin.org 4.1.3 Dealing with students’ sexuality in PE Possibilities for dealing with this central problem are discussed controversially in the internet forums within a framework of partly contradictory values and norms. One dominant norm is the shifting of sexuality into the private sphere. Sexuality is declared to be an “intimate private matter” that is inappropriate in the public sphere of PE. This norm is expressed, for instance, in calls for refraining from sexualized acts (“In PE, it’s about doing sports and nothing else”) or for hiding signs of sexual arousal (“Just put on a tight pair of underpants under your boxer shorts, this will prevent your pants dancer from going upwards”). These calls for discretion transport the overarching goal of not appearing as a sexually active being to others. Failing to be discrete is depicted as a disruptive factor in the lessons and as an unacceptable act of (violent/powerful) transgression of the privacy of others. In accordance with this norm, persons that publicly exhibit sexuality in PE are called disgusting, perverted, or encroaching on others: “Disgusting, especially if he still thinks it’s cool to have a hard-on.” This norm of privacy and discretion is, however, not absolute. Rather, the discursive inventory of this interpretive scheme contains different relativizations of this norm and even counter-norms. For example, responding to the question if it is appropriate to look at the naked upper body of others in the locker room, a user states: “Of course, looking at it intensively and deliberately would be more conspicuous, but if it’s inconspicuous, it’s okay.” In the public sphere of the locker room, looking – as a form of acting on one’s sexual impulses – is declared acceptable if it is done secretly enough. A different example are repeated calls for mildness and relaxation: “He cannot help it [erection during swimming lessons] and it’s already embarrassing for him. I would not make a drama out of it.” Drawing on the notion of expressions of sexuality (here: an erection during swimming lessons) being natural biological reactions, students are (partly) relieved of the responsibly to control them, and others are advised to tolerate or ignore them. Departing from this notion as well, few posts also encourage students to handle their sexually acting bodies self-confidently: “Wear it [the erection] with pride! 4.1.4 Social differentiations of studentshf motivated “gawking.” Most importantly, these actions and phenomena are naturalized and normalized as an age-specific search movement in the context of finding sexual identity or developing sexual interest: “Looking at other people’s butt is normal at your age - a lot of people do that to compare themselves with others,” “As a student, I could not take my eyes off the girls either!.” Underlying this interpretation is the idea that bodies react to other bodies quasi-automatically through some sort of stimulus– response chains. This idea delegates sexuality and the primary responsibility for it to the realm of biology. At the same time, posts call on individuals to deal with this according to the social conventions and rules that apply in PE classes. Thus, the central problem that is negotiated within this interpretative scheme is the necessity and, at the same time, the limited possibilities to control one’s bodily sexual impulses and expressions. The social differentiations that are produced discursively within this interpretive scheme construct sexuality, sex, and gender as an inseparable constellation. When addressing the phenomena, problems, and norms of students’ sexuality just described, many posts differentiate between students according to their sex/gender. Thereby, they (re-)produce specific gendered power relations. For example, this interpretative scheme contains the idea that pubescent boys are more strongly bound to sexual drives compared to girls. They are pictured as “testosterone-controlled” boys whose sexual receptivity poses a problem for PE classes. According to this interpretation, phenomena such as being aroused by others to the point where you cause distractions in the lesson are viewed as being a male problem: “Boys are not easy to handle in puberty in the presence of girls (...) especially in PE, when girls dress rather skimpily.” Girls, on the other hand, are predominantly positioned as causes of boys’ arousal and distraction, and they are at times addressed as being responsible for limiting these effects, e.g., through their clothing choices. Additionally, female students are positioned as victims vis-à-vis “libidinously” acting boys/ men. A different example of social differentiations along the category gender, which are part of the inventory of this interpretative scheme, is that female students are attributed certain traits, such as being naturally oversensitive. They are assumed to prematurely interpret certain physical actions by classmates or teachers as sexual, particularly at a certain age: “Are you in middle school? 4.1.3 Dealing with students’ sexuality in PE The girls may giggle, but of course they like to see something like that.” Within the interpretative scheme, counter norms like this, which distance themselves from the norm of privacy and discretion, at the same time stabilize the dominant interpretation of sexual acts by students being considered failures of self-control and therefore embarrassing, cringeworthy, and out of place in PE classes. 4.1.4 Social differentiations of studentshf That’s where female students see this kind of thing particularly often, sometimes unjustly.” As this quote illustrates, this can result in accusatory posts that downplay actions that were perceived as inappropriate. Other posts also open avenues for female students to deconstruct or invert these gendered power relations. They discuss possibilities for girls to use male sexual receptivity for their own purposes, for instance, by influencing a teacher’s grading through a revealing appearance: “What can I wear to get more attention from my PE teacher?” While opening different subject positions for female students, these posts still adhere to the interpretative scheme of viewing sexuality as a natural- biological phenomenon whose bodily impulses can be expressed and acted upon differently by males and females. 4.2 Sexuality as a cultural phenomenon that is part of students’ identity The second interpretative scheme offers a fundamentally different discursive inventory. In this scheme, sexuality is understood as a cultural, socio-historically variable phenomenon that is part of student’s identity. As such, sexuality is not conceived as stable and singular but rather as a spectrum of sexualities that people choose from and actively shape. 4.1.2 Manifestations of students’ sexuality in PEhi 4.1.2 Manifestations of students sexuality in PE The specific actions and phenomena that are interpreted as manifestations of students’ sexuality in everyday PE classes include flirting, covert but also undisguised sexualized actions such as glances or touching as well as bodily signs of sexual arousal such as a “boner” becoming visible in shorts. In posts that draw on this interpretative scheme, these actions and phenomena are naturalized, e.g., by being declared to be expressions of a natural sexual drive that students are almost powerless against. PE is constructed as a field in which these expressions are provoked but also must be controlled. Some posts, for example, describe that students at a particular age show a pronounced interest in the bodies of other students or teachers, or more specifically in their intimate body parts such as breasts, buttocks, or genitals. The posts describe that this becomes virulent or is even stimulated in characteristic situations of PE classes, such as situations where individuals present movements in front of others or in changing and showering situations. Some posts describe students engaging in body comparisons in these situations while others problematize the phenomenon of sexually These results are now present in detail. We present them in an aggregated form that includes illustrative quotes from the data material. Our presentation starts out with a summary of the interpretative inventory of each scheme, which is then elaborated along our research sub-questions: What fundamental idea of sexuality is conveyed? How does the phenomenon of students’ sexuality manifest itself in PE classes? What are the ways, norms, and values of 05 frontiersin.org Böhlke et al. 10.3389/fsoc.2024.1374488 Frontiers in Sociology frontiersin.org 4.2.2 Manifestations of students’ sexuality in PE 4.2.2 Manifestations of students sexuality in PE Practices of bodily self-staging, desire in an overarching sense (e.g., sexually motivated behaviors in movement situations, glances at intimate body parts), and relationships in PE (e.g., student-teacher) are manifestations of students’ sexuality in PE that are discussed within this interpretative scheme. For instance, PE is associated with a particular type of clothing (lightweight, freedom of movement) that is discussed with regard to issues of bodily exposure. Several posts discuss the possibilities of students, especially female ones, deliberately staging themselves physically in front of others to be perceived as attractive or “sexy,” for instance by wearing hot pants or crop tops. The discussions revolve around the legitimacy of this self-staging. In some posts it is encouraged and viewed as a modern self-confident approach to one’s body and sexuality: “Do it! Quietly play with the charms and enjoy the looks,” “Come on, we live in the 20th century! It should be okay to wear belly-free.” In other posts, physically revealing self- portrayal is negotiated as an everyday beauty code with clothing being interpreted as a sign of one’s own youth cultural positioning: “Stay cool and wear what you like, as long as the teachers do not say anything.” Again, other posts feature derogating comments about females engaging in this self-staging, especially if they explicitly express the intention of attracting others’ attention: “Do you want to look like a bitch?.” However, such defamation is also called out as “slut shaming.” The central problem that is discussed is how to deal with the diversity of sexuality-related options that students face in different situations. Some posts cover fundamental questions, e.g., about sexual orientation, while others touch on topics such as belonging to a certain social group, for instance the “mature” that engage in sexual relationships vs. the “young ones” that are not interested in sexuality. These phenomena and issues are not discussed from an “anything goes” perspective but rather against the backdrop of specific notions of legitimacy and particular moral concepts that have a strong normative impact. y Practices of bodily self-staging, desire in an overarching sense (e.g., sexually motivated behaviors in movement situations, glances at intimate body parts), and relationships in PE (e.g., student-teacher) are manifestations of students’ sexuality in PE that are discussed within this interpretative scheme. 4.2.2 Manifestations of students’ sexuality in PE For instance, PE is associated with a particular type of clothing (lightweight, freedom of movement) that is discussed with regard to issues of bodily exposure. Several posts discuss the possibilities of students, especially female ones, deliberately staging themselves physically in front of others to be perceived as attractive or “sexy,” for instance by wearing hot pants or crop tops. The discussions revolve around the legitimacy of this self-staging. In some posts it is encouraged and viewed as a modern self-confident approach to one’s body and sexuality: “Do it! Quietly play with the charms and enjoy the looks,” “Come on, we live in the 20th century! It should be okay to wear belly-free.” In other posts, physically revealing self- portrayal is negotiated as an everyday beauty code with clothing being interpreted as a sign of one’s own youth cultural positioning: “Stay cool and wear what you like, as long as the teachers do not say anything.” Again, other posts feature derogating comments about females engaging in this self-staging, especially if they explicitly express the intention of attracting others’ attention: “Do you want to look like a bitch?.” However, such defamation is also called out as “slut shaming.” 4.2.4 Social differentiations of studentsh The manifestations and norms of students’ sexuality in PE that shape the discursive inventory of this interpretative scheme lead to different social differentiations. One is the difference between hetero- and homosexuality. Several posts specifically deal with issues of homosexuality in PE, for instance an interest in the bodies of same-sex classmates. These issues are often normalized or even prioritized with reference to developments toward openness and sexual diversity in society: “Being gay is completely normal today.” Secondly, as already mentioned above, actors are differentiated according to their institutional roles in PE. Choosing, exploring, and expressing one’s sexual identity is discussed differently for students and teachers. For teachers, the norm of keeping sexuality out of PE is made uncompromisingly binding, with posts referring to role-specific requirements such as the duty of care and ethical considerations, for instance, regarding age differences. Thirdly, gender related differentiations come into play again. These touch on issues also mentioned in the presentation of the first interpretative scheme above, such as a presumed heightened sensitivity of female students. Here, this sensitivity is interpreted to be a social rather than a natural- developmental phenomenon, for instance by being attributed to social movements in current society: “One wrong saying and you are already a pervert as a man today. Just because this stupid me-too movement is trendy these days.” In these discussions, supposedly over-sensitive female students are, on the one hand, called upon to question their assessments or to refrain from rash actions such as publicly accusing a teacher. On the other hand, the discussions, among others, also feature constant appeals to girls to defend themselves, communicate their discomfort to others, and empower each other in the sense of female self-emancipation (“Defend yourself! Your teacher has no right to grab you at any time”). The central problem that is discussed is how to deal with the diversity of sexuality-related options that students face in different situations. Some posts cover fundamental questions, e.g., about sexual orientation, while others touch on topics such as belonging to a certain social group, for instance the “mature” that engage in sexual relationships vs. the “young ones” that are not interested in sexuality. These phenomena and issues are not discussed from an “anything goes” perspective but rather against the backdrop of specific notions of legitimacy and particular moral concepts that have a strong normative impact. 4.2.1 Basic idea of sexuality Erotic preferences, sexual interests, and subjective forms of desire are the central reference themes within this interpretative scheme. They are discussed with regard to individual intentions, attitudes, and ideas that inform sexually motivated practices but also with regard to social conditions. Posts that draw on this scheme of interpretation associate sexuality with aspects of self-determination or freedom of choice within a given diversity of options, for instance regarding forms of desire, relationship constellations, and sexual practices: “I’m gay 06 frontiersin.org Böhlke et al. 10.3389/fsoc.2024.1374488 consistently dismissed in such posts: “Teacher with student does not work. End.” According to this norm, teachers’ actions toward students that are perceived as sexual, such as physical touching of intimate body parts during assistance, are unacceptable: “Report immediately! Pedos do not belong in school!” In other posts, this norm of self-regulation is expressed with regard to students’ clothing choices: “If I were you, I’d rather wear something discreet in PE!” The morality and character of individuals who do not adhere to the notion of school and PE as sexuality-free spaces are judged accordingly: “I think girls who come to PE with a deep neckline are just bxxx.” This norm may seem similar to the calls for decency and self-control mentioned in the context of the first interpretative scheme above. However, the posts mentioned above draw on the notion of sexuality as natural and biological impulses that should be controlled. In contrast, when analyzed in their discursive context, the posts mentioned here draw on a notion of sexuality as the free choice and self-determined expression of sexual identity, which should not be practiced in school or during PE. and I find my classmates’ upper bodies interesting, what difference does it make? 😊.” Additionally, such posts draw on publicly disseminated knowledge about (youth) trends, dress codes (“Belly- free is the trend today after all!),” body and gender politics as well as discourses about the current state of society. The central idea is that people, specifically students, actively (co-)construct, shape, and also control their sexuality as part of a self-determinant and self- responsible lifestyle in modern society. Frontiers in Sociology 5 Discussion Our main finding is that discursive constructions of students’ sexuality in PE predominantly draw on – and by that reproduce – a ‘nature’ and a ‘culture’ perspective on constellations of body, sex, gender, and sexuality. That is, the interpretative schemes we have identified reflect the central perspectives of biological-natural scientific as well as cultural and social scientific approaches (e.g., Wrede, 2000; Dannecker, 2017) that traditionally structure the sexual science discourse. While these perspectives typically exist independently within different disciplinary communities and are often placed in opposition to one another (e.g., Benkel and Lewandowski, 2021), our study demonstrates that in everyday online discourse, individual interpretations and arguments from each perspective are taken up and related to each other in complex ways. Within the online threats, each perspective features specific knowledge about human nature, the body, adolescence, or PE under current social conditions. From each perspective, different everyday phenomena and problems in PE and different norms for dealing with these phenomena and problems become important. Similar phenomena, such as the presumed heightened sensitivity of female students, and norms, such as a call for self-regulation, are interpreted and explained differently. Many posts draw on a ‘nature’ or ‘culture’ perspective with absolute claims to truth, devaluing or delegitimizing post and arguments from the other perspective. At the same time, other posts do not adhere to this binary logic. Given the multifaceted and complex nature of the online discussions about students’ sexuality in PE, our findings thus provide a better understanding of the overall discursive context and the basic interpretative inventory in which individual posts or articulations in internet forums are situated, and they reveal these online discussions as a site of cultural wrestles over the dominance of certain forms of knowledge and subjectivity between ‘nature” and “culture” two schemes of interpretating sexuality. The limitations of our study pertain to its focus on the German- speaking context, on students’ sexuality, on internet forums, and on current PE-related communication. Since the social construction of sexuality is always situated in a specific cultural and societal context, in which it is specifically positioned (Fitzpatrick and McGlashan, 2016), future research should include online forums from other national school systems and cultural contexts. It should take the phenomenon of teachers’ sexuality more prominently into account. 5 Discussion of female heightened sensitivity as a current social phenomenon. However, especially the ‘culture’ scheme of interpretation features knowledge and “truths”, e.g., about choosing and expressing sexual preferences autonomously, that also offer non-binary and non-heteronormative perspectives. From a discourse analytical perspective, sexuality is a multifaceted phenomenon that is shaped, de- and reconstructed in various discourses of modern societies. In this paper, we employed this perspective to conceive of PE as a field in which such discourses intersect to help inform the practices, experiences, and realities of this field. We identified internet forums as spaces in which active negotiation and (re-)production of discourses on sexuality in PE happen intensely and broadly. Focusing on the sexuality of students in PE, our study was interested in exploring which dominant schemes of interpretation underly and structure the negotiations of this phenomenon in German-speaking internet forums. Another aspect discussed in existing research is the elimination of sexuality from PE (Sykes, 2003; Landi, 2019a). Sykes describes sexual desire between students and teachers as a “trope of silence” (Sykes, 2001, p. 14) and states that, in the heteronormative context of PE, teachers can only succeed if they adhere to the normative expectation of suppressing or concealing desire. Our study confirms these results regarding online communication about students’ sexuality in PE, and it expands on them by revealing their constitutive discursive context und underlying interpretative schemes. From their respective perspectives, both interpretative schemes work on eliminating sexuality from PE. While they explain and locate sexuality differently (nature vs. culture, bodily impulses vs. personal choice), they both take the individuals to be responsible for keeping sexuality out of PE. For this, sexuality is perceived as a private matter in the naturalistic perspective, while in a sociocultural perspective, it is tabooed within the context of institutional roles and relationships. Additionally, we were able to surface that these discursive schemes also offer possibilities to counter or relativize this norm of silence. For example, in the “nature” scheme, it is assumed that students have limited ability to control their pubescent bodies, while in the “culture” scheme, sexualized self-staging through wearing revealing clothes is legitimated with common clothing styles in (western) modern societies.h Our study reconstructed two dominant schemes of interpreting students’ sexuality in PE, both of which are differentiated in complex ways. 4.2.3 Dealing with students’ sexuality in PE In this interpretative scheme, the dominant norm for dealing with students’ sexuality in PE refers to a notion of school as an asexual place. Actors in school are reduced to their roles as teachers and students, with the expectation that they should subordinate their personal preferences and qualities to these roles. This specifically includes the expectation of self-regulating one’s sexuality, as any kind of sexuality – except for sex education work – is seen as not belonging in school. This regulative norm is articulated, for instance, through posts that interpret sexually motivated acts in PE as inappropriate: “He [student who lets his pants down in PE class] has to behave appropriately in public and refrain from doing that.” Other posts assert normative truths about sexual relationships, citing the institutional roles of the individuals involved. As a result, the possibility of discussing such matters in online forums is These examples show how controversial discussions and very different positions in internet forums about sexual orientations, roles, and gender relations in PE draw on a common, underlying notion of sexuality as an elementary and omnipresent feature of adolescent life. Within this interpretative scheme, skillful handling of this feature ensures social acceptance among peers by creating group affiliations and showing boundaries, e.g., by illustrating a modern way of thinking. 07 frontiersin.org 10.3389/fsoc.2024.1374488 Böhlke et al. frontiersin.org Frontiers in Sociology 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. References heteronormative secondary physical education contexts. Phys. Educ. Sport Pedagog. 23, 103–116. doi: 10.1080/17408989.2017.1341477 heteronormative secondary physical education contexts. Phys. Educ. Sport Pedagog. 23, 103–116. doi: 10.1080/17408989.2017.1341477 Böhlke, N., and Zander, B. (2022). Sexualität von schüler*innen im sportunterricht. Ergebnisse einer diskursanalyse in onlineforen [Students` sexuality in physical education. Results of a discourse analysis in online forums]. Ger. J. Exerc. Sport Res. 52, 529–538. doi: 10.1007/s12662-021-00775-x Döring, N. (2013). “Medien und Sexualität” [media and sexuality] in Enzyklopädie Erziehungswissenschaft online EEO/Fachgebiet Medienpädagogik/Aktuelle Diskurse [encyclopedia of educational science online EEO/subject area media education/current discourses], eds D. Meister, GrossF. Von and U. Sander (Weinheim: Beltz). Döring, N. (2013). “Medien und Sexualität” [media and sexuality] in Enzyklopädie Erziehungswissenschaft online EEO/Fachgebiet Medienpädagogik/Aktuelle Diskurse [encyclopedia of educational science online EEO/subject area media education/current discourses], eds D. Meister, GrossF. Von and U. Sander (Weinheim: Beltz). Böhlke, N., Zander, B., and Braun, E. M. (2022). Sexualität und sexualisierte Körper im Sportunterricht. Ein Thema für jede Sportlehrkraft! [Sexuality and sexualised bodies in PE lessons. A topic for every PE teacher!]. doi: 10.13140/ RG.2.2.24787.04644 Eysenbach, G., and Till, J. E. (2001). Ethical issues in qualitative research on internet communities. Br. Med. J. 323, 1103–1105. doi: 10.1136/bmj.323.7321.1103 Benkel, T., and Lewandowski, S. (2021). Kampfplatz Sexualität - Normalisierung – Widerstand [sexuality as a battleground - normalisation – resistance]. Bielefeld: transcript. Benkel, T., and Lewandowski, S. (2021). Kampfplatz Sexualität - Normalisierung – Widerstand [sexuality as a battleground - normalisation – resistance]. Bielefeld: transcript. Ferfolja, T., and Ullman, J. (2020). Gender and sexuality diversity in a culture of limitation: Student and teacher experiences in schools. Abingdon: Routledge Fitzpatrick, K., and Enright, E. (2016). “Gender sexuality and physical education” in Routledge handbook of physical education pedagogies. ed. C. D. Ennis (London: Taylor & Francis), 319–331. Berg, P., and Kokkonen, M. (2022). Heteronormativity meets queering in physical education: the views of PE teachers and LGBTIQ+ students. Phys. Educ. Sport Pedagog. 27, 368–381. doi: 10.1080/17408989.2021.1891213 Berg, P., and Kokkonen, M. (2022). Heteronormativity meets queering in physical education: the views of PE teachers and LGBTIQ+ students. Phys. Educ. Sport Pedagog. 27, 368–381. doi: 10.1080/17408989.2021.1891213 Fitzpatrick, K., and McGlashan, H. (2016). ““Rethinking “straight pedagogy”: Gender, sexuality and physical education”” in Social justice in physical education: Critical reflections and pedagogies for change. eds. D. B. Robinson and L. Randall (Toronto: Canadian Scholar Press Inc), 102–121. Clarke, G. (2006). “Sexuality and physical education” in Handbook of physical education. eds. D. M. Data availability statement The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. 5 Discussion It should investigate other discourse spaces, both online and offline, with the aim of mapping what is and can be said about sexuality in PE by whom and in which spaces. It should trace in more detail which larger discourses are referenced or cited. One possibility would be following up with a survey or face to face interviews. Additionally, further research should also take a historical perspective to reconstruct sexuality in PE as a socio-historical phenomenon. Our discourse analytical focus on interpretative schemes and the two interpretative schemes that we were able to reconstruct in this study may serve as important reference points for such future research. Focusing on specific phenomena of sexuality in PE, such as homoerotic desire, being in love with the teacher, or clothing styles in PE, future research could investigate how “nature” and “culture” interpretations are actually invoked and (inter-)related in discourses on these phenomena. Previous research shows that social constructions of sexuality in sport continue to reinforce binary and heteronormative discourses (Sykes, 2003; Fitzpatrick and McGlashan, 2016; Landi, 2019a,b), with heteronormative ideas being defined in particular by the naturalization of heterosexuality and dichotomous gender. Our study confirms this to some degree regarding the social constructions of students’ sexuality in PE in internet forums. Both reconstructed interpretative schemes provide knowledge about manifestations of “male” and “female” sexuality of students in PE that supports heteronormative views. The “nature” scheme of interpretation, for instance, draws on a quasi-objective knowledge about the human body and its biology, e.g., regarding male students’ sexual drive. The “culture” scheme of interpretation draws on knowledge, e.g., about the social production For pedagogical practice, our study suggests that it is important to address sexuality in a subject-specific approach and take the discursive knowledge and fundamental patterns of interpretation into account that shape the (im-)possibilities of addressing sexuality in PE. As a school subject, PE features several characteristics that distinguish it from other subjects, chief among them its pronounced focus on the body (Landi, 2019a; Berg and Kokkonen, 2022). This makes PE a very specific field for students and teachers to make sense of various facets of sexuality. Our study highlights that this sensemaking includes navigating different and partly conflicting knowledge, interpretations, and norms that 08 Böhlke et al. 10.3389/fsoc.2024.1374488 Funding 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. Author contributions converge in two basic interpretative schemes. Following Van der Steeg et al. (2021), we find it important to create ethical concepts of positive sexual integrity that not only protects students from harm, but also proactively contributes to healthy sexual development. We support existing sport pedagogical approaches that focus on questioning binary and limiting norms regarding gender, sexuality, and body to promote sexuality/gender/body-related diversity (e.g., Larsson et al., 2014), for example in the context of transformative pedagogies of physical education (Fitzpatrick and Enright, 2016). NB: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing. BZ: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing. DR: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing. Particularly, our study supports approaches that try to make PE a diversity-sensitive environment (Ruin and Stibbe, 2023) in which non-binary, non-heteronormative, inclusive interpretations of sexuality can be explored (e.g., Landi, 2018). This includes providing immediate support services for students and teachers who are vulnerable to (allegations of) sexual harassment, abuse, or exclusion, such as anonymous complaint channels or information and counseling services. In addition, long-term prevention measures are necessary, such as mandated workshops by external and explicitly trained sexual educators that could be incorporated into the curriculum or no-go and best-practice examples that are integrated into teacher training to enhance the awareness of current and future physical educators (e.g., Böhlke et al., 2022). Yet, pedagogical approaches should also go beyond a focus on negative phenomena such as harassment or exclusion and conceptualize PE as a space particularly suited for discussing topics such as desire, physicality, closeness, or intimacy and questions related to clothing, relationship building, or group belonging with students. This should also include reflecting on the discursive “truths” and patterns of interpretation that are reproduced or challenged in such discussions. After all, talking about sexuality is a powerful practice that shapes realities of PE. Frontiers in Sociology References doi: 10.18420/muc2019-ws-258-02 Ruin, S., and Stibbe, S. (2023). Sportdidaktik und Schulsport – Zentrale Themen einer diversitätssensiblen Fachdidaktik [sport didactics and school sports. Central topics of diversity-sensitive specialised didactics]. Schorndorf: Hofmann Schmidt-Lux, T., and Wohlrab-Sahr, M. (2020). Qualitative online-Forschung. Methodische und methodologische Herausforderungen [qualitative online research. Methodological and methodological challenges]. Zeitschrift für qualitative Forschung 21, 3–11. doi: 10.3224/zqf.v21i1.01 Holtz, P., Kronberger, N., and Wagner, W. (2012). Analyzing internet forums: a practical guide. J. Media Psychol. 24, 55–66. doi: 10.1027/1864-1105/a000062 Keller, R. (2013). Doing discourse research: an introduction for social scientists. Thousand Oaks, CA: Sage. Sciberras, R., and Tanner, C. (2023). ‘Sex is so much more than penis in vagina’: sex education, pleasure and ethical erotics on Instagram. Sex Educ., 1–14. doi: 10.1080/14681811.2023.2199976 Keller, R. (2018). ““The sociology of knowledge approach to discourse. An introduction” in the sociology of knowledge approach to discourse” in Investigating the politics of knowledge and meaning-making. eds. R. Keller, A. K. Hornidge and W. J. Schünemann (Abingdon: Routledge), 16–47. Smedley, R. M., and Coulson, N. S. (2021). A practical guide to analysing online support forums. Qual. Res. Psychol. 18, 76–103. doi: 10.1080/14780887.2018.1475532 Kirk, D., Macdonald, D., and O’Sullivan, M. (2006). The handbook of physical education. London: Sage. Smithson, J. (2015). “Using discourse analysis to study online forums for young people who self-harm” in The Palgrave handbook of child mental health. eds. M. O’Reilly and J. N. Lester (London: Palgrave Macmillan) Kosciw, J. G., Clark, C. M., Truong, N. L., and Zongrone, A. D. (2020). The 2019 National School Climate Survey. The experiences of lesbian, gay, bisexual, transgender, and queer youth in our nation's schools. New York: GLSEN. Sykes, H. (2001). Understanding and overstanding: Feministpoststructural life histories of physical education teachers. Int. J. Qual. Stud. Educ. 14, 13–31. doi: 10.1080/0269172001009744 Landi, D. (2018). Toward a queer inclusive physical education. Phys. Educ. Sport Pedagog. 23, 1–15. doi: 10.1080/17408989.2017.1341478 Sykes, H. (2003). The angel’s playground: same-sex desires of physical education teachers. J. Gay Lesbian Issues Educ. 1, 3–31. doi: 10.1300/J367v01n01_02 Landi, D. (2019a). LGBTQ youth in physical education and sexuality education: Affect, curriculum, and (new) materialism. [dissertation thesis]. [Auckland]: University of Auckland. Sykes, H. (2011). Queer bodies: Sexualities, genders, and fatness in physical education. New York: Peter Lang. Landi, D. (2019b). Queer men, affect, and physical education. Qual. Res. Sport, Exerc. Health 11, 168–187. References Kirk and M. O’Sullivan (London: Sage) Clarke, G. (2006). “Sexuality and physical education” in Handbook of physical education. eds. D. M. Kirk and M. O’Sullivan (London: Sage) Dannecker, M. (2017). Faszinosum Sexualität: Theoretische, empirische und sexualpolitische Beiträge [the fascination of sexuality: Theoretical, empirical and sexual- political contributions]. Gießen: Psychosozial-Verlag. Dannecker, M. (2017). Faszinosum Sexualität: Theoretische, empirische und sexualpolitische Beiträge [the fascination of sexuality: Theoretical, empirical and sexual- political contributions]. Gießen: Psychosozial-Verlag. Foucault, M. (1983). Sexualität und Wahrheit 1: Der Wille zum Wissen [sexuality and truth 1: The will to know]. Frankfurt am Main: Suhrkamp. Dekker, A. (2013). Was heißt: Sexualität ist bio-psycho-sozial? [what means: sexuality is bio-psycho-social?]. Zeitschrift für Sexualforschung [J. Sex. Res.] 26, 34–43. doi: 10.1055/s-0033-1335069 Dekker, A. (2013). Was heißt: Sexualität ist bio-psycho-sozial? [what means: sexuality is bio-psycho-social?]. Zeitschrift für Sexualforschung [J. Sex. Res.] 26, 34–43. doi: 10.1055/s-0033-1335069 Franzke, A.S., Bechmann, A., Zimmer, M., and Ess, C., and the Association of Internet Researchers (2020). Internet Research: Ethical Guidelines 3.0. Available at: https://aoir. org/reports/ethics3.pdf Devís-Devís, J., Pereira-García, S., López-Cañada, E., Pérez-Samaniego, V., and Fuentes-Miguel, J. (2018). Looking back into trans persons’ experiences in Glaser, B. G., and Strauss, A. L. (1998). Grounded theory: Strategien qualitativer Forschung [grounded theory: Strategies of qualitative research]. Bern: Hans Huber. Devís-Devís, J., Pereira-García, S., López-Cañada, E., Pérez-Samaniego, V., and Fuentes-Miguel, J. (2018). Looking back into trans persons’ experiences in 09 frontiersin.org Böhlke et al. Böhlke et al. 10.3389/fsoc.2024.1374488 10.3389/fsoc.2024.1374488 Gorely, T., Holroyd, R., and Kirk, D. (2003). Muscularity, the habitus and the social construction of gender: toward a gender-relevant physical education. Br. J. Educ. 24, 428–448. doi: 10.1080/01425690301923 Pérez-Samaniego, V., Fuentes-Miguel, J., Pereira-García, S., and Devís-Devís, J. (2016). Abjection and alterity in the imagining of transgender in physical education and sport: a pedagogical approach in higher education. Sport Educ. Soc. 21, 985–1002. doi: 10.1080/13573322.2014.981253 Gråstén, A., and Kokkonen, M. (2022). Associations between teacher- and student- directed sexual and physical violence in physical education. J. Interpers. Violence 37, NP4372–NP4392. doi: 10.1177/0886260520959640 Piedra, J., Ramírez-Macías, G., Ries, F., Rembrandt Rodríguez-Sánchez, A., and Phipps, C. (2016). Homophobia and heterosexism: Spanish physical education teachers’ perspectives. Sport Soc. 19, 1156–1170. doi: 10.1080/17430437.2015.1096257 Heibges, M., Mörike, F., and Feufel, M. A. (2019). Wann braucht Ethnografie eine Einverständniserklärung? Praktische Antworten auf ethische Fragen zu ethnografischen Methoden in der HCI-Forschung [when does ethnography need informed consent? Practical answers to ethical questions about ethnographic methods in HCI research]. Bonn: Gesellschaft für Informatik e.V. References doi: 10.1080/2159676X.2018.1504230 van der Steeg, E., Schipper-van Veldhoven, N., Cense, M., Bellemans, T., and de Martelaer, K. (2021). A Dutch perspective on sexual integrity in sport contexts: definition and meaning for practice. Phys. Educ. Sport Pedagog. 26, 460–482. doi: 10.1080/17408989.2020.1823955 Larsson, H., Quennerstedt, M., and Öhman, M. (2014). Heterotopias in physical education: towards a queer pedagogy? Gend. Educ. 26, 135–150. doi: 10.1080/09540253.2014.888403 Lauritsalo, K., Sääkslahti, A., and Rasku-Puttonen, H. (2012). Student’s voice online: experiences of PE in Finnish schools. Adv. Phys. Educ. 2, 126–131. doi: 10.4236/ ape.2012.23022 Varea, V., and Öhman, M. (2022). “Break the rules or quit the job”: physical education teachers’ experiences of physical contact in their teaching practice. Sport Educ. Soc. 28, 395–406. doi: 10.1080/13573322.2022.2036119 Löw, M. (2008). “Sexualität” in [Sexuality] in Handbuch Soziologie [handbook of sociology]. eds. N. Baur, H. Körte, M. Löw and M. Schroer (Wiesbaden: Springer) Wagner, I., and Knoke, C. (2022). Sexualisierte Grenzverletzungen durch Lehrkräfte im Sportunterricht. [sexualized boundary violations by teachers in physical education]. Ger. J. Exerc. Sport Res. 52, 539–549. doi: 10.1007/s12662-022-00806-1 Müller, J., and Böhlke, N. (2021). Physical education from LGBTQ+ students’ perspective. A systematic review of qualitative studies. Phys. Educ. Sport Pedagog. 28, 601–616. doi: 10.1080/17408989.2021.2014434 Wrede, B. (2000). “Was ist Sexualität? Sexualität als Natur, als Kultur und als Diskursprodukt [what is sexuality? Sexuality as nature, as culture and as a product of discourse]” in Sexuelle Szenen [Sexual scenes]. eds. C. Schmerl, S. Soine and M. Stein- Hilbers (Opladen: Budrich) Penney, D. (2002). Gender and physical education: Contemporary issues and future directions. Abingdon: Routledge. 10 10 Frontiers in Sociology frontiersin.org
https://openalex.org/W4386283560	https://www.nature.com/articles/s41598-023-41429-2.pdf	English	null	Restoration of soils contaminated with PAHs by the mixture of zeolite composites mixed with exogenous organic matter and mineral salts	Scientific reports	2,023	cc-by	10,144	www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Restoration of soils contaminated with PAHs by the mixture of zeolite composites mixed with exogenous organic matter and mineral salts OPEN Justyna Szerement 1, Adam Kowalski 2, Jakub Mokrzycki 3, Lidia Marcińska‑Mazur 4 & Monika Mierzwa‑Hersztek 4,5 The major cause of soil degradation (contamination, erosion, compaction) is closely linked to agriculture, i.e., unsustainable agriculture practices, which are reflected in the depletion of the soil organic carbon pool, loss in soil biodiversity, and reduction of C sink capacity in soils. Therefore, the agricultural practice of applying carbon-rich materials into the soil is an attractive solution for climate change mitigation and soil ecosystem sustainability. The paper aimed to evaluate the effectiveness of the addition of organic-mineral mixtures to the mineral salts (NPK), including the exogenous organic matter (lignite) mixed with zeolite-carbon (NaX-C) or zeolite-vermiculite (NaX-Ver) composites in the restoration of soils contaminated with PAHs. The addition of zeolite composites to fertilizer resulted in a significant reduction in soil PAH levels and a corresponding reduction in plant tissue content, without compromising yields, compared to the control and separate application of NPK. A Significant correlation between PAHs and pHH2O, pHKCl, EC and dehydrogenase activity (DhA) was found in soils. The addition of zeolite composites with lignite significantly reduced the content of PAHs in straws, especially following the application of NaX-C. However, in the case of grains, the highest percentage reduction in comparison to NPK was observed for the highest dose of NaX-Ver. 1 entific Reports \| (2023) 13:14227 \| https://doi.org/10.1038/s41598-023-41429-2 Abbreviations BaA Benz(a)anthracene B(a)Peq Benzo(a)pyrene-equivalent BbF Benzo(b)fluoranthene BaP Benzo(a)pyrene BC Black carbon C3L3 3% NaX-C + 3% lignite + 94% NPK C9L6 9% NaX-C + 6% lignite + 85% NPK V3L3 3% NaX-Ver + 3% lignite + 94% NPK V9L6 9% NaX-Ver + 6% lignite + 85% NPK Chr Chrysene DhA Dehydrogenase activity d.m. Dry mass HMW High molecular weight (Kow) Octanol–water partitioning coefficient LMW Low molecular weight NaX-C Zeolite–carbon composite NaX-Ver Zeolite–vermiculite composite 1Department of Radiochemistry and Environmental Chemistry, Maria Curie-Sklodowska University, 3 Maria Curie‑Skłodowska Square, 20‑031 Lublin, Poland. 2Department of Environmental Analysis, Geological Mapping and Economic Geology, AGH University of Science and Technology, Mickiewicza 30 Av., 30‑059 Kraków, Poland. 3Department of Coal Chemistry and Environmental Sciences, Faculty of Energy and Fuels, AGH University of Science and Technology, Mickiewicza 30 Av., 30‑059 Kraków, Poland. 4Department of Mineralogy, Petrography and Geochemistry, AGH University of Science and Technology, Mickiewicza 30 Av., 30‑059 Kraków, Poland. 5Department of Agricultural and Environmental Chemistry, University of Agriculture in Krakow, Mickiewicza 21 Av., 31‑120 Kraków, Poland. Restoration of soils contaminated with PAHs by the mixture of zeolite composites mixed with exogenous organic matter and mineral salts OPEN email: jszerement@gmail.com; justyna.szerement@mail.umcs.pl Abbreviations BaA Benz(a)anthracene B(a)Peq Benzo(a)pyrene-equivalent BbF Benzo(b)fluoranthene BaP Benzo(a)pyrene BC Black carbon C3L3 3% NaX-C + 3% lignite + 94% NPK C9L6 9% NaX-C + 6% lignite + 85% NPK V3L3 3% NaX-Ver + 3% lignite + 94% NPK V9L6 9% NaX-Ver + 6% lignite + 85% NPK Chr Chrysene DhA Dehydrogenase activity d.m. Dry mass HMW High molecular weight (Kow) Octanol–water partitioning coefficient LMW Low molecular weight NaX-C Zeolite–carbon composite NaX-Ver Zeolite–vermiculite composite 1Department of Radiochemistry and Environmental Chemistry, Maria Curie-Sklodowska University, 3 Maria Curie‑Skłodowska Square, 20‑031 Lublin, Poland. 2Department of Environmental Analysis, Geological Mapping and Economic Geology, AGH University of Science and Technology, Mickiewicza 30 Av., 30‑059 Kraków, Poland. 3Department of Coal Chemistry and Environmental Sciences, Faculty of Energy and Fuels, AGH University of Science and Technology, Mickiewicza 30 Av., 30‑059 Kraków, Poland. 4Department of Mineralogy, Petrography and Geochemistry, AGH University of Science and Technology, Mickiewicza 30 Av., 30‑059 Kraków, Poland. 5Department of Agricultural and Environmental Chemistry, University of Agriculture in Krakow, Mickiewicza 21 Av., 31‑120 Kraków, Poland. email: jszerement@gmail.com; justyna.szerement@mail.umcs.pl \| https://doi.org/10.1038/s41598-023-41429-2 Scientific Reports \| (2023) 13:14227 www.nature.com/scientificreports/ PAHs Polycyclic aromatic hydrocarbons Σ16 PAHs The sum of 16 analysed PAHs TOC Total organic carbon TN Total nitrogen UFA/SFA Unsaturated fatty acids/saturated fatty acids PAHs Polycyclic aromatic hydrocarbons Σ16 PAHs The sum of 16 analysed PAHs TOC Total organic carbon TN Total nitrogen UFA/SFA Unsaturated fatty acids/saturated fatty acids It is estimated that a third of the world’s soil is moderately to highly degraded1. Of all the types of soil degrada- tion, chemical soil degradation (caused by the presence of heavy metals, organic contaminants etc.) is recognised as one of the most prevalent worldwide2, and its growing number is closely linked to unsustainable agriculture practices reflected in the depletion of the soil organic carbon (SOC) pool, loss in soil biodiversity, and decrease in soil fertility and elemental imbalance3. Polycyclic aromatic hydrocarbons (PAHs) are a large group of persistent, hydrophobic organic compounds containing two or more aromatic rings4. They can be classified according to their benzene ring numbers into two groups: 2–3-rings for low molecular weight (LMW) and 4-,5-, and 6-rings for high molecular weight (HMW)5. Extensive accumulation of PAHs in soils leads to serious agricultural and environmental issues around the world6. PAHs in crops may directly exert adverse impacts on the quality and safety of agricultural products and result in potential risks to human health7. Restoration of soils contaminated with PAHs by the mixture of zeolite composites mixed with exogenous organic matter and mineral salts OPEN These pollutants are highly toxic to soil microorganisms8. Microorganisms and soil enzymes can decompose benzene ring chains in PAHs9. For example, the dehydrogenase activity (DhA) can be used to evaluate the degradation performance10. It was also evidenced that microorganisms can take part in the regulation of SOC decomposition and storage, thus playing an important role in organic matter turnover and nutrient cycling11. Highly contaminated soils are usually poor in soil organic matter (SOM) and microbial activity. SOM, often estimated and expressed as SOC12, acts as a large carbon sink, and carbon farming as one of the land management practices that reduce greenhouse gas emissions and increase the sequestration and storage of carbon in soils and vegetation13,14. Additionally, it is one of the most important components of soil, essential for sustaining high levels of food production15. Unfortunately, the current rate of carbon loss due to. unsustain- able agriculture practices corresponds to 1.5 (1.0/1.8) GT carbon per year16. To restore the appropriate agricultural suitability of soils, degraded soil must be remediated and conserved using simple and cost-effective approaches17. These approaches should also include sustainable agriculture, which recommends reducing the quantity of chemical fertilizers used in their sector without compromising yields and promoting techniques that generate co-benefits in terms of adaptation, mitigation, and increased food production18.f One of the promising approaches recommended to offset the loss of SOM content and crop productivity is the application of exogenous organic materials containing organic carbon such as lignite and nutrients19. As an organic additive, lignite is rich in humic and fulvic acids 20. It was also proved that zeolite (natural and synthetic) as an additive to fertiliser can improve the physical, chemical, and biological properties of soil21–24. Previous studies have shown that adding exogenous organic matter mixed with zeolite-carbon composite (NaX-C) and zeolite-vermiculite composite (NaX-Ver) can stimulate soil microbial activity25–27 and also have a positive effect on root morphological parameters28. However, information about the application of these additives to the soil contaminated with PAHs is limited. Restoration of soils contaminated with PAHs by the mixture of zeolite composites mixed with exogenous organic matter and mineral salts OPEN It was evidenced that NaX-C and NaX-Ver mixed with leonardite reduced the content of PAHs in soil and tissues of maize29; however, there is no information on the effect of zeolite com- posites combined with other exogenous organic matter, like lignite, on the content of PAHs in soils and their plant accumulation.hf p The aim of the paper was the evaluate the effectiveness of the addition of organic-mineral mixtures to the mineral salts (NPK), including the lignite mixed with NaX-C or NaX-Ver composites on changes in content of total organic carbon (TOC), DhA, pH, EC, total N (TN), level of the degradation of PAHs, and uptake and distribution of PAHs in maize plant cultivated on PAHs-contaminated soil. Results S il Soil properties. According to the World Base for Soil Resources (WRB), the soil was Eutric Cambisol (CM- eu), with 85%, 12% and 3% of sand, silt and clay (loamy sand), respectively (Table 1). The soil was acidic, with a pH value of 5.24. The content of TOC was 6.34 g kg−1. Based on the European classification system of soil contamination30, the sum of 16 analysed PAHs (Σ16 PAHs) in soils represents heavy pollution. The content of Cd can be defined as a I degree of soil contamination, and on the basis of Zn and Pb, as a II degree of soil contamination31. The value of the pH significantly decreased in fertilized objects in comparison to the control; however, no significant differences (p > 0.05) were observed between types of fertilization (Table 2). The EC value was the lowest for C9L6 (305.25 ± 60.52 µS cm−3). Generally, in all soils with fertilization, the DhA was lower compared to the control (0.85 μg TPF g−1 h−1), except for C9L6. There were no significant differences in the TOC between control and fertilized objects, except C3L3. Additionally, there was a positive correlation between DhA and TOC (0.60, p < 0.05). The BC varied in soils from 5.49 ± 0.39 for V3L3 to 6.50 ± 0.42 for C3L3. The TN did not vary between variants. The ratio TOC:TN was higher for all variants with fertilization in comparison to the control, with the highest value for V3L3. Pearson’s correlation coefficients of the pH, EC, BC, TOC, DhA and 2, 3, 4, 5, and 6-rings PAHs are summarised in Table 3. Content of PAHs in soils. The concentration of 2-, 3-, 4-, 5-, and 6-rings PAHs in soils is presented in Fig. 1a. The content of individual PAHs is also shown in Table S1. All soil samples contained all rings of PAHs, with HMW PAHs contributing the highest percentage of total PAHs (ranging from 73.7 to 84.0%). The contri- bution of HMW and LMW differed among objects with zeolite composites amendments, and the lowest ratio between HMW and LMW was observed for the application of Ver3L3 (3.18). In fertilized soils, the Σ16 PAHs https://doi.org/10.1038/s41598-023-41429-2 Scientific Reports \| (2023) 13:14227 \| www.nature.com/scientificreports/ Table 1. Selected properties of the soil used in the experiment. Results S il Soil parameter Value Unit Sand 85 % Silt 12 % Clay 3 % pHH2O 5.24 – pHKCl 5.03 – EC 273 μS cm−1 BC 0.65 % TOC 6.34 g kg−1 d.m Ntotal 0.40 Ctotal 5.74 Stotal 0.118 Pbtotal 188 mg kg−1 d.m Cdtotal 1.15 Zntotal 267 PAHs 1.3 Table 1. Selected properties of the soil used in the experiment. Table 1. Selected properties of the soil used in the experiment. Table 1. Selected properties of the soil used in the experiment. Table 2. Selected soil characteristics after the experiment. Data represent means ± SD of four replications. Different letters in the rows indicate a significant difference (p < 0.05) according to the HSD Tukey test for α = 0.05. Variants C NPK C3L3 C9L6 V3L3 V9L6 Properties pHH2O 5.99 ± 0.06a 5.62 ± 0.17b 5.57 ± 0.29b 5.58 ± 0.03b 5.49 ± 0.39b 5.81 ± 0.61b pHKCl 5.26 ± 0.07a 4.81 ± 0.08b 4.87 ± 0.08b 4.89 ± 0.05b 4.88 ± 0.01b 4.94 ± 0.13b EC, μS cm−1 398.25 ± 45.41cd 416.67 ± 55.44bcd 531.33 ± 82.68ab 305.25 ± 60.52d 561.00 ± 35.36a 476.00 ± 41.82abc DhA, μg TPF g−1 h−1 0.85 ± 0.14a 0.37 ± 0.09b 0.21 ± 0.04b 0.69 ± 0.07a 0.35 ± 0.02b 0.29 ± 0.09b TOC, g kg−1 d.m 6.08 ± 0.35a 5.61 ± 0.36a 5.18 ± 0.20b 5.97 ± 0.20a 5.96 ± 0.29a 6.21 ± 0.31a BC, g kg−1 6.39 ± 0.22ab 6.37 ± 0.35ab 6.50 ± 0.42a 6.33 ± 0.45ab 5.49 ± 0.39b 5.81 ± 0.61ab TN, g kg−1 0.5 ± 0.05 0.42 ± 0.09 0.42 ± 0.01 0.42 ± 0.08 0.40 ± 0.10 0.47 ± 0.06 TOC:TN 12.3 17.9 16.9 17.1 19.4 16.8 Cdtotal, mg kg−1 d.m 1.06 ± 0.08ab 0.79 ± 0.08c 0.89 ± 0.14c 0.91 ± 0.03abc 0.89 ± 0.01bc 1.07 ± 0.04a Zntotal, mg kg−1 d.m 241.53 ± 16.1ab 174.88 ± 23.7c 220.30 ± 18.35b 213.16 ± 5.59b 210.43 ± 6.90b 255.33 ± 12.45a Pbtotal, mg kg−1 d.m 172.69 ± 10.28a 154.39 ± 5.94bc 165.96 ± 8.60ab 165.30 ± 8.67ab 144.10 ± 1.44c 179.53 ± 8.04a Table 2. Selected soil characteristics after the experiment. Data represent means ± SD of four replications. Different letters in the rows indicate a significant difference (p < 0.05) according to the HSD Tukey test for α = 0.05. 3 Vol.:(012 14227 \| https://doi.org/10.1038/s41598-023-41429-2 Table 3. Pearson’s correlation coefficients between PAHs content and soil characteristics. Table 3. Pearson’s correlation coefficients between PAHs content and soil characteristics. Asterisks indicate a significant difference (p < 0.05). Scientific Reports \| (2023) 13:14227 \| Results S il Asterisks indicate a significant difference (p < 0.05). Soil parameter 2-rings 3-rings 4-rings 5-rings 6-rings pHH2O 0.73 0.58* 0.53* 0.59* 0.61* pHKCl 0.61* 0.49* 0.55* 0.68* 0.75* EC − 0.45* − 0.62* − 0.66* − 0.57* − 0.57* DhA 0.65* 0.60* 0.71* 0.74* 0.78* TOC 0.09 0.06 0.34 0.43* 0.42* TN − 0.46* − 0.67* − 0.64* − 045* − 0.52* BC 0.32 0.27 0.15 0.15 0.17 Cdtotal 0.19 0.06 0.31 0.40 0.51* Zntotal 0.11 − 0.07 0.24 0.34 0.44* Pbtotal 0.47* 0.38 0.45* 0.42* 0.53* Soil parameter 2-rings 3-rings 4-rings 5-rings 6-rings pHH2O 0.73* 0.58* 0.53* 0.59* 0.61* pHKCl 0.61* 0.49* 0.55* 0.68* 0.75* EC − 0.45* − 0.62* − 0.66* − 0.57* − 0.57* DhA 0.65* 0.60* 0.71* 0.74* 0.78* TOC 0.09 0.06 0.34 0.43* 0.42* TN − 0.46* − 0.67* − 0.64* − 045* − 0.52* BC 0.32 0.27 0.15 0.15 0.17 Cdtotal 0.19 0.06 0.31 0.40 0.51* Zntotal 0.11 − 0.07 0.24 0.34 0.44* Pbtotal 0.47* 0.38 0.45* 0.42* 0.53* Table 3. Pearson’s correlation coefficients between PAHs content and soil characteristics. Asterisks indicate significant difference (p < 0.05). https://doi.org/10.1038/s41598-023-41429-2 Scientific Reports \| (2023) 13:14227 \| www.nature.com/scientificreports/ Figure 1. Concentration of 2, 3, 4, 5, 6-rings PAHs in soils (a), roots (b), straws (c) and grains (d) of maize (in mg kg−1) and mass of roots, straws, and grains (in g pot−1, black square with SD). Different letters on bars indicate a significant difference in Σ16 PAHs (p < 0.05) according to the HSD Tukey test for α = 0.05. Figure 1. Concentration of 2, 3, 4, 5, 6-rings PAHs in soils (a), roots (b), straws (c) and grains (d) of maize (in mg kg−1) and mass of roots, straws, and grains (in g pot−1, black square with SD). Different letters on bars indicate a significant difference in Σ16 PAHs (p < 0.05) according to the HSD Tukey test for α = 0.05. were significantly lower (p > 0.05) in comparison to the control. (1.45 ± 0.17) and ranged from 0.79 ± 0.05 mg kg−1 for C3L3 to 1.24 ± 0.29 mg kg−1 for V9L6. Content of PAHs in plant tissues. The concentration of 2-, 3-, 4-, 5-, and 6-rings PAHs in roots (Fig. 1b), straws (Fig. 1c) and grains (Fig. 1d) is presented in Fig. 1 and Table S1. The content of Σ16 PAHs changed in the following order: straws > roots > grains. Results S il Percentage reduction of the content of 2, 3, 4, 5, and 6-rings PAHs in roots (a), straws (b), and grains (c) of maize after application of zeolite composites mixed with lignite against the PAHs content in maize tissues for the application of the conventional fertilization NPK. Figure 2. Percentage reduction of the content of 2, 3, 4, 5, and 6-rings PAHs in roots (a), straws (b), and grains (c) of maize after application of zeolite composites mixed with lignite against the PAHs content in maize tissues for the application of the conventional fertilization NPK. Figure 2. Percentage reduction of the content of 2, 3, 4, 5, and 6-rings PAHs in roots (a), straws (b), and grains (c) of maize after application of zeolite composites mixed with lignite against the PAHs content in maize tissues for the application of the conventional fertilization NPK. Table 4. Content of BaP and value of the BaPeq in maize grains. Data represents means ± SD of four replications. Different letters in the columns indicate a significant difference (p < 0.05) according to the HSD Tukey test for α = 0.05. BaP (μg kg−1) BaPeq ( μg-BaP kg−1) NPK 1.45 ± 0.05a 1.59 ± 0.05a V3L3 0.89 ± 0.03c 1.08 ± 0.04c V9L6 0.82 ± 0.03c 0.89 ± 0.03d C3L3 0.86 ± 0.03c 0.97 ± 0.03d C9L6 1.36 ± 0.04b 1.49 ± 0.05b Table 4. Content of BaP and value of the BaPeq in maize grains. Data represents means ± SD of four replications. Different letters in the columns indicate a significant difference (p < 0.05) according to the HSD Tukey test for α = 0.05. BaP (μg kg−1) BaPeq ( μg-BaP kg−1) NPK 1.45 ± 0.05a 1.59 ± 0.05a V3L3 0.89 ± 0.03c 1.08 ± 0.04c V9L6 0.82 ± 0.03c 0.89 ± 0.03d C3L3 0.86 ± 0.03c 0.97 ± 0.03d C9L6 1.36 ± 0.04b 1.49 ± 0.05b Table 4. Content of BaP and value of the BaPeq in maize grains. Data represents means ± SD of four replications. Different letters in the columns indicate a significant difference (p < 0.05) according to the HSD Tukey test for α = 0.05. Table 5. Bioaccumulation factors (BFs) of 2, 3, 4, 5, and 6-rings PAHs for roots, straws and grains of maize. Data represents means ± SD of four replications. Results S il The 4-ringed PAHs were the most predominant group, and their high- est content was observed in the straws (from about 72.2% in the C9L6 to 90.0% in the control). The application of both zeolite composites mixed with lignite significantly reduced the Σ16 PAHs in maize roots (from 8.21 to 30.5% and from 17.5 to 37.5% in comparison to control and NPK, respectively) with simultaneously no reduc- tion in mass of roots. The application of both zeolite composites mixed with lignite significantly reduced the content of 6-rings of PAHs in roots by about 78.84% for C3L3 to 87.18% for V9L6 compared to NPK. In straws, the highest reduction of 4-, 5-, and 6-rings PAHs was observed for application of NaX-C, especially when applied at a higher dose (69.26%, 66.13%, 59.44%). For grains, the lowest content of Σ16 PAHs was observed for V9L6 (0.12 ± 0.02 mg kg−1). There was no grain yield in the control variant. ( g g )h g y Figure 2 shows the reduction of content of the 2-, 3-, 4-, 5-, and 6-rings PAHs in roots, straws, and grains compared to results obtained after NPK fertilization. In roots, the highest reduction was observed for 6-rings PAHs (Fig. 2a) (from 78.84% for C3L3 to 83.70% for V3L3), whereas a significant increase of 5-rings PAHs was observed (up to 52.17% for C3L3). In the case of straws, the highest reduction HMW was observed for NaX-C (more than 52%). Contrasting results were obtained for grains, where the highest decrease of HMW was observed for NaX-Ver, but only for the higher dose of mineral-organic mixture. The application of V9L6 reduced the level of 4-, 5, and 6-rings PAHs by 30.33%, 42.44%, and 40.51%, respectively, whereas the application of C9L6 increased the level of these PAHs by 26.26%, 11,09%, and 18.24%. The lowest content of BaPeq and BaP with the most carcinogenic properties in grains was observed in V9L6 (0.89 ± 0.03 and 0.82 ± 0.03) (Table 4.). Bioaccumulation factors (BFs). The mobility of PAHs in soil and their availability in plants were calcu- ated as bioaccumulation factors (BFs). The bioaccumulation factors of 2, 3, 4, 5, and 6-rings PAHs for maize https://doi.org/10.1038/s41598-023-41429-2 Scientific Reports \| (2023) 13:14227 \| www.nature.com/scientificreports/ Figure 2. Discussionh Microorganisms rapidly consume nutrients released after the first stages of hydrocarbon degradation38; hence, at the medium and long-term phases, when nutrients release is reduced considerably, carbon and nutrient concentrations in hydrocarbon-polluted soils may decrease significantly. The DhA in soils are usually the most sensitive indicator of environmental changes, and their activities are always affected by soil conditions through shifting the synthesis and structure of local microorganisms39. It was also evidenced that zeolites, due to their high porosity and well-developed specific surface area, can improve the soil’s microbial activity26,27, 40. The DhA in amended soils was lower compared to the control, except for C9L6. Additionally, there were no statistical dif- ferences between fertilization. The lower DhA in soils after the maize harvest might have resulted from a lower availability of soluble organic carbon41. Additionally, the pot experiment took nearly 5 months, and the highest DhA was observed after 3 months in the incubation experiment (data not shown), particularly for the application of both zeolite composites mixed with lignite. It was also evidenced that NaX-C and NaX-Ver combined with leonardite or lignite stimulated the microbial activity of unpolluted soils26,42, 43.f g y p Soil amendments (organic and inorganic) may change the soil pH, which can have an indirect effect on the bioavailability of PAHs and control the soil’s biology and biological processes44. A significant positive correla- tion between soil pHH2O/pHKCl and PAHs suggested that the soil’s pH was a key factor in the soil PAHs levels. The statistical negative correlation of all rings PAHs with EC suggest that this factor also indirectly affects the degradation of PAHs in soils45. The same results were observed for the application of zeolite composites mixed with leonardite29.i In the presented study, the 2-, 3-, 4-, 5-, and 6-rings PAHs were significantly correlated with Pb, and for 6-rings PAHs with Zn and Cd. Heavy metals can make cation-π-bonds with PAHs46. Results showed by Oste et al.47 indicated that the alkaline pH of the synthetic zeolites caused an increase in the content of the dissolved organic matter, affecting improved sorption of HMs by the solid phase.i f Generally, applying both zeolite composites combined with lignite reduced the level of PAHs significantly in roots, straws and grains (except C9L6) compared to NPK applied separately. The highest values of the RBFs, SBFs, and GBFs were obtained for 4-rings PAHs, with pyrene being the most dominant. Discussionh The degradation of PAHs in soils is a complex process and depends on many factors, such as soil type, tempera- ture, pH, and soil organic matter content32. In the present study, the application of both zeolite composites mixed with lignite generally decreased the level of all the rings-PAHs in soils in comparison to the control. However, the ratio HMW:LMW for these soils increased, suggesting that LMW PAHs were more easily degraded than HMW PAHs. The HMW PAHs are more resistant to degradation by bacterial or litter-decomposing fungi than LMW PAHs31,32. Additionally, the HMW PAHs represented the majority of the total PAHs in tested soils (from 73.7 to 84.0%), which is in line with the results obtained by Ukalska-Jaruga et al.33. In the presented study, a significant positive correlation between 5-, and 6-rings PAHs and TOC was found to be consistent with the results shown by Duan et al.34. The PAHs with a higher number of rings have a higher octanol–water partitioning coefficient (logKow) compared to PAHs with a lower number of rings, resulting in a lower tendency to dissolve in water and more tendency to adsorb onto the organic matter35.hf y g There were no statistical differences between the content of TN in tested soils. For better characterisation of the quality of SOM, the TOC/TN ratio was calculated (Table 2). The potential decomposition rate of organic C and potentially mineralizable N was higher in soils with high organic matter input. The TOC/TN ratio was higher in soils with all fertilization and showed diversified values from 12.3 for the control to 17.1 for V3L3, which indi- cates a varying degree of organic matter decomposition in tested soils. It was also found that the content of the TN in soils had a significant negative correlation with the content of the 2-, 3-, 4-, 5-, and 6-rings PAHs, which is opposite to results reported by Han et al.36, where TN had significant positive correlation with HMW PAHs.h pp p y gi p The addition of mineral-organic mixtures, rich in organic carbon, did not change the level of TOC in tested soils except for C3L3, where the level of the TOC decreased statistically. Generally, the TOC levels change slowly and are insensitive to vegetation restoration over short periods in natural conditions37. However, the exogenous addition of SOM to the soil can stimulate soil microorganisms, which decompose benzene ring chains in PAHs 9. Results S il Different letters in the columns indicate a significant difference (p < 0.05) according to the HSD Tukey test for α = 0.05. Object 2-rings 3-rings 4-rings 5-rings 6-rings RBFs NPK 1.43 ± 0.31a 1.34 ± 0.24ab 2.20 ± 0.21ab 0.15 ± 0.01b 0.541 ± 0.041a V3L3 1.76 ± 0.10a 1.57 ± 0.25a 1.45 ± 0.10c 0.17 ± 0.01b 0.074 ± 0.064bc V9L6 1.32 ± 0.16a 1.07 ± 0.24b 1.23 ± 0.11c 0.16 ± 0.02b 0.057 ± 0.007c C3L3 0.81 ± 0.04b 1.18 ± 0.21ab 2.28 ± 0.09a 0.21 ± 0.01a 0.106 ± 0.007b C9L6 0.50 ± 0.22b 1.18 ± 0.14ab 1.94 ± 0.03b 0.21 ± 0.01a 0.090 ± 0.001bc SBFs NPK 0.78 ± 0.15b 1.36 ± 0.23ab 7.53 ± 0.78a 0.64 ± 0.07a 1.029 ± 0.091a V3L3 0.39 ± 0.04c 1.85 ± 0.30a 7.55 ± 0.52a 0.49 ± 0.04b 0.895 ± 0.077a V9L6 0.40 ± 0.08c 1.31 ± 0.30b 5.70 ± 0.56b 0.48 ± 0.08b 0.504 ± 0.069bc C3L3 0.69 ± 0.06bc 1.10 ± 0.21b 2.69 ± 0.12c 0.27 ± 0.01c 0.638 ± 0.039b C9L6 1.35 ± 0.33a 1.10 ± 0.14b 2.60 ± 0.08c 0.26 ± 0.02c 0.393 ± 0.007c GBFs NPK 0.77 ± 0.15b 0.25 ± 0.05bc 0.26 ± 0.03b 0.014 ± 0.001b 0.100 ± 0.009b V3L3 0.69 ± 0.08bc 0.35 ± 0.06ab 0.28 ± 0.02b 0.013 ± 0.001b 0.084 ± 0.007c V9L6 0.40 ± 0.08 cd 0.19 ± 0.04c 0.16 ± 0.01c 0.007 ± 0.001d 0.049 ± 0.006d C3L3 0.30 ± 0.02d 0.32 ± 0.06b 0.25 ± 0.01b 0.009 ± 0.001c 0.081 ± 0.005c C9L6 1.13 ± 0.27a 0.45 ± 0.05a 0.41 ± 0.01a 0.016 ± 0.001a 0.121 ± 0.001a Table 5. Bioaccumulation factors (BFs) of 2, 3, 4, 5, and 6-rings PAHs for roots, straws and grains of maize. Data represents means ± SD of four replications. Different letters in the columns indicate a significant difference (p < 0.05) according to the HSD Tukey test for α = 0.05. https://doi.org/10.1038/s41598-023-41429-2 Scientific Reports \| (2023) 13:14227 \| www.nature.com/scientificreports/ plant tissues are presented in Table 5. The highest value of root bioconcentration factors (RBFs) and straw bio- concentration factors (SBFs) for 6-rings PAHs was observed for NPK. The lowest value of grain bioconcentra- tion factors (GBFs) for 2- (0.40 ± 0.08), 3- (0.19 ± 0.04), 4- (0.16 ± 0.01), 5- (0.007 ± 0.001), and 6-rings PAHs 0.049 ± 0.006) was observed after the application of V9L6. Results S il plant tissues are presented in Table 5. The highest value of root bioconcentration factors (RBFs) and straw bio- concentration factors (SBFs) for 6-rings PAHs was observed for NPK. The lowest value of grain bioconcentra- tion factors (GBFs) for 2- (0.40 ± 0.08), 3- (0.19 ± 0.04), 4- (0.16 ± 0.01), 5- (0.007 ± 0.001), and 6-rings PAHs 0.049 ± 0.006) was observed after the application of V9L6. Materials and methods Chemical reagents. Ammonium nitrate (NH4NO3), calcium dihydrogenphosphate monohydrate (Ca(H2PO4)2 H2O); potassium chloride (KCl), sodium sulfate (Na2SO4), acetone, anhydrous sodium sulfate (Na2SO4), triphenyltetrazolium chloride (TTC), acetone were purchased from S.A. POCH, Poland. N-Hexane (HEX), acetone (ACE), dichloromethane (DCM), and methanol (MeOH) with purity > 99.9% used for chro- matographic analyses were purchased from Chemsolute. Standard of 16 PAHs in a 2000 μg ml−1 mixture solu- tion in DCM (CRM47930), deuterated PAHs internal standard solutions (phenanthrene-d10 at concentration 2000 μg ml−1 in DCM) were obtained from Sigma-Aldrich. Standard working solutions of PAHs mixture, inter- nal standard mixture and phenanthrene-d10 were diluted properly with dichloromethane (DCM) and prepared freshly before the analysis. Characteristics of soil samples. The soil (upper soil layer of 0–30 cm) used in the experiment was sam- pled from an agriculture field located near a coniferous forest in South Malopolska, Poland (50°05′35.9" N 19°39′52.9" E). Next, the soil was air-dried, sieved (2 mm) and manually homogenized. Pot experiment and sampling. A pot experiment was carried out in 2020 in the vegetation hall with a transparent glass roof to prevent precipitation but ensure natural light and ventilation (Faculty of Agriculture and Economics of the University of Agriculture in Krakow-Mydlniki). PVC pots (25 cm height, 22 cm diameter) were filled with 9 kg of air-dried soil. During the experiment, the soil water content was maintained at the maxi- mum water holding capacity using a Rain Bird sprinkler system (Rain Bird Inc., Tucson, USA) equipped with a soil moisture sensor. Soil moisture was controlled using a portable probe with an ECH2O EC5 sensor (Decagon Devices, Pullman, Washington, USA).hfh g The soils were mixed with six different treatments in four independent replications. The reference variant was a control object without fertilization: C—control soil (without fertilization), and NPK, 100% NPK—soil with an addition of mineral salts: N—NH4NO3; P—Ca(H2PO4)2 H2O; K—KCl in doses of 0.20 g N kg−1, 0.10 g P kg−1, and 0.25 g K kg−1 for N, P, K, respectively. www.nature.com/scientificreports/ as the most dangerous pollutant due to its high carcinogenic and mutagenic character52. The application of both zeolite composites mixed with lignite significantly reduced the level of BaP and BaPeq in maize grains compared to NPK. In the case of BaP, the best results were obtained for the lower dose of NaX-C (C3L3) and both doses of NaX-Ver (V3L3, V9L6), i.e., reduction of BaP by approximately 40.69%, 43.51, and 40.65, respectively in comparison to NPK. These trends are in line with results obtained for the application of the zeolite composites mixed with leonardite29. However, applying the lower dose of zeolite-carbon composite mixed with leonardite has about 33% better effect on the reduction of BaP than lignite used in the current experiment. f Based on the obtained results, it can be concluded that adding a mixture of zeolite composites mixed with lignite and mineral salts can be an innovative and effective way to restore soil contaminated with PAHs and limit their uptake by plants. Another advantage of their application is that they can reduce the use of mineral fertiliz- ers, which is one of the main strategies of sustainable agriculture. However, the main disadvantage is their high salinity and low structure stability at the lower pHs. Thus long-term studies are needed to better understand their impact on soil changes53. Discussionh Pyrene was mainly accumu- lated in straws of maize, in contrast to wheat cultivated in a hydroponic system, where pyrene was accumulated primarily in roots48. The lower SBFs for 4-, 5- and 6- rings PAHs for both zeolite composites mixed with lignite in comparison to NPK (more than two times lower in the case of zeolite-carbon composites) suggested that less HMW PAHs were transported from soil to straws of maize which is in line with the results showed by Tao et al.49 for wheat roots, and Szerement et al.29 for maize. The addition of both zeolite composites combined with lignite changed the properties of the soil, which could have affected the composition of root exudates (low-molecular- weight organic acids)50. Thus, the lower uptake of PAHs from soil can also be connected to mechanisms between microorganisms and plant abilities to stimulate microbial PAH degradation by root exudates. However, the role of root exudates in the degradation of PAHs is still not fully understood51. g y Grains exhibited the lowest concentration of PAHs compared to straws and roots. Similarly to the results obtained for zeolite composites mixed with leonardite (− 0.81, p < 0,05)29, a significant negative correlation was found between the content of PAHs in soil and grains (− 0.58, p < 0.05). Thus, it was evidence that PAHs in grains were mainly accumulated from the air; however, depending on applied zeolite composites, the level of PAHs in grains varied. It is further confirmation that the type and amount of zeolite composites play a significant role in altering soil properties and the maize plant’s ability to accumulate PAHs. According to US-EPA, BaP is recognized Scientific Reports \| (2023) 13:14227 \| https://doi.org/10.1038/s41598-023-41429-2 www.nature.com/scientificreports/ Materials and methods More details about mineralogical composition and structural and textural characterization of applied zeolite composites are provided in the paper by Mokrzycki et al.55 Lignite was sup- plied by the Sieniawa Coal Mine (Poland). Extraction and analysis of PAHs. Determination of PAHs in plant and soil materials. Homogenized, air-dried soil samples (10 g) were mixed with DCM (100 mL) at room temperature and extracted using Soxtec with the following steps: (i) boiling in solvent for 90 min at 180 °C, (ii) rising step for 60 min, (iii) evaporation/ solvent recovery for 15 min. Deuterated PAHs internal standard solutions (100 μL of phenanthrene-d10 at a concentration of 40 μg mL−1) were added to all samples. The solutions after Soxtec extraction were concentrated to a volume of 0.5 mL under nitrogen evaporation. Plant materials. Ground roots, straw and grains (5 g) were mixed with 100 mL of solvent (HEX : DCM, 1:1, v/v) with the addition of anhydrous Na2SO4 (previously dried at 600 °C) and spiked with a labelled surrogated standard of phenanthrene-d10 (100 μL at a concentration of 40 μg mL−1)56. The extraction was performed using the Soxtec method (as described above). After conditioning the column with two portions of HEX (8 mL for each portion), samples were air-dried, solved with 4 mL of HEX and transferred into the column. The extract was eluted with two portions (8 mL) of a mixture of DCM and HEX (1:1, v/v). The extracts were purified on a column containing activated silica gel (10 g, Silica 100, Merck) topped with anhydrous Na2SO4 (5 g). The column was conditioned twice with 8 mL of hexane. The air-dried samples after Soxtec extraction were solved with 4 mL of HEX and transferred into the conditioned column. The samples of grains were additionally eluted with a two- portion (8 mL) of MeOH for fatty acids (FAs) analysis. Determination of PAHs in plant and soil materials. The samples were concentrated to 0.5 mL under nitro- gen evaporation. The content of 16 priority PAHs including naphthalene, acenaphthene, acenaphthylene, flu- orene, phenanthrene, anthracene, fluoranthene, pyrene, benzo(a)anthracene, chrysene, benzo(b)fluoranthene, benzo(k)fluoranthene, benzo(a)pyrene, indeno(1,2,3-cd)pyrene, dibenzo(a,h)anthracene and benzo(g,h,i) per- ylene were determined using gas chromatography-mass spectrometry (GC/MS) (Agilent 7890A GC with 5975C MSD, Agilent Technology). The injection was done in on-column mode. The transfer line was set to 300 °C. Materials and methods The objects with the addition of the mineral-organic mixtures (zeolite com- posites mixed with lignite) were as follows: V3L3—soil with a mixture of the 3% of zeolite-vermiculite composite, 3% of lignite, and 94% NPK; V9L6—soil with a mixture of the 9% of zeolite-vermiculite composite, 6% of lignite, and 85% NPK; C3L3—soil with a mixture of the 3% of zeolite-carbon composite, 3% of lignite, and 94% NPK; C9L6—soil with a mixture of the 9% of zeolite-carbon composite, 6% of lignite, and 85% NPK. Each pot had 15 seeds of maize variety of Kosynier (provided by Centrala Nasienna Krakow) sown and then left to 5 seedlings after germination. Due to visible symptoms of N deficiency during vegetation, the supplementary NH4NO3 at a dose of 0.02 g N kg−1 dry mass (d.m.) of soil was applied. Soil moisture during plant vegetation was maintained at 40% to 60% of the maximum water capacity of the soil (depending on the development phase of the plant). p y p g p p p In the fully ripe stage of the maize—on day 126 post-planting, the plants were harvested, and the roots were collected from the soil and washed with distilled water to remove soil and other debris. The dry mass was cal- culated after drying straws (stalks + leaves), roots and cobs at 65 °C for 24 h. Soil samples were collected from each pot, sieved and stored for further analyses at 4 °C and 25 °C for biological and physicochemical analyses, respectively.h p y The temperature and humidity were recorded from March to September outside the vegetation hall (Table 6) Table 6. Average monthly temperature and air humidity. Month Temperature (oC) Air humidity (%) IV 10.1 67.9 V 13.9 64.5 VI 18.4 85.7 VII 19.4 76.7 VIII 20.7 76.2 IX 17.1 83.1 Table 6. Average monthly temperature and air humidity. Table 6. Average monthly temperature and air humidity. https://doi.org/10.1038/s41598-023-41429-2 Scientific Reports \| (2023) 13:14227 \| www.nature.com/scientificreports/ Amendments. NaX-C were synthesized according to methods described by Panek et al.54, whereas NaX- Ver were synthesized with the addition of vermiculite (the Namekara mine, Mbale, Uganda). The selected prop- erties of both zeolite composites are presented in Table 7. The specific surface area (SBET) of the NaX-C was about 1.3 times higher than NaX-Ver. Materials and methods The temperature program was: 99 °C for initial temperature, 2 °C min−1 to 310 °C for 34 min. Gas flow (He) 1.1 mL min −1. The analysis was conducted with a 60 m column (DB-5MS, 250 μm, 0.25 μm), using QTM 16-PAHs mix (CRM47930, Sigma-Aldrich) as a standard. The LODs and LOQs of the 16 PAHs were 0.07– 0.15 μg mL−1 and 0.20–0.45 μg mL−1, respectively. To ensure the accuracy and precision of this study’s individual PAHS analysis process, a seven-point calibration standard (linearity R2 > 0.99; 0.5–12.5 μg mL−1) in solution, detection limits and procedural blank were carried out. Each calibration standard and sample contained an internal standard (100 μL of phenanthrene-d10 at a concentration of 40 μg mL−1). The recoveries ranged from 76 to 102% for individual PAHs. The reported results have been corrected for losses. Physicochemical characteristics of soils. The following analyses were carried out for soils: TOC, black carbon (BC), pH, EC, contents of Ntotal (TN) and heavy metals: Cd, Zn, and Pb. The content of TOC was meas- ured according to Tiurin’s method57. The BC and TN analyses were conducted using CHNS-analyzer (Carlo Erba EA 1108). The soil materials were combusted briefly in a tube furnace at 375 °C for 18–24 h. Then, the sam- ples were cooled, and about 25 mg of the materials were weighed and placed into silver tin capsules. To remove any remaining inorganic carbonates, two drops of 1 M HCl were added (HCl:water ratio of 1:1, v:v)29. The pHH2O (distilled water), pHKCl (1 M KCl)58 and EC (distilled water) were measured at a soil:solution ratio of 1:2.5 using ELMETRON CX-502 (Elmetron, Poznan, Poland). Dehydrogenase activity (DhA). Dehydrogenase activity (DhA) was carried out according to the Thal- mann method (1968)59, using triphenyltetrazolium chloride (TTC) as the electron acceptor. After incubation at 37 ± 2 °C for 24 h with TTC, samples were filtrated to separate the solution from soil and measured at 546 nm wavelength using a UV–VIS spectrophotometer (HITACHI U-5100, Hitachi High-Tech Science Corporation, Tokyo, Japan). The results are given as mean values from four analyses. Determination of Cd, Zn, and Pb concentration. The soil material (0.5 g) was mineralized using a mixture of 9 mL HNO3 and 3 mL HCl. The total concentration of Cd, Zn, and Pb were analyzed using 8 6 8 ) Scientific Reports \| (2023) 13:14227 \| https://doi.org/10.1038/s41598-023-41429-2 Table 7. Selected properties of zeolite composites. Materials and methods PAHs concentrations in all samples were calculated on the RBFs, SBFs and GBFs as follows (2) RBFs = Croots Csoils , RBFs = Croots Csoils , (2) RBFs = Croots Csoils , (3) SBFs = Cstraws Csoils , (4) GBFs = Cgrains Csoils , (2) (3) SBFs = Cstraws Csoils , (3) SBFs = Cstraws Csoils , (4) GBFs = Cgrains Csoils , (3) (4) GBFs = Cgrains Csoils , (4) where Csoils , Croots , Cstraws , Cgrains represent the PAHs concentration in soils, roots, straws and grains, respectively. where Csoils , Croots , Cstraws , Cgrains represent the PAHs concentration in soils, roots, straws and grains, respectively where Csoils , Croots , Cstraws , Cgrains represent the PAHs concentration in soils, roots, straws and grains, respectively. Statistical analysis. Statistical analyses were processed using Statistica version 13.0 software (StatSoft Inc., Poland). A one-way analysis of variance and Tukey’s post-hoc test (p > 0.05) were used to explore differences between the samples. Pearson’s correlation coefficients were also calculated. All of the figures were prepared using OriginPro2022 (OriginLab Corporation). Ethical statement. The study comply with relevant institutional, national, and international guidelines and legislation. Conclusionsh The cultivation of maize on PAHs-degraded soils increases the risk of contaminating the pollutants in the edible parts of crops. The addition of zeolite composites mixed with lignite caused a marked decrease in Σ16 PAH levels in the soil and plant tissues (except for C9L6 in the grain) when compared to the control and NPK applied separately. It was evidenced that adding exogenous organic matter like lignite mixed with zeolite composites had a beneficial role in the degradation of PAHs in soils. In soils, the content of PAHs was correlated with pHH2O/ pHKCl, EC, DhA, TN, and TOC for 5- and 6-rings PAHs. The lowest content of PAHs in straws was observed after the application of C3L3 and C9L6; however, in the case of grains, the best results were obtained for V3L3. Lower level of Σ16 PAHs in plant tissues was observed for NaX-C, which represented a higher specific surface area in comparison to NaX-Ver. Additionally, it could be concluded that the type of material used for zeolite- composites synthesis and the dose of applied additives influenced the accumulation of PAHs in particular parts of plants. Long-term stability tests and the impacts of applied zeolites on soils and soil micro-organisms should be evaluated in the future. Data availability Th d d y The datasets generated and/or analysed during the current study are not publicly available but may be obtained from the corresponding author on reasonable request. Received: 29 April 2023; Accepted: 26 August 2023 Received: 29 April 2023; Accepted: 26 August 2023 Materials and methods pH (–) EC (–) K (mg g−1 d.m) Mg (mg g−1 d.m) Ca (mg g−1 d.m) P (mg g−1 d.m) Na (mg g−1 d.m) Fe (mg g−1 d.m) S (mg g−1 d.m) Zn (mg g−1 d.m) Σ PAHs (µg g−1 d.m) NaX-C 10.30 ± 0.05 1.58 ± 0.02 7.05 ± 2.15 6.55 ± 1.60 8.30 ± 2.71 0.21 ± 0.07 23.96 ± 6.72 15.00 ± 2.31 1.05 ± 0.35 0.12 ± 0.04 36.11 ± 0.32 NaX-Ver 10.87 ± 0.09 2.01 ± 0.05 20.62 ± 3.90 27.70 ± 2.84 11.23 ± 1.50 0.18 ± 0.01 31.50 ± 2.24 7.03 ± 1.48 0.13 ± 0.01 0.02 ± 0.01 3.18 ± 0.02 Table 7. Selected properties of zeolite composites. pH (–) EC (–) K (mg g−1 d.m) Mg (mg g−1 d.m) Ca (mg g−1 d.m) P (mg g−1 d.m) Na (mg g−1 d.m) Fe (mg g−1 d.m) S (mg g−1 d.m) Zn (mg g−1 d.m) Σ PAHs (µg g−1 d.m) NaX-C 10.30 ± 0.05 1.58 ± 0.02 7.05 ± 2.15 6.55 ± 1.60 8.30 ± 2.71 0.21 ± 0.07 23.96 ± 6.72 15.00 ± 2.31 1.05 ± 0.35 0.12 ± 0.04 36.11 ± 0.32 NaX-Ver 10.87 ± 0.09 2.01 ± 0.05 20.62 ± 3.90 27.70 ± 2.84 11.23 ± 1.50 0.18 ± 0.01 31.50 ± 2.24 7.03 ± 1.48 0.13 ± 0.01 0.02 ± 0.01 3.18 ± 0.02 https://doi.org/10.1038/s41598-023-41429-2 www.nature.com/scientificreports/ inductively-coupled plasma optical emission spectrometry (Optima 7300DC Perkin Elmer, USA). Multi-ele- mental solutions of 1.000 mg dm−3 ICP Standard Certipur® (Merck, Darmstadt, Germany) containing the ana- lysed elements were used for calibration. inductively-coupled plasma optical emission spectrometry (Optima 7300DC Perkin Elmer, USA). Multi-ele- mental solutions of 1.000 mg dm−3 ICP Standard Certipur® (Merck, Darmstadt, Germany) containing the ana- lysed elements were used for calibration. Estimation of cancer risk. The BaP equivalent (BaPeq), based on the measured 4PAHs [benz(a)anthracene (BaA), benzo(b)fluoranthene (BbF), and chrysene (Chr)] concentrations and the corresponding toxic equiva- lence factors (TEFs), were calculated using the following equation: (1) BaPeq = n i=1 Ci · TEFi, (1) where BaPeq is the total equivalent concentration of 4PAHs in maize grain (μg kg−1 d.m.); Ci is the concentration of 4PAHs in the maize grain sample; and TEFi, is the corresponding toxic equivalency factor (BaP = 1, Chr = 0.01, BaA = 0.1, BbF = 0.1)60. Data analysis. www.nature.com/scientificreports/ Głąb, T., Gondek, K., Marcińska-Mazur, L., Jarosz, R. & Mierzwa–Hersztek, M. Effect of organic/inorganic composites as soil amendments on the biomass productivity and root architecture of spring wheat and rapeseed. J. Environ. Manag. 344, 118628 (2023).l 9. Szerement, J. et al. Zeolite composites from fly ashes mixed with leonardite as a useful addition to fertilizer for accelerating the PAHs degradation in soil. Soil Tillage Res. 230, 105701 (2023).h g g 30. Klimkowicz-Pawlas, A., Smreczak, B. & Ukalska-Jaruga, A. The impact of selected soil organic matter fractions on the PAH accu- mulation in the agricultural soils from areas of different anthropopressure. Environ. Sci. Pollut. Res. 24, 10955–10965 (2017). b d d l l d l ( ) f . Kabata-Pendias, A. & Pendias, H. Trace Elements in Soils and Pl 2. Guarino, C. et al. Investigation and assessment for an effective approach to the reclamation of polycyclic aromatic hydrocarbon (PAHs) contaminated site: SIN Bagnoli, Italy. Sci. Rep. 9, 1–12 (2019).f 3. Ukalska-Jaruga, A., Smreczak, B. & Klimkowicz-Pawlas, A. Soil organic matter composition as a factor affecting the accumulation of polycyclic aromatic hydrocarbons. J. Soils Sediments 19, 1890–1900 (2019). 33. Ukalska-Jaruga, A., Smreczak, B. & Klimkowicz-Pawlas, A. Soil organic matter composit of polycyclic aromatic hydrocarbons. J. Soils Sediments 19, 1890–1900 (2019). 4. Duan, Y. et al. Characteristics of polycyclic aromatic hydrocarbons in agricultural soils at a typical coke production base in Shanxi China. Chemosphere 127, 64–69 (2015).hfif p ( ) 35. Sheng, Y. et al. The partitioning behavior of PAHs between settled dust and its extracted water phase: Coefficients and effects of the fluorescent organic matter. Ecotoxicol. Environ. Saf. 223, 112573 (2021). 36. Han, L. et al. Polycyclic aromatic hydrocarbons (PAHs) in surface soils from reclaimed and ditch wetlands along a 100-year chron- osequence of reclamation in a Chinese estuary: Occurrence, sources, and risk assessment. Agric. Ecosyst. Environ. 286, 106648 (2019).l 37. Wu, X., Fu, D., Duan, C., Huang, G. & Shang, H. Distributions and influencing factors of soil organic carbon fractions unde ferent vegetation restoration conditions in a subtropical mountainous area, SW China. Forests 13, 629 (2022). g 38. Zhang, X., Liu, Z., Luc, N. T., Liang, X. & Liu, X. Dynamics of the biological properties of soil and the nutrient release of Amorpha fruticosa L. litter in soil polluted by crude oil. Environ. Sci. Pollut. Res. 22, 16749–16757 (2015). 9. Kaczyńska, G., Borowik, A. & Wyszkowska, J. www.nature.com/scientificreports/ www.nature.com/scientificreports/ 7. Abdel-Shafy, H. I. & Mansour, M. S. M. A review on polycyclic aromatic hydrocarbons: Source, environmental impact, effect on human health and remediation. Egypt. J. Pet. 25, 107–123 (2016). gyp 8. Li, X. et al. New insights into the responses of soil microorganisms to polycyclic aromatic hydrocarbon stress by combining enzyme activity and sequencing analysis with metabolomics. Environ. Pollut. 255, 113312 (2019).hf y q g y 9. Kwiatkowska-Malina, J. Functions of organic matter in polluted soils: The effect of organic amendments on phytoavailability o heavy metals. Appl. Soil Ecol. 123, 542–545 (2018).hi y pp 0. Lu, H., Sun, J. & Zhu, L. The role of artificial root exudate components in facilitating the degradation of pyrene in soil. Sci. Rep. 7 1–10 (2017).fi ( ) 11. Soares, M. & Rousk, J. Microbial growth and carbon use efficiency in soil: Links to fungal-bacterial dominance, SOC-quality and stoichiometry. Soil Biol. Biochem. 131, 195–205 (2019).h y ( ) 12. Biswas, B. et al. The fate of chemical pollutants with soil properties and processes in the climate change paradigm—A review. Soil Syst. 2, 1–20 (2018).if y 3. Tisserant, A. & Cherubini, F. Potentials, limitations, co-benefits, and trade-offs of biochar applications to soils for climate change mitigation. Land 8, 179 (2019).f 14. Usowicz, B. & Lipiec, J. Determining the effect of exogenous organic materials on spatial distribution of maize yield. Sci. Rep. 9, 1–11 (2019). k Th l l f l l f l d b l ( ) ( ) 15. Gerke, J. The central role of soil organic matter in soil fertility and carbon storage. Soil Syst. 6, 33 (2022). h 16. Hu, X. et al. Recent global land cover dynamics and implications for soil erosion and carbon losses from deforestation. Anthropocene 34, 100291 (2021). 17. Kumar, M. et al. Remediation of soils and sediments polluted with polycyclic aromatic hydrocarbons: To immobilize, mobilize, or degrade? J. Hazard. Mater. 420, 4 (2021). g 8. Ye, L. et al. Bio-organic fertilizer with reduced rates of chemical fertilization improves soil fertility and enhances tomato yield and quality. Sci. Rep. 10, 1–11 (2020).i q y p 9. Garratt, M. P. D. et al. Enhancing soil organic matter as a route to the ecological intensification of European arable systems. Eco- systems 21, 1404–1415 (2018). y 20. Doskočil, L., Burdíková-Szewieczková, J., Enev, V., Kalina, L. & Wasserbauer, J. References f p g ( ) 2. Tetteh, R. N. Chemical soil degradation as a result of contamination: A review. J. Soil Sci. Environ. Manag. 6, 301–308 (2015 3. Kopittke, P. M., Menzies, N. W., Wang, P., McKenna, B. A. & Lombi, E. Soil and the intensification of agriculture for global f p g ( ) 2. Tetteh, R. N. Chemical soil degradation as a result of contamination: A review. J. Soil Sci. Environ. Manag. 6, 301–308 (2015). 3 Kopittke P M Menzies N W Wang P McKenna B A & Lombi E Soil and the intensification of agriculture for global foo f p g 2. Tetteh, R. N. Chemical soil degradation as a result of contamination: A review. J. Soil Sci. Environ. Manag. 6, 301–308 (2015). 3. Kopittke, P. M., Menzies, N. W., Wang, P., McKenna, B. A. & Lombi, E. Soil and the intensification of agriculture for global food security. Environ. Int. 132, 105078 (2019). f g 2. Tetteh, R. N. Chemical soil degradation as a result of contamination: A review. J. Soil Sci. Environ. Manag. 6, 301–308 (2015). 3. Kopittke, P. M., Menzies, N. W., Wang, P., McKenna, B. A. & Lombi, E. Soil and the intensification of agriculture for global food security. Environ. Int. 132, 105078 (2019). y , ( ) 4. Kuppusamy, S. et al. Remediation approaches for polycyclic aromatic hydrocarbons (PAHs) contaminated soils: Technological constraints, emerging trends and future directions. Chemosphere 168, 944–968 (2017). 4. Kuppusamy, S. et al. Remediation approaches for polycyclic aromatic hydrocarbons (PAHs) contaminated soils: Technological constraints, emerging trends and future directions. Chemosphere 168, 944–968 (2017). g g p esus, F. et al. A review on polycyclic aromatic hydrocarbons distribution in freshwater ecosystems and their toxicity to benthic auna. Sci. Total Environ. 820, 153282 (2022). 6. Cui, Z., Wang, Y., Du, L. & Yu, Y. Contamination level, sources, and health risk of polycyclic aromatic hydrocarbons in suburban vegetable field soils of Changchun, Northeast China. Sci. Rep. 12, 1–9 (2022). 6. Cui, Z., Wang, Y., Du, L. & Yu, Y. Contamination level, sources, and health risk of polycyclic aromatic hydrocarbons in suburban vegetable field soils of Changchun, Northeast China. Sci. Rep. 12, 1–9 (2022). https://doi.org/10.1038/s41598-023-41429-2 Scientific Reports \| (2023) 13:14227 \| www.nature.com/scientificreports/ Spectral characterization and comparison of humic acids isolated from some European lignites. Fuel 213, 123–132 (2018). p g ( ) 1. Szerement, J., Szatanik-Kloc, A., Jarosz, R., Bajda, T. & Mierzwa-Hersztek, M. Contemporary applications of natural and synthetic zeolites from fly ash in agriculture and environmental protection. J. Clean. Prod. 311, 127461 (2021).h l 22. Jarosz, R., Szerement, J., Gondek, K. & Mierzwa-Hersztek, M. The use of zeolites as an addition to fertilisers—A review. Catena 213, 106125 (2022).fl 23. Gondek, K., Mierzwa-hersztek, M. & Jarosz, R. Effect of willow biochar and fly ash-derived zeolite in immobilizing heavy metals and promoting enzymatic activity in a contaminated sandy soil. CATENA 232, 107429 (2023).f 4. Szatanik-Kloc, A., Szerement, J., Adamczuk, A. & Józefaciuk, G. Effect of low zeolite doses on plants and soil physicochemica properties. Materials Basel 14, 2617 (2021).t p p 25. Justyna, J. et al. Heavy metals and polycyclic aromatic hydrocarbons in soils and plants cultivated on contaminated soil after application of fertilizers with the addition of zeolite composites mixed with leonardite. in Proceedings of the 8th World Congress on New Technologies. 170–171. https://doi.org/10.11159/icepr22.170 (2022).f g p g p ( ) 26. Wolny-Koładka, K., Jarosz, R., Marcińska-Mazur, L., Lošák, T. & Mierzwa-Hersztek, M. Effect of mineral and organic additions on soil microbial composition. Int. Agrophys. 36, 131–138 (2022). p g p y 7. Wolny-Koładka, K., Jarosz, R., Juda, M. & Mierzwa-Hersztek, M. Distinct changes in abundance of culturable microbial community d i i i i i i i l i i li i i i d il S t i bilit 14 15004 (2022) 27. Wolny-Koładka, K., Jarosz, R., Juda, M. & Mierzwa-Hersztek, M. Distinct changes in abundance of culturable microbial community and respiration activities in response to mineral-organic mixture application in contaminated soil. Sustainability 14, 15004 (2022). 28 Gł b T G d k K M iń k M L J R & Mi H k M Eff f i /i i i il 27. Wolny-Koładka, K., Jarosz, R., Juda, M. & Mierzwa-Hersztek, M. Distinct changes in abundance of culturable microbial community and respiration activities in response to mineral-organic mixture application in contaminated soil. Sustainability 14, 15004 (2022). 28. Głąb, T., Gondek, K., Marcińska-Mazur, L., Jarosz, R. & Mierzwa–Hersztek, M. Effect of organic/inorganic composites as soil amendments on the biomass productivity and root architecture of spring wheat and rapeseed. J. Environ. Manag. 344, 118628 (2023).l 28. www.nature.com/scientificreports/ Jia, J., Bi, C., Zhang, J., Jin, X. & Chen, Z. Characterization of polycyclic aromatic hydrocarbons (PAHs) in vegetables near industria areas of Shanghai, China: Sources, exposure, and cancer risk. Environ. Pollut. 241, 750–758 (2018). Acknowledgements g (1) Article was conducted under the project POIR.04.04.00-00-14E6/18-00 which is carried out within the TEAM-NET programme of the Foundation for Polish Science co-financed by the European Union under the European Regional Development Fund. (2) The research work was financed by the Ministry of Science and Higher Education of the Republic of Poland and Innovation Center of the University of Agriculture in Krakow Sp. z o. o. www.nature.com/scientificreports/ www.nature.com/scientificreports/ 7. Oste, L. A., Lexmond, T. M. & Van Riemsdijk, W. H. Metal immobilization in soils using synthetic zeolites. J. Environ. Qual. 31 813–821 (2002). 8. Sobhani, A., Salehi-Lisar, S. Y., Motafakkerazad, R. & Movafeghi, A. Uptake and distribution of phenanthrene and pyrene in roots and shoots of wheat (Triticum aestivum L.). Polycycl. Aromat. Compd. 42, 543–550 (2022). y y p 9. Tao, Y., Zhang, S., Zhu, Y.-G. & Christie, P. Uptake and acropetal translocation of polycyclic aromatic hydrocarbons by wheat (Triticum aestivum L.) grown in field-contaminates soil. Environ. Sci. Technol. 43, 3556–3560 (2009).i i 0. Maurer, D. et al. Interactive regulation of root exudation and rhizosphere denitrification by plant metabolite content and soi properties. Plant Soil 467, 107–127 (2021).ff 1. Zhang, L. et al. Root exuded low-molecular-weight organic acids affected the phenanthrene degrader differently: A multi-omics study. J. Hazard. Mater. 414, 125367 (2021). study. J. Hazard. Mater. 414, 125367 (2021). y ( ) 52. Zena Bukowska, B., Mokra, K. & Michałowicz, J. Benzo[a]pyrene-environmental occurrence, human exposure, and mechanisms of toxicity. Int. J. Mol. Sci. 23, 6348 (2022).lfi 52. Zena Bukowska, B., Mokra, K. & Michałow of toxicity. Int. J. Mol. Sci. 23, 6348 (2022). y 3. Mokrzycki, J. et al. Copper ion-exchanged zeolite X from fly ash as an efficient adsorbent of phosphate ions from aqueous solutions J. Environ. Chem. Eng. 10, 6 (2022).l g ( ) 54. Panek, R. et al. Simultaneous removal of Pb2+ and Zn2+ heavy metals using fly ash Na-X zeolite and its carbon Na-X(C) composite. Materials (Basel). 14, 1–18 (2021).l Materials (Basel). 14, 1–18 (2021). ( ) ( ) 5. Mokrzycki, J. et al. Zeolite composite materials from fly ash : An assessment of physicochemical and adsorption properties. Materi- als (Basel) 16, 2142 (2023). 6. Zhang, S. et al. Uptake and translocation of polycyclic aromatic hydrocarbons (PAHs) and heavy metals by maize from soil irrigated with wastewater. Sci. Rep. 7, 1–11 (2017). p 57. PN-ISO 14235. Jakość gleby—Oznaczanie zawartości węgla organicznego przez utlenianie dwuchromianem (VI) w środowisku kwasu, Warsaw, Poland siarkowego (VI) (2003). g 58. PN-EN ISO 10390. Soil, Treated Biowaste and Sludge—Determination of pH (Polish Committee for Standardizations, 2022). Th l h d f d h d d h h l l hl d ( ) d h 9. Thalmann, A. Methods of dehydrogenase activity determination with triphenyl tetrazolium chloride (TTC). Landwirtsch. Forsch 21, 249–258 (1968). 0. www.nature.com/scientificreports/ Soil dehydrogenases as an indicator of contamination of the environment with petroleum products. Water Air Soil Pollut. 226, 372 (2015).f p p 0. Wu, S., Zhang, Z., Li, J., Wu, T. & Jiao, X. An experimental study of paddy drainage treatment by zeolite and effective microorgan- isms (EM). Sustain. 14, 1–11 (2022).f 41. Sahoo, S. et al. Tillage and N-source affect soil fertility, enzymatic activity, and crop yield in a maize–rice rotation system in the Indian Terai zone. Front. Environ. Sci. 10, 1–17 (2022). 42. Wolny-Koładka, K., Jarosz, R., Juda, M. & Mierzwa-Hersztek, M. Distinct changes in abundance of culturable microbial community and respiration activities in response to mineral–organic mixture application in contaminated soil. Sustain. 14, 15004 (2022). 3 W l K ł dk K l Eff f il li i f li b i l di d li i h i i E l g 3. Wolny-Koładka, K. et al. Effect of soil application of zeolite–carbon composite, leonardite and lignite on the microorganisms. Ecol Chem. Eng. S 29, 553–563 (2022).h g 44. Neina, D. The role of soil pH in plant nutrition and soil remediation. Appl. Environ. Soil Sci. 2019, 15–18 (2019). g 44. Neina, D. The role of soil pH in plant nutrition and soil remed h h h 5. Wang, Y., Li, B., Ma, Y., Yang, L. & Song, X. Characterizing the variation of dissolvable pahs in receiving water in a reclaimed water irrigation region. Water (Switzerland) 12, 1–14 (2020). g g 46. Zhu, D., Herbert, B. E., Schlautman, M. A., Carraway, E. R. & Hur, J. Cation–π bonding: A new perspective on the sorption of polycyclic aromatic hydrocarbons to mineral surfaces. J. Environ. Qual. 33, 1322 (2004). https://doi.org/10.1038/s41598-023-41429-2 Scientific Reports \| (2023) 13:14227 \| Author contributions Conceptualization: J.S.; Methodology: J.S., A.K.; Validation: J.S., A.K.; Formal analysis: J.S.; Investigation: J.S., R.J., J.M., Resources: M.M.-H., A.K.; Data Curation: J.S., J.M.; Writing—Original Draft: J.S.; Writing—Review & Editing: J.S., A.K., J.M., R.K., M.M.-H.; Visualization: J.S.; Supervision: M.M.-H.; Project administration: M.M.-H.; Funding acquisition: M.M.-H. Competing interests h p g The authors declare no competing interests. © The Author(s) 2023 Additional informationh Additional information Supplementary Information The online version contains supplementary material available at https://doi.org/ 10.1038/s41598-023-41429-2. Additional information Supplementary Information The online version contains supplementary material available at https://doi.org/ 10.1038/s41598-023-41429-2. Correspondence and requests for materials should be addressed to J.S. Correspondence and requests for materials should be addressed to J.S. Reprints and permissions information is available at www.nature.com/reprints. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 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/. 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/. https://doi.org/10.1038/s41598-023-41429-2 Scientific Reports \| (2023) 13:14227 \|
https://openalex.org/W2998804910	https://repositorio.iscte-iul.pt/bitstream/10071/20361/1/PPM_2019_04_Costa.pdf	English	null	The impact of artificial intelligence on commercial management	Problems and perspectives in management/Problems & perspectives in management	2,020	cc-by	8,422	“The impact of artificial intelligence on commercial management” AUTHORS Renato Costa https://orcid.org/0000-0001-9364-534X Álvaro Dias https://orcid.org/0000-0003-4074-1586 http://www.researcherid.com/rid/T-7293-2019 Leandro Pereira https://orcid.org/0000-0002-4920-0498 http://www.researcherid.com/rid/Z-4046-2019 José Santos https://orcid.org/0000-0003-3543-3570 André Capelo ARTICLE INFO Renato Costa, Álvaro Dias, Leandro Pereira, José Santos and André Capelo (2019). The impact of artificial intelligence on commercial management. Problems and Perspectives in Management, 17(4), 441-452. doi:10.21511/ppm.17(4).2019.36 DOI http://dx.doi.org/10.21511/ppm.17(4).2019.36 RELEASED ON Wednesday, 08 January 2020 RECEIVED ON Monday, 09 September 2019 ACCEPTED ON Tuesday, 03 December 2019 LICENSE This work is licensed under a Creative Commons Attribution 4.0 International License JOURNAL "Problems and Perspectives in Management" ISSN PRINT 1727-7051 ISSN ONLINE 1810-5467 PUBLISHER LLC “Consulting Publishing Company “Business Perspectives” FOUNDER LLC “Consulting Publishing Company “Business Perspectives” NUMBER OF REFERENCES 23 NUMBER OF FIGURES 1 NUMBER OF TABLES 6 © The author(s) 2020. This publication is an open access article. “The impact of artificial intelligence on commercial management” AUTHORS Renato Costa https://orcid.org/0000-0001-9364-534X Álvaro Dias https://orcid.org/0000-0003-4074-1586 http://www.researcherid.com/rid/T-7293-2019 Leandro Pereira https://orcid.org/0000-0002-4920-0498 http://www.researcherid.com/rid/Z-4046-2019 José Santos https://orcid.org/0000-0003-3543-3570 André Capelo ARTICLE INFO Renato Costa, Álvaro Dias, Leandro Pereira, José Santos and André Capelo (2019). The impact of artificial intelligence on commercial management. Problems and Perspectives in Management, 17(4), 441-452. doi:10.21511/ppm.17(4).2019.36 DOI http://dx.doi.org/10.21511/ppm.17(4).2019.36 RELEASED ON Wednesday, 08 January 2020 RECEIVED ON Monday, 09 September 2019 ACCEPTED ON Tuesday, 03 December 2019 LICENSE This work is licensed under a Creative Commons Attribution 4.0 International License JOURNAL "Problems and Perspectives in Management" ISSN PRINT 1727-7051 ISSN ONLINE 1810-5467 PUBLISHER LLC “Consulting Publishing Company “Business Perspectives” FOUNDER LLC “Consulting Publishing Company “Business Perspectives” NUMBER OF REFERENCES 23 NUMBER OF FIGURES 1 NUMBER OF TABLES 6 © The author(s) 2020. This publication is an open access article. “The impact of artificial intelligence on commercial management” © The author(s) 2020. This publication is an open access article. © The author(s) 2020. This publication is an open access article. businessperspectives.org businessperspectives.org businessperspectives.org Problems and Perspectives in Management, Volume 17, Issue 4, 2019 Renato Costa (Portugal), Álvaro Dias (Portugal), Leandro Pereira (Portugal), José Santos (Portugal), André Capelo (Portugal) Renato Costa (Portugal), Álvaro Dias (Portugal), Leandro Pereira (Portugal), José Santos (Portugal), André Capelo (Portugal) This is an Open Access article, distributed under the terms of the Creative Commons Attribution 4.0 International license, which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited. JEL Classification M15, M19, M30 JEL Classification M15, M19, M30 Abstract The essence of this research is to shed light on use and importance of artificial intel- ligence (AI) in commercial activity. As such, the objective of the present study is to understand the impact of AI tools on the development of business functions and if they can be affirmed as a means of help or as a substitute for these functions. In-depth interviews were conducted with 15 commercial managers from technological SMEs. The results indicate that all the participants use AI systems frequently, that these tools assist in developing of their functions, allowing having more time and better prepar- ing to solve the commercial problems. The findings also indicate that the tools used by commercials are still somewhat limited, and companies should focus on their training and development in AI, as well as the training of their commercials. Furthermore, the results show that firms intend to use the data collection and the analytical tool that en- able real-time response and customization according to customer needs. LLC “СPС “Business Perspectives” Hryhorii Skovoroda lane, 10, Sumy, 40022, Ukraine Received on: 9th of September, 2019 Accepted on: 3rd of December, 2019 Received on: 9th of September, 2019 Accepted on: 3rd of December, 2019 artificial intelligence, decision-making, analytical tools, consultancy, SMEs, CRM artificial intelligence, decision-making, analytical tools, consultancy, SMEs, CRM Keywords © Renato Costa, Álvaro Dias, Leandro Pereira, José Santos, André Capelo, 2019 © Renato Costa, Álvaro Dias, Leandro Pereira, José Santos, André Capelo, 2019 JEL Classification M15, M19, M30 INTRODUCTION Renato Costa, Ph.D., Professor, Higher Institute of Labour and Business Sciences - University Institute of Lisbon (ISCTE-IUL), Portugal. Renato Costa, Ph.D., Professor, Higher Institute of Labour and Business Sciences - University Institute of Lisbon (ISCTE-IUL), Portugal. In the current context, there is clear importance that AI has assumed in several areas, such as radiology (Paiva & Prevedello, 2017), cardiol- ogy (Mesquita, 2017), data analysis (Sharma, Mithas, & Kankanhalli, 2014), marketing and sales (Martínez-Lopez & Casillas, 2013). AI is seen as a powerful tool in the development of daily tasks in each of these areas, helping stakeholders make better decisions and optimiz- ing the response time of professionals. However, some questions arise: what kind of intelligent machines and what functions will they be able to perform, and above all, what place will the human being occupy when this happens? Is it just a means of help, or can AI become a sub- stitute for some functions in the future? As such, the main motivation for this article is to try to find some answers to the questions posed, as well as to understand this theme so in, and that is present through the most varied forms in our daily lives. Furthermore, this article aims to understand what type of AI systems’ are used in commercial area, and how these tools can be used for development of daily tasks. Álvaro Dias, Ph.D., Professor, Lusófona University, and Higher Institute of Labour and Business Sciences - University Institute of Lisbon (ISCTE-IUL), Portugal. Álvaro Dias, Ph.D., Professor, Lusófona University, and Higher Institute of Labour and Business Sciences - University Institute of Lisbon (ISCTE-IUL), Portugal. Leandro Pereira, Ph.D., Professor, Higher Institute of Labour and Business Sciences - University Institute of Lisbon (ISCTE-IUL), Portugal. André Capelo, Ph.D. Student, Researcher, Higher Institute of Labour and Business Sciences - University Institute of Lisbon (ISCTE-IUL), Portugal. It will also be one of the objectives of this research to understand how business professionals look at this type of AI system in the future. In other words, the intention is to understand if they value these systems, how they view them for the future of their function and the organization itself, and what perception they have about the way companies look at these AI tools. 1.1. AI concept and evolution According to Lustosa (2004), the term AI first appeared in 1956. The idea was that machines have the same human intelligence capabilities and could reproduce intelligent behavior. Gunkel (2012) stated that the concept of AI was first de- fined and characterized by Alan Turing in 1950. The term AI is related to the development of ex- pert systems to solve the problems in specific ar- eas, such as medicine, which has benefited greatly from AI developments (Mendes, 1997). The incor- poration of a natural language into the expert sys- tem is simple, and that obeys the semantic rules (Mendes, 1997). The author argues that AI and ANN are very use- ful techniques for the study of zootechnics, as they allow more effective by processing real the in- formation through simulations. However, Costa (2009) argues that the zootechnician cannot be abolished from the equation, as it is this subject who inserts the data into the machine and who oversees the entire process. Costa (2009) stated that AI includes the methods, tools, and systems for solving the problems that typically require the use of human intelligence. Other living systems also have intelligence and complex problem-solving capabilities, adapting to new realities. Thus, AI can also be considered as the ability to systematize the problem-solving by observing the complex systems existing in living nature. Sarfati (2016) states that AI is machine learning and believes that both AI and the collaborative economy will have a major impact on social rela- tionships. Without broadening the concept of col- laborative economics, as this is not the objective of this paper, it refers to a type of business that involves peer to peer or P2P exchanges, such as the platform. Sarfati (2016) refers to Tesla S model that, in addition to the constant innovations that are made in the automotive industry year after year, has incorporated an autopilot system fueled by AI algorithms. He also states that the system can learn and improve its performance after each trip and make safer decisions than a human being. According to Costa (2009), when living systems identify a problem, they react adaptively way by performing three complex tasks: pattern recogni- tion in images and sounds, language processing, and action planning and prediction. Costa (2009) also argues that heuristics is closely related to problem-solving. This is an Open Access article, distributed under the terms of the Creative Commons Attribution 4.0 International license, which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited. Considering the literature, more and more authors, such as Ahearne (2017), Nartissa (2012), Steenburgh and Ahearne (2012), and Palmatier 441 http://dx.doi.org/10.21511/ppm.17(4).2019.36 Problems and Perspectives in Management, Volume 17, Issue 4, 2019 (2017), argue that there must be a partnership between universities and firms, to solve everyday-life problems. And this is only likely to happen if companies provide real data that impact the studies per- formed. Another objective of this research will be to understand how professionals in the commercial area look at this type of partnership, value training and how, if exists, they seek knowledge to find the solutions in their daily lives. This paper is divided into two parts: theoretical and an empirical part. The first part is divided into con- cept and framework, studies conducted in the commercial area, and studies linking AI and the com- mercial area. The second part concerns the research methodology, the results, and the discussion. In the end, the conclusions are presented. 1. LITERATURE REVIEW be to integrate the heuristics by establishing the mathematical and algorithmic methods, enabling its use in a computer system. Still, according to Costa (2009), an artificial neural network (ANN) is a machine designed to model the way the brain performs its tasks. These resemble the brain be- cause knowledge comes from experience through a learning process, and the connecting forces be- tween neurons allow accumulating the acquired knowledge. http://dx.doi.org/10.21511/ppm.17(4).2019.36 1.1. AI concept and evolution This is because heuristics, which means discovery, is based on experience, ration- al ideas and rules, despite the common sense as- sociated with the concept. Thus, the author con- siders that one of the major challenges of AI will Sarfati (2016) also warns that AI is moving to- wards a future replacement of human beings in routine and repetitive tasks. According to Sarfati (2016), AI can assert itself as a substitute for less- 442 http://dx.doi.org/10.21511/ppm.17(4).2019.36 http://dx.doi.org/10.21511/ppm.17(4).2019.36 Problems and Perspectives in Management, Volume 17, Issue 4, 2019 skilled jobs by creating cheaper and more effi- cient automated solutions, confirming the above with the following example: “In the last 15 years, the US economy has grown 15% and the employ- ment level fell 1%.” Given a more recent perspec- tive, Mesquita (2017) says that AI is defined as a set of items (e.g., algorithms, robotics, and neural networks), which allows the software to have intel- ligence capabilities compared to that of a human being, including the ability to learn with minimal human interference. man user, while always monitoring the surround- ing environment, generating the information that can be further analyzed by other intelligent sys- tems or even humans. Lustosa (2004) states that the evolution that com- puters have been undergoing since they appeared was astounding, and questions what might hap- pen or become possible if AI progresses to the same extent and at the same pace. What kind of intelligent machines and what functions will they be able to perform, and most of all, what place will a human being occupy when this happens? The subject that has been approached so far can be found in many different forms and sciences. It can be evidenced that it is approached in various con- texts and occasions. Recent studies that have most motivated the enthusiasm of the scientific com- munity and medicine concern AI and the concept of machine learning, which means the ability of a system or computer to learn over time (Mesquita, 2017). Dornelles (2018) carried out a study that aimed to use ANN-based AI and genetic algorithms, to predict oat grain yield (oat sativa) and to optimize seeding density in the main succession systems of Southern Brazil. Dornelles (2018) concludes that the use of AI, through ANN techniques and ge- netic algorithms, allows efficiently simulating oat grain yield with better optimization of sowing density when compared to traditional polynomial regression models. http://dx.doi.org/10.21511/ppm.17(4).2019.36 1.1. AI concept and evolution Obermayer and Emanuel (2016) suggest that in a digital age, where information is directly collected from patients through examinations (e.g. radiolo- gy), the need for medics increases. Machine learn- ing contributes once again to automated deci- sion-making, thus adding the ability to collect the data without human intervention automatically. The bottom line is autonomous and more effective machine to make decisions better than humans. Within the study by the above author, other stud- ies with a similar basis have been developed. It can be said in a study conducted 14 years earlier, where it can be understood that the basis is simi- lar, since it addresses the theme of ANN (Poersch, 2004). Connectionism, which is based on a paral- lel distribution processor, seeks to design comput- ers inspired by the human brain. The number of neurons that make up the neural network is close- ly related to the learning algorithm designed to train the network. This neural network consists of three layers: an input neuron layer, an intermedi- ate neuron layer, and an output layer. The middle layer is responsible for the network learning pro- cess (Poersch, 2004). Unlike Obermayer and Emanuel (2016), medi- cine is considered an area where specialists are extremely difficult to replace (Mesquita, 2017). However, the use of these machines makes perfect sense and that it improves the doctor’s ability to make a better decision and with a significant re- duction in the analysis process (Mesquita, 2017). From a corporate perspective, Lustosa (2004) states that AI is already present in many compa- nies through data mining and is a fundamental part of the decision-making process because it generates strategic information not always recog- nized by human analysts. Poersch (2004) also states that cognition science is the study of intelligence and computational pro- cesses. According to neuroscience, it is the area of knowledge that studies the input, storage, pro- cessing, and retrieval of knowledge, both declara- tive and procedural, whether natural or comput- er simulated. Poersch (2004) argues that a neural network is constituted as a machine designed to simulate the way the brain performs a certain task or function. According to Lustosa (2004), AI is also present in network management systems, e-commerce, and distance education. 1.1. AI concept and evolution Intelligent agent technology allows negotiating between the system and the hu- 443 Problems and Perspectives in Management, Volume 17, Issue 4, 2019 Thus, for Paiva and Prevedello (2017), AI will bring professional changes for radiologists, just as it has changed some aspects of people’s lives, such as electricity, internet, thus improving people’s qual- ity of life. But it is not only in radiology that AI has had an impact, in medicine, this has also hap- pened, as has been the development of software to identify the malignant dermatological lesions. Lab exams are also analyzed almost automatical- ly. Paiva and Prevedello (2017) consider that these technological advances are mainly due to three factors: the abundance of existing data, the devel- opment of ANN, and reduced hardware cost. is access to quality business data, and the need to identify the issues that are truly impacting for the academic community and sales directors at the same time. According to Ahearne (2017) for decades, business schools have been conducting the investigations, mainly in the areas of finance and marketing, in order to improve the student education. However, Kumar (2017) says that research that is not based on evidence makes it difficult for students to ap- ply what is learned in the real world, due to its extremely theoretical nature. As suggested by Wetherbe and Eckhardt (2014), many business schools conduct a great deal of research, which generates a great deal of interest from the academ- ic community, but of little interest in the business world. Taking the example of medicine, which conducts research that can be applied in practice by health care professionals, much of the research carried out at management level lacks applicability in practice. If this happens, Ahearne (2017) consid- ers that companies may become more interested. Regarding the development of neural networks, they permit an increase in efficiency, recogniz- ing, in some studies, that the effectiveness of the machine surpassed the effectiveness of humans (Paiva & Prevedello, 2017). According to Paiva and Prevedello (2017), the im- pact of AI on the routine of radiologists must be progressive because there is the software that pro- vides the data that cannot be extracted from the images, but the importance of radiologists in in- tegrating the information obtained through the data from AI. 1.1. AI concept and evolution Lilien (2017) also states that to teach effectively, it is necessary to “tackle” real problems, have a high degree of expertise, access the data, and have re- sources to get started. Paiva and Prevedello (2017) also think that radi- ologists should integrate these techniques into their daily lives in order to provide better service to patients. Palmatier (2017) states that the most successful re- search centers have five key factors: strong leader- ship, production of relevant research, strong rela- tionships with firm directors, a diverse and well-co- ordinated team of students, researchers, theorists, and teachers, and a related academic community. For Nartissa (2012), most microenterprises are con- centrated on direct management and production, not on research. They do not reach their full poten- tial due to lack of knowledge about method, skills, and new trends in science and research. 1.2. Studies carried out in the commercial area Increasingly, companies are seeking to build close relationships with research centers and business schools. This trend is the result of research that has been done that has helped companies evolve the way business is done today, and it also leads to a natural evolution of theoretical knowledge (Ahearne, 2017; Nartissa, 2012; Steenburgh & Ahearne, 2012). Steenburgh and Ahearne (2018) presuppose un- derstanding how to sell new products by the prop- er training of salespeople, or massively betting on product demos. Steenburgh and Ahearne (2018) suggest that the most successful sellers are those who constantly want to learn because custom- ers want to know exactly the history of the prod- ucts they buy. Therefore, it is necessary to create a mindset of demand, research and development in corporate salespeople. Despite the advances made in the research con- ducted in the area of marketing, in the sales area, advances are scarcer (Ahearne, 2017). Most like- ly because sales executives wonder what the real impact sales studies can have. One of the barri- ers that most sales researchers have encountered 444 http://dx.doi.org/10.21511/ppm.17(4).2019.36 http://dx.doi.org/10.21511/ppm.17(4).2019.36 Problems and Perspectives in Management, Volume 17, Issue 4, 2019 Steenburgh and Ahearne (2018) state that the time spent in each customer is longer for a seller selling a new product than a seller who sells reg- ular goods. The authors also suggest that compa- nies should have a candidate selection system that includes an analysis of their skills and market knowledge. Another point is the training program that the company must provide to the seller of a new product, which should be based on the acqui- sition of new skills, as well as personal growth, as selling new products puts the sellers’ confidence to the test. Thyagarajan, & Seetharaman, 2003). Khatibi et al. (2003) also conclude that most of the compa- nies interviewed consider that e-commerce is a real competitive advantage and that competing companies that ignore this technological advance will eventually close down. The companies inter- viewed consider e-commerce to be the future, and it is a benefit to providing the information, spread- ing the company’s image, improving the business processes, and customer service. On the other hand, Sharma et al. 1.2. Studies carried out in the commercial area (2014), state that in recent years, interest in the subject of big da- ta and analytics has increased, by companies and researchers in the areas of AI and management, due to its potential to increase the organizational performance. According to Sharma et al. (2014), at the same time that automated AI systems process the data, they also generate a significant amount of information. This information can and should be used to make better decisions. Steenburgh and Ahearne (2018) say that the man- agers’ thinking must also change in order to have the same attitude of seeking knowledge, as well as applying and trying new strategies and approach- es to the market. In other words, basically putting themselves on the line and putting the seller on the line, not just blaming the product when it does not fit the market at some point. Finally, Steenburgh and Ahearne (2018) conclude that the companies questioned admit that invest- ing in research and development is not sufficient, there must be a commitment to commercializa- tion (e.g., effective seller selection, training and coaching processes), and not only in launching the innovative ideas and products. Managers and analysts nowadays have at their disposal a huge amount of analytical tools such as data analysis, data mining, and data visualiza- tion. However, in order to be able to process the data and gather the information from these sys- tems, analysts and managers are critical because they must constantly enter the data into the sys- tem so that it can evolve at the informational level (Sharma et al., 2014). http://dx.doi.org/10.21511/ppm.17(4).2019.36 1.3. AI trends in the commercial area Zoltners, Sinha, and Lorimer (2018) address- ing the issue of decreasing sales forces, state that many industries have had to decrease their sales force. They indicate several reasons for these cuts, such as changes in a dynamic market, as well as consumer needs, new sales channels, and market growth slowdown; changing company strategy, including new priority and more specialized prod- ucts/markets; the desire to increase productivity by eliminating unproductive and inefficient sales times, reducing the cost of sales. A sales force de- crease is always a stressful and cumbersome event (Zoltners et al., 2018). These cuts do not always happen for the right reasons and in the best way, leading to the loss of key customers and a decrease in sales team performance. Only in this way, as mentioned above, is informa- tion about the business processed, which allows increasing the organizational performance, as well as operational level, in the employees’ daily tasks (Sharma et al., 2014). Its main objective is to optimize the work of each one, either in a man- agement function (e.g., costs and income), which is more of analysis and planning, or in a more op- erational function, such as the salesperson. Sharma et al. (2014) affirm that the fact that these systems produce the information alone is not enough to improve the performance. Dealing with this type of systems needs proper training and know-how to interpret and value the informa- tion obtained to make good decisions. Therefore, the authors state that the use of these analysis systems depends largely on each person. Sharma et al. (2014) state that the use of business intelli- Furthermore, the internet has transformed the tra- ditional marketing model and its system (Khatibi, 445 Problems and Perspectives in Management, Volume 17, Issue 4, 2019 ence sample, constituted according to the availa- bility and accessibility of the elements addressed (Carmo & Ferreira, 1998). Fifteen interviews were conducted with commercials from various com- panies. However, it is important to note that these were intentional to constitute the sample since the participants who best represented the investigat- ed the phenomenon in terms of knowledge were selected. Although the response rate is consid- ered satisfactory, the findings of this investigation should be carefully taken from a small sample. 1.3. AI trends in the commercial area Thus, given the impossibility of making generali- zations, this factor presents itself as the main limi- tation of this investigation, with the due exception that generalization was not a primary objective either. gence enables the companies to better understand their business problems, as well as the market in which they operate, identifying the opportunities through analysis of current operations, and which may lead to new forms of financial return or sav- ings costs. Sharma et al. (2014) conclude that analysts and managers should pay particular attention to infor- mation that creates value while ignoring less rele- vant ones. By value creation, the authors mean in- formation that is important to make strategic and operational, day-to-day decisions. 2. METHODOLOGY This research was based on a pragmatic or induc- tive character, and a non-probabilistic conveni- Regarding the methodology applied, this research was based on a set of primary sources, from the Figure 1. Categorization and coding of the interview corpus for qualitative analysis Generic categories Subcategories Main category 1.1. AI current trends in the commercial area 1.2. E-commerce, data analytics and business intelligence 1.1.1. Perception about the impact of the AI in the process of commercial decision 1.2.1. AI tools might replace the commercials 1.2.2. AI tools as competitive advantage 1.2.4. AI tools as used by commercials and commercial managers 1.2.3. How companies look at AI tools from the commercial’s perspective 1. The impact of the AI in the commercial area Generic categories Subcategories Main category 1.1.1. Perception about the impact of the AI in the process of commercial decision 1.1. AI current trends in the commercial area 1.2.1. AI tools might replace the commercials 1. The impact of the AI in the commercial area 1.2.2. AI tools as competitive advantage 1.2. E-commerce, data analytics and business intelligence 1.2.3. How companies look at AI tools from the commercial’s perspective 1.2.4. AI tools as used by commercials and commercial managers Figure 1. Categorization and coding of the interview corpus for qualitative analysis 446 http://dx.doi.org/10.21511/ppm.17(4).2019.36 Problems and Perspectives in Management, Volume 17, Issue 4, 2019 Table 1. Framework of the research objectives and research questions Objective Research questions Framework Analyze the role of AI in the commercial area, its impact on the performance of commercial employees, and in the decision-making process Q1 – What is the impact of AI tools (e.g., data analysis and processing systems) on the daily commercial decision-making process? Martinez and Casillas (2013), Sharma et al. (2014), Lustosa (2004), Mesquita (2017), Dornelles (2018) Verify which AI tools are used by commercials, and to understand the perception about the use of these tools Q2 – Are AI tools a substitute for business professionals, or will they help in the development of their functions? Martinez and Casillas (2013), Sharma et al. (2014), Zoltners et al. (2018), Khatibi et al. (2003), Costa (2009), Sarfati (2016), Lustosa (2004) Q3 – How do companies view these types of AI tools? Sharma et al. (2014), Costa (2009), Sarfati (2016), Obermayer and Emanuel (2016), Mesquita (2017), Paiva and Prevedello (2017) Q4 – Are AI tools seen as a competitive advantage? 2. METHODOLOGY Martinez and Casillas (2013), Sharma et al. (2014), Zoltners et al. (2018), Khatibi et al. (2003) Q5 – What type of AI tools might be useful for commercials and business managers to use? Nartissa (2012), Steenburgh and Ahearne (2018), Zoltners et al. (2018) Table 1. Framework of the research objectives and research questions At the beginning of the interviews, all the varia- bles were collected that could statistically charac- terize the sample, especially regarding its demog- raphy, age group, gender, qualifications, and aca- demic background, in order to understand the ex- isting sample regarding its nature and dimension of experience and professional knowledge (Freitas, 2013). Finally, we proceeded to the content analy- sis of the answers in order to obtain the data that later allowed to draw theoretical and empirical conclusions. application of surveys on semi-structured in- terviews to commercials from various compa- nies, and from secondary sources, through bib- liographic research and information processing, comprised in the systematized study developed in books, magazines, scientific articles, and electron- ic networks. Regarding the qualitative analysis technique used to interpret the data reproduced in the interviews, it was translated into a content analysis, trying to relate the semantic (significant) structures with the sociological (meaning) structures, in order to articulate the surface of the texts with the factors that determine their characteristics (psychosocial variables, cultural context and context, process- es, and message reproduction) (Duriau, Reger, & Pfarrer, 2007). Figure 1 details the categorization and codification of the in-depth interview that gave rise to the qualitative analysis. From the total number of interviewees, 20% were men and 80% women. Regarding the academ- ic qualifications, five respondents (33%) have no higher education, 6 (40%) has a BA degree, 5 (33%) have a MsC degree. Regarding the period in their company, the average years is 3.8 years, and the average time of the interviewees in the commer- cial area is 6.5 years. This distribution is represent- ative of the Portuguese labor market on the com- mercial area. According to the ESC (2014) report, the proportions in gender and education are very close to the sample. 2. METHODOLOGY From the voice reproductions, the process, sys- tematization and expression of message content, promoted by content analysis, was organized in accordance with the three chronological poles of Bardin (1977), in other words, a first phase, where ideas were organized and systematized, and a sec- ond phase, in which all the material was explored, and the treatment and its interpretations of the re- sults obtained were performed at the end. http://dx.doi.org/10.21511/ppm.17(4).2019.36 3.1. Perception of the impact of AI on commercial decision-making By analyzing Table 2, two types of impacts are con- sidered by respondents relating to the role that AI tools have in the decision-making of commercials. These findings are aligned with Lustosa (2004), Mosque (2017), and Dornelles (2018) regarding In Table 1, on the other hand, it is possible to analyze the relationship between the study ob- jectives, the research questions elaborated, and their connection with the literature review pre- viously made. 447 Problems and Perspectives in Management, Volume 17, Issue 4, 2019 the facilitation of AI systems in decision-making. the facilitation of AI systems in decision-making. placement by AI tools in certain functions. In this particular case, the respondents consider that there are social skills in the commercial function and techniques (e.g., negotiation) that are difficult to reproduce by an AI system. Hence the vast ma- jority of respondents consider that hardly an AI system can replace a commercial. Most respondents find that the AI tools they use in their daily lives help them negotiate better with their customers and, therefore, make better deci- sions in line with the argument of Martinez and Casillas (2013) considering AI tools to produce the automated decisions. On the other hand, we can see that 2 respondents state that, in some areas and industries where work is more routine, substitution may occur, while in others, it will remain as a means of help. This per- spective partially supports the view of Obermayer and Emanuel (2016) concerning the replacement of more automated functions. Although they do not mention it directly, when respondents talk about a possible replacement in some industries, this will eventually lead to a decrease in the sales force. In this case, the theory of Zoltners et al. (2018), when it states that the emergence of AI sys- tems is generating the increasing interest and that this has led to the decrease of some sales forces. During the interviews, respondents mentioned the quality and quantity of information coming from the tools they use, as we can see in Table 2. However, giving more value to the quality of in- formation, rather than quantity. One can also ob- serve that four of the respondents affirm that AI tools allow them to have access to comparative market data and define the action plans, thus giv- ing them a greater ability to argue with the client. 3.1. Perception of the impact of AI on commercial decision-making These data allow partially confirming Sharma et al.’s (2014) argument, where AI tools generate a quantity and quality of information that has a great impact on the performance of companies and their employees. Table 3. Commercial perspective about the future impact of IA tools in the commercial area Table 2. Impact AI tools have on commercial Table 2. Impact AI tools have on commercial work work Key ideas Frequency Respondents AI can help on routine tasks 1 1 Help in commercial decision-making 11 2, 3, 6 to 15 The amount of information coming from AI tools facilitates client negotiation 8 2, 4, 5, 6, 7, 8, 9, 11 AI tools allows comparing market data, with greater argumentative capacity 4 4, 8, 9, 10 AI tools have been changing the functions of commercials 4 5, 9, 12, 14 Key ideas Frequency Respondents Key ideas Frequency Respondents AI tools are and will continue to be a help in developing business functions 13 1, 2, 3, 5 to 15 It is difficult for an AI tool to perform trading functions 3 1, 7, 8 The commercial function still has a large weight in the human relationship with the customer 8 2, 5, 6, 8, 10, 11, 13, 14 In some industries where work is more routine, AI tools may become a substitute, in others where it is not, AI tools will be an aid 2 4, 12 http://dx.doi.org/10.21511/ppm.17(4).2019.36 3.4. How companies look at AI tools from a commercial perspective Looking at Table 5, all respondents consider that there is a clear advantage in the use of AI tools by commercials and companies. These data support the theory of Martinez and Casillas (2013), Sharma et al. (2014), Zoltners et al. (2018), Khatibi et al. (2003), when they state that the use of these tools was a great advance for companies. Reinforcing the point above, a third of respond- ents say that AI tools are the future and that if companies that want to evolve and stay on trend, have to keep up with those same trends, and in- creasingly use these tools. A third of respondents also say that in more routine roles, the company may consider replacing, but even with a more au- tonomous tool, human supervision will be needed. Respondents consider that the use of these tools to- day should be a priority in any company, as it assists the commercial in the development of their daily tasks, either in the information collection and deci- sion-making, as Martinez and Casillas (2013) argue. Respondents also value information allowing them to access more and better information. Sharma et al.’s (2014) perspective about AI tools and their tech- nological advances, transforming employee’s roles in companies is supported. In this case, the respondents believe that a partial substitution may happen, meeting what was veri- fied in the literature review in the articles by Costa (2009), Sarfati (2016), Mosque (2017), Lustosa (2004), Paiva and Prevedello (2017) who consider that they concluded that professionals in other ar- eas could not be replaced by an AI system, as they still need human supervision. Hierarchically, in the table we can also see that the most cited justification by respondents to sup- port their response, is that AI tools allow more re- al-time access to customer information. The sec- ond most mentioned justification has to do with the possibility that AI tools make it possible to make a more conscious decision, given the data presented. And lastly, three of the respondents jus- tify that AI tools are a competitive advantage over other companies that do not use them, stating that there is a clear advantage for companies that fol- low technology development. As for the e-commerce issue, it was never men- tioned in this part of the interview by the respond- ents. Regarding the point defended by Martinez and Casillas (2013), Sharma et al. 3.3. AI tools as competitive advantage Looking at Table 3, it can be seen that 13 respond- ents consider that AI tools will continue to be a means of assisting in the development of daily commercial tasks. To justify this point of view, 8 respondents also state that the commercial func- tion still has a large weight, regarding the human relationship that is created with the customer. About 53% of respondents (Table 4) consider that AI tools may replace commercial functions that will depend on business-to-business strat- egy, vision, and profitability. On the other hand, 53% of respondents also mention that companies view AI tools as a means of assisting in the devel- opment of daily tasks, as advocated by Lustosa (2004), Mosque (2017), Dornelles (2018). On this vein, respondents show some reticence regarding this theme, when asked about the possible view This perspective reinforces what we are told in the literature by Costa (2009) and Paiva and Prevedello (2017), regarding the difficulty of re- 448 http://dx.doi.org/10.21511/ppm.17(4).2019.36 Problems and Perspectives in Management, Volume 17, Issue 4, 2019 Table 4. Companies perspective, according to commercials, about the future impact of AI tools on business Key ideas Frequency Respondents Very helpful, due to the difficulty of AI in negotiation 4 1, 7, 8, 14 There may be more automated AI tools (under the supervision of a human-being) 5 1, 6, 7, 11, 14 It may depend on B2B strategy, vision, and profitability 8 2, 4, 8, 9, 10, 11, 12, 15 AI as a mean for the development of business tasks 8 2, 4, 5, 6, 8, 9, 13, 14 Companies that want to evolve will have to follow the trend as traditional methods are falling further behind 5 3, 5, 8, 9, 15 Some companies pay little attention to AI 2 6, 12 ble 4. Companies perspective, according to commercials, about the future impact of AI tools o usiness depending on their business vision and company profitability. that the company has about its functions. On the one hand, they consider that their function is ir- replaceable; however, they admit that the perma- nence or not of their function in the future may have to do with the company’s view of its business and profitability. http://dx.doi.org/10.21511/ppm.17(4).2019.36 3.5. AI tools used by commercials and commercial managers Considering the results, it was possible to veri- fy through the literature review the importance that AI has assumed in several areas. However, the studies that have been developed in the commercial area, including the AI theme, are very incipient. Looking at Table 6, it can be seen that most of the respondents, about one-third, do not know or are unaware of what type of AI tools could be useful for their daily tasks. These results suggest that firms’ should invest in AI training. Nartissa (2012) talks about research and training partnerships to improve the employees’ performance. According to Steenburgh and Ahearne (2018), the best sellers are the ones who research the most and invest the most in training. The objective of this work was, therefore, to make a contribution, both professional and ac- ademic, where commercial area and AI could be integrated. And so, try to understand how AI influences the commercial area. Thus, AI is present and influences the daily life of the com- mercials that were interviewed, either in their decision-making or in the data collection, which eventually are relevant to their decision-making. On the other hand, another group of 5 respond- ents indicated that it would be interesting for their daily life to have a tool that would allow data col- lection and analysis, while, in real time, generate a solution that best suits their customer. In other words, part of the respondents considers that the tools they use can still be improved and evolve to another level. In this case, the companies where these employees work should invest a little more in the area of AI and try to provide other solutions to their commercials. Based on the question of Lustosa’s (2004) article which is, what kind of intelligent machines and what functions will they be able to perform, and above all what place will the human being occu- py when this happens? Is it just a means of help, or can AI become a substitute for some func- tions in the future? The table also shows that at least three respond- ents do not use any type of Customer Relationship Management (CRM) tool or use a poorly devel- oped tool. Being CRM, as stated by Zoltners et al. (2018), an essential tool for companies and em- ployees to have access to all customer information. 3.4. How companies look at AI tools from a commercial perspective (2014), Zoltners et al. (2018), Khatibi et al. (2003), Costa (2009), and Sarfati (2016), it is found that respondents consid- er their function difficult to replace by AI systems because they consider the commercial function still highly dependent on customer relationship. However, respondents feel that companies, de- pending on the industry in which they operate, may think of a partial or even total replacement, 449 Problems and Perspectives in Management, Volume 17, Issue 4, 2019 Table 5. Commercial perspective on competitive advantage in using AI tools Key ideas Frequency Respondents AI tools develop the competitive advantage 15 All respondents More real-time access to customer information. 7 1, 5, 7, 8, 10, 13, 14 It allows a more conscious decision-making, given the data presented. 4 3, 4, 7, 10 Advantage of keeping up with technological development 3 8, 12, 15 3.5. AI tools used by commercials and commercial managers These turned out to be the basic questions of this research. The interviewees, in general, con- sider that AI enhances the competitive advan- tage, however, they consider that the relation- ship with the customer is still very important in Table 6. Commercial perspective on the most beneficial tools in the future Table 6. Commercial perspective on the most beneficial tools in the future Key ideas Frequency Respondents Use of a collection and analysis tool integrated with other areas 2 1, 7 Using a data collection and analysis tool that enables real-time solution to be tailored to customer needs 5 2, 7, 9, 12, 15 Increasingly gradual use of e-commerce 2 2, 12 Use of a tool that allows to automating processes 1 3 CRM tool used for real customer management 3 4, 5, 10 Does not know or does not have the knowledge 5 6, 8, 11, 13, 14 p pii Key ideas Frequency Respondents Use of a collection and analysis tool integrated with other areas 2 1, 7 Using a data collection and analysis tool that enables real-time solution to be tailored to customer needs 5 2, 7, 9, 12, 15 Increasingly gradual use of e-commerce 2 2, 12 Use of a tool that allows to automating processes 1 3 CRM tool used for real customer management 3 4, 5, 10 Does not know or does not have the knowledge 5 6, 8, 11, 13, 14 450 Problems and Perspectives in Management, Volume 17, Issue 4, 2019 the negotiation process and difficult to replace by AI. and Palmatier (2017), they argue that there must be a partnership between universities and firms, promoting the knowledge transfer, as well as a search for knowledge is important for the evolu- tion of the professionals. Considering Ahearne (2017), Nartissa (2012), Steenburgh and Ahearne (2012), Kumar (2017), CONCLUSION Based on the results, some conclusions can be considered. First, commercials use AI tools, but their knowledge on this subject is still scarce, which can give us two indicators, or lack of training by com- panies in the area of AI, or lack of investment and training in the commercial area. In either case, the solution that seems most valid will be a strong investment from the companies or even from the profes- sionals if their companies do not have this opportunity, both in the commercial area and in the AI area. Second, in the commercial area, the use of tools such as e-commerce or data analytics systems has in- creased in recent years (Sharma et al., 2014). However, results also showed that firms are investing on data analytics systems, and reducing e-commerce efforts, which can generate an important return for companies, for non-key customers. Third, it was interesting to realize that very few commercials mentioned the fact that e-commerce could be a substitute for commercial functions and could lead to the extinction of some jobs. Most of the respondents’ discourse was directed at the importance that data analysis tools have in their daily lives. Fourth, the participants do not believe that a machine can produce the same work they do, with the same effectiveness and efficiency, especially with regard to negotiation techniques or customer relation- ship. However, there is a small group of respondents who believe that the AI tools could lead to a total or partial replacement of professionals in this area. This, according to the respondents, in less qualified and more automated functions, and depending on the company and the business area in which they are, as also defended by Sarfati (2016) who argues that AI can assert itself as a substitute in less skilled jobs by creating cheaper and more efficient automated solutions. Finally, it was also found out that AI systems have a great impact on the professional of the commercial area, because, as it was mentioned, it allows access to a quantity and quality of information in real time, which ultimately better prepares the professionals working in this area regarding negotiation processes with their clients, as defended by Paiva and Prevedello (2017) in their article on the impact that AI has on the daily work of radiologists. http://dx.doi.org/10.21511/ppm.17(4).2019.36 2. Costa, E. (2009). Inteligência artificial aplicada à Zootecnia. Revista Brasileira de Zootecnia, 28, 390-396. Retrieved from http://www. dx.doi.org/10.1590/1807-1929/ agriambi.v22n3p183-188 scielo.br/scielo.php?script=sci_ abstract&pid=S1516- 35982009001300038&lng=en&nr m=iso&tlng=pt 3. Dornelles, E. F., Kraisig, A. R., Silva, J. A G., Sawicki, S., Roos- Frantz1, F., & Carbonera, R. (2018). Artificial intelligence in seeding density optimization and yield simulation for oat. Revista Brasileira de Engenharia Agrícola e Ambiental, 22(3), 183-188. http:// dx.doi.org/10.1590/1807-1929/ agriambi.v22n3p183-188 4. Duriau, V. J., Reger, R. K., & Pfarrer, M. D. (2007). A content analysis of the content analysis literature in organization studies. Research themes, data sources, and methodological refinements. Organizational research methods, 10(1), 5-34. https://doi.org /10.1177%2F1094428106289252 REFERENCES 1. Ahearne, M. (2017). Research centers, business schools, and the world of sales. Journal of the Acad- emy of Marketing Science, 45(4), 461-464. Retrieved from https:// link.springer.com/article/10.1007/ s11747-017-0536-7 2. Costa, E. (2009). Inteligência artificial aplicada à Zootecnia. Revista Brasileira de Zootecnia, 28, 390-396. Retrieved from http://www. scielo.br/scielo.php?script=sci_ abstract&pid=S1516- 35982009001300038&lng=en&nr m=iso&tlng=pt 3. Dornelles, E. F., Kraisig, A. R., Silva, J. A G., Sawicki, S., Roos- Frantz1, F., & Carbonera, R. (2018). Artificial intelligence in seeding density optimization and yield simulation for oat. Revista Brasileira de Engenharia Agrícola e Ambiental, 22(3), 183-188. http:// dx.doi.org/10.1590/1807-1929/ agriambi.v22n3p183-188 4. Duriau, V. J., Reger, R. K., & Pfarrer, M. D. (2007). A content analysis of the content analysis literature in organization studies. Research themes, data sources, and methodological refinements. Organizational research methods, 10(1), 5-34. https://doi.org /10.1177%2F1094428106289252 1. Ahearne, M. (2017). Research centers, business schools, and the world of sales. Journal of the Acad- emy of Marketing Science, 45(4), 461-464. Retrieved from https:// link.springer.com/article/10.1007/ s11747-017-0536-7 1. Ahearne, M. (2017). Research centers, business schools, and the world of sales. Journal of the Acad- emy of Marketing Science, 45(4), 461-464. Retrieved from https:// link.springer.com/article/10.1007/ s11747-017-0536-7 2. Costa, E. (2009). Inteligência artificial aplicada à Zootecnia. Revista Brasileira de Zootecnia, 28, 390-396. Retrieved from http://www. 2. Costa, E. (2009). Inteligência artificial aplicada à Zootecnia. Revista Brasileira de Zootecnia, 28, 390-396. Retrieved from http://www. 451 http://dx.doi.org/10.21511/ppm.17(4).2019.36 Problems and Perspectives in Management, Volume 17, Issue 4, 2019 5. ESC (2014). European Sector Skills Council Commerce Report 2014. European Skills Council. Retrieved from https://www.eu- rocommerce.eu/media/113775/ European%20Commerce%20 Skills%20Council%20Report%20 2014.pdf 23(4), 433-441. Retrieved from https://link.springer.com/ar- ticle/10.1057/ejis.2014.17 Intelig%C3%AAncia-Artificial- e-Machine-Learning-em- %C2%AD-Uma-Mesquita/de76b1ff- 87b1ac90ded12329c58ad69e- aff0f772 Intelig%C3%AAncia-Artificial- e-Machine-Learning-em- %C2%AD-Uma-Mesquita/de76b1ff- 87b1ac90ded12329c58ad69e- aff0f772 21. Steenburgh, T., & Ahearne, M. (2018). How to Sell New Products Focus on learning, not performance. Harvard Business Review, 96(6), 92-101. 14. Nartissa, I. (2012). Openness and knowledge as leading tenden- cies in development of micro enterprises. Economics and Management, 17(4), 1579-1584. https://doi.org/10.5755/j01. em.17.4.3032 6. Gunkel, D. (2017). Comunicação e inteligência artificial: novos desafios e oportunidades para a pesquisa em comunicação. Galaxia, 34, 5-19. http:// dx.doi.org/10.1590/1982- 2554201730816 22. Wetherbe, J., & Eckhardt, J. (2014). Making business school research more relevant. Harvard Business Review. Retrieved from https://hbr.org/2014/12/making- business-school-research-more- relevant 15. Obermeyer, Z., & Emanuel, E. J. (2016). Predicting the future – big data, machine learning, and clinical medicine. The New England Journal of Medicine, 375(13), 1216. https://doi. org/10.1056/NEJMp1606181 7. Khatibi, A., Thyagarajan, V., & Seetharaman, A. (2003). REFERENCES E- commerce in Malaysia: perceived benefits and barriers. Interfaces, 28(3), 77-82. https://doi.org/ 10.1177%2F0256090920030307 23. Zoltners, A. A., Sinha, P., & Lorimer, S. E. (2018). How to Downsize Your Sales Force. Harvard Business Review, 4. Retrieved from https://hbr. org/2018/04/how-to-downsize- your-sales-force 16. Paiva, O., & Prevedello, L. (2017). O potencial impacto da inteligência artificial na radiologia. Ra- diol Bras, 50(5), 5-6. http:// dx.doi.org/10.1590/0100- 3984.2017.50.5e1 8. Kumar, V. (2017). The role of university research centers in promoting research. Journal of the Academy of Market- ing Science, 45(4). https://doi. org/10.1007/s11747-016-0496-3 9. Lilien, G. (2017). Perspectives on university research centers: Lessons from the ISBM. Journal of the Academy of Market- ing Science, 45(4). https://doi. org/10.1007/s11747-017-0537-6 17. Palmatier, R. W. (2017). Market- ing research centers: community, productivity, and relevance. Jour- nal of the Academy of Marketing Science, 45(4), 465-466. https:// doi.org/10.1007/s11747-017- 0538-5 10. Lustosa, V. (2004). O Estado da Arte em Inteligência Artificial. Revista Digital da CVA – Ricesu, 2(8). 18. Poersch, J. M. (2004). Simulações conexionistas: a inteligência artificial moderna. Linguagem em (Dis) curso, 4(2), 441-458. Retrieved from http://www. portaldeperiodicos.unisul.br/ index.php/Linguagem_Discurso/ article/view/273 11. Martínez-López, F., & Casillas, J. (2013). Artificial intelligence- based systems applied in indus- trial marketing: an historical overview, current and future insights. Industrial Marketing Management, 42, 489-495. https://doi.org/10.1016/j.indmar- man.2013.03.001 19. Sarfati, G. (2016). Prepare-se para a revolução: economia colaborativa e inteligência artificial. GV Executivo, 15(1), 25-28. Retrieved from https:// rae.fgv.br/gv-executivo/vol15- num1-2016/prepare-se-para-rev- olucao-economia-colaborativa- inteligencia-artificial 12. Mendes, R. (1997). Inteligência artificial: sistemas especialistas no gerenciamento da informação. Brasilia, 26(1). http://dx.doi.org/10.1590/S0100- 19651997000100006 20. Sharma, R., Mithas, S., & Kank- anhalli, A. (2014). Transforming decision-making processes: a re- search agenda for understanding the impact of business analyt- ics on organisations. European Journal of Information Systems, 13. Mesquita, C. (2017). Inteligência Artificial e Machine Learning em Cardiologia – Uma Mudança de Paradigma. International Journal of Cardiovascular Sciences, 30(3), 187-188. Retrieved from https:// www.semanticscholar.org/paper/ 452 http://dx.doi.org/10.21511/ppm.17(4).2019.36 http://dx.doi.org/10.21511/ppm.17(4).2019.36
https://openalex.org/W1183216895	https://ojs.ihar.edu.pl/index.php/biul/article/download/1231/1099	Polish	null	Zmienność wielkości szkód wyrządzanych przez dziki w zróżnicowanych strukturach agrocenoz	Biuletyn Instytutu Hodowli i Aklimatyzacji Roślin	2,010	cc-by-sa	4,186	DOI: 10.37317/biul-2010-0042 DOI: 10.37317/biul-2010-0042 NR 256 BIULETYN INSTYTUTU HODOWLI I AKLIMATYZACJI ROŚLIN MARIAN FLIS Zakład Ekologii i Hodowli Zwierząt Łownych Uniwersytet Przyrodniczy w Lublinie Differentiation of damages caused by wild boars under different agrocenose structures Celem badań była analiza wielkości i rozkładu przestrzennego oraz czasowego szkód wyrządzanych przez dziki, w graniczących ze sobą dwóch obwodach łowieckich charakteryzujących się zróżnicowaną strukturą agrocenoz, w odstępie 6 lat. Obydwa obwody łowieckie położone są w rejonie Wyżyny Lubelskiej i dzierżawione są przez to samo koło łowieckie, co sprawia, że zabiegi związane z prowadzeniem gospodarki łowieckiej są zbliżone. W ocenianym okresie na terenie jednego obwodu łowieckiego zarówno liczba, jak i areał szkód ogółem utrzymywały się na tym samym poziomie, zaś na terenie drugiego z obwodów stanowiących teren badań, liczba szkód wzrosła w tym samym okresie blisko 6-krotnie, przy jednoczesnym 3,5-krotnym wzroście powierzchni szkód. W okresie badań, nastąpił blisko 1,5-krotny wzrost liczebności dzików oraz kilkukrotny wzrost eksploatacji łowieckiej tego gatunku. Zróżnicowanie występowania szkód w cyklu rocznym w poszczególnych rodzajach upraw wskazuje na ścisłe ich powiązanie z cyklem rozwojowym roślin. W uprawach zbożowych szkody występowały zwłaszcza w okresie wiosennym oraz tuż przed zbiorem, co można bezpośrednio powiązać z dostępnością w tych okresach ziarniaków, zbóż w postaci materiału siewnego oraz dojrzewających kłosów. Podobna sytuacja występowała w uprawach kukurydzy, w których największe nasilenie szkód wystąpiło w maju, tuż po siewie tego gatunku oraz w sierpniu, wrześniu i październiku, czyli w okresie osiągania dojrzałości ziarniaków u tego gatunku. W uprawach ziemniaków najwięcej szkód, niezależnie od terenu badań, wystąpiło w marcu i kwietniu, a w kolejnych miesiącach wielkość szkód poczynionych przez dziki w ocenianych obwodach łowieckich była różna. Najbardziej preferowanymi gatunkami roślin przez zwierzynę były kukurydza oraz ziemniaki, zaś najmniej uprawy zbożowe. Słowa kluczowe: dzik, preferencje żerowe, szkoda łowiecka, uprawy rolnicze Słowa kluczowe: dzik, preferencje żerowe, szkoda łowiecka, uprawy rolnicze The survey was aimed at analyzing the extent as well as spatial and time distribution of damages caused by wild boars at a six-year interval in two adjacent hunting areas characterized by various agrocenose structures. The two hunting areas are localized in the Lublin Upland and are leased by the same hunting society, which makes similar the operations referring to the hunting management. In one of the hunting areas both the number and area of damages done by wild boars remained at the same level over the period of survey, whereas in the other area almost 6-fold and 3.5-fold increase in these parameters, respectively, was observed. Over the survey period, the wild boar population rose one and 193 Marian Flis half times, and hunting exploitation of this species increased several times. Differentiation of damages in particular crops within an annual cycle indicates their close association with stages of plant development. The damages to cereal crops were mostly done by wild boars in spring and just before harvest, which can be directly attributed to the availability of kernels, cereal seeding material and maturing ears. A similar situation was observed in maize plantations, where the greatest intensity of damages was recorded in May (just after sowing), and then in August, September and October, when maize kernels reach physiological maturity. In potato fields, the damages caused by wild boars in March and April were at high level in the two hunting areas, whereas those done in the succeeding months varied in damage size between the surveyed areas. The prey preference index values indicate that wild boars more often searched for food in maize and potato plantations than in cereal crops. Key words: agricultural cultivations, game damage, prey preference, wild boar MATERIAŁ I METODY Analizę przeprowadzono w oparciu o badania dotyczące wielkości szkód wyrządzonych przez dzikie zwierzęta na terenie dwóch obwodów łowieckich, dzierżawionych przez Koło Łowieckie nr 155 „Sokół” w Stawach. W ocenie uwzględniono również zmiany liczebności, a tym samym i zagęszczenia dzików jak również poziom łowieckiej eksploatacji populacji tego gatunku. Badania prowadzono w sezonach łowieckich 2003/2004–2008/2009. W obwodzie łowieckim nr 136 o powierzchni 5000 hektarów, powierzchnia leśna stanowi 26,6%, a tym samym w kategoryzacji obwód ten ujmowany jest jako polny. Drugi obwód stanowiący teren badań (nr 137), to obwód o charakterze leśnym, gdyż na ogólną powierzchnię 7000 hektarów, lasy stanowią w nim 57% powierzchni. Miejsce występowania poszczególnych uszkodzeń roślin w uprawach polowych, zgłaszane było dzierżawcy obwodów, przez posiadaczy uszkodzonych upraw. Przedstawiciele dzierżawcy dokonywali szacowania wstępnego oraz ostatecznego powstałych szkód celem ustalenia wielkości przysługujących z tego tytułu odszkodowań (Ustawa Prawo łowieckie, 1995; Rozp. Min. Środ. z 15 lipca 2002; Flis, 2008). Podczas ostatecznego szacowania każdej szkody, celem precyzyjnego określenia zniszczeń ustalana jest tzw. powierzchnia zredukowana szkody, czyli teoretycznie wyliczona powierzchnia, na której rośliny zniszczone są w 100% (szkoda całkowita). W niniejszej pracy posłużono się kryterium powierzchni zredukowanej jako najbardziej obiektywnym i porównywalnym wskaźnikiem tego typu analiz. Celem określenia preferencji żerowych zwierzyny, obliczono wskaźnik atrakcyjności żerowej poszczególnych rodzajów roślin. Wskaźnik ten obliczono jako iloraz różnicy procentowego udziału zniszczonej uprawy i procentowego udziału danej uprawy w strukturze zasiewów, do procentowego udziału danej uprawy w strukturze zasiewów. Obwody stanowiące teren badań położne są w północnej części Wyżyny Lubelskiej i oddzielone są od siebie drogą krajową Lublin-Warszawa. Również obydwa dzierżawione są przez to samo koło łowieckie, co sprawia, że intensywność zabiegów związanych z ich zagospodarowaniem, w tym również stosowaniem środków profilaktycznych jest zbliżona. W obydwu terenach badań, gospodarkę rolną cechuje typowe dla Lubelszczyzny rozdrobnienie upraw. Dodatkowo ze względu na fakt, że w obydwu obwodach tereny północnej ich części położne są w pradolinie rzeki Wieprz, która jednocześnie stanowi północną ich granicę administracyjną, w strukturach agrocenoz dość duży udział stanowią użytki zielone. WSTĘP W ciągu ostatnich kilku lat obserwowane jest zjawisko gwałtownego wzrostu szkód wyrządzanych w uprawach rolnych przez dzikie zwierzęta. Pomimo znacznego zróżnicowania tego typu szkód, problem ten osiągnął rangę ogólnopolską. Jako główną przyczynę wzrostu szkód w uprawach rolniczych wymienia się najczęściej gwałtowny wzrost liczebności dzików, które poprzez swoiste zmiany behawioralne, doskonale zaadoptowały się do użytkowania rozległych upraw polowych. W połączeniu ze zmianami siedliskowymi związanymi z intensyfikacją rolnictwa, głównie pod postacią wzrostu areału upraw wielko łanowych, jak również coraz powszechniejszego wykorzystywania kukurydzy jako rośliny paszowej, bytowanie dzików w strukturach agrocenoz zasobnych w wysokoenergetyczną bazę żerową pociągnęło za sobą zmiany parametrów rozrodu u tego gatunku. Zmiany te, to przede wszystkim zwiększenie potencjału rozrodczego populacji, poprzez większe liczebnie mioty oraz przystępowanie młodszych samic do rozrodu jak również swoiste rozregulowanie cyklu płciowego u tego gatunku, skutkujące wydawaniem potomstwa w zróżnicowanych porach roku (Kozdrowski i Dubiel, 2004; Flis, 2009 a). Wszystkie te elementy w sposób bezpośredni jak również pośredni, kompleksowo wpływają na wzrost szkód wyrządzanych przez ten gatunek w uprawach rolniczych. Zróżnicowanie wielkości i rozmieszczenia przestrzennego oraz rozkładu szkód w cyklach rocznych, związane jest bezpośrednio z liczebnością dzików w danym terenie, jak również zależne jest od struktury agrocenoz w postaci mozaikowatości środowisk oraz wielkości kompleksów leśnych. Dodatkowo uzależnione jest od dostępności żeru w postaci buczyny i żołędzi w przyległych kompleksach leśnych. Samo nasilenie szkód w strukturach lokalnych wynika z cyklów rozwojowych roślin uprawnych oraz rodzaju i intensywności zabiegów profilaktycznych, mających na celu ich ograniczanie (Mackin, 1970; Drozd, 1988; Flis, 2009 b). Celem niniejszej pracy była analiza wielkości i rozkładu przestrzennego oraz czasowego szkód wyrządzanych przez dziki, w dwóch obwodach łowieckich, charakteryzujących się dość zróżnicowaną strukturą agrocenoz, w sześcioletnim odstępie czasowym. 194 Marian Flis WYNIKI I DYSKUSJA Kształtowanie się wielkości szkód w uprawach zbożowych, w ocenianym okresie, wykazywało znaczne zróżnicowanie w poszczególnych obwodach jak i gatunkach zbóż (tab. 1). W obwodzie łowieckim nr 136, niezależnie od okresu dominowały szkody w uprawach pszenicy i pszenżyta. Z kolei na terenie obwodu łowieckiego nr 137, w sezonie łowieckim 2003/2004 w uprawach zbożowych szkód nie odnotowano, a sześć lat później liczba szkód wynosiła 19, zaś wielkość uszkodzonej powierzchni wynosiła 3,04 ha, z czego 195 Marian Flis 60% szkód wystąpiło w uprawach pszenicy, a dalsze 30% w uprawach owsa. W uprawach kukurydzy, w obwodzie łowieckim nr 136, w okresie badań liczba oraz powierzchnia szkód uległa zmniejszeniu. WYNIKI I DYSKUSJA Wielkość szkód w uprawach ziemniaków w obwodzie łowieckim nr 136, w okresie oceny uległa znacznemu zmniejszeniu, przy symbolicznym wręcz spadku liczby szkód w tego typu uprawach. W obwodzie tym wystąpił też spadek udziału szkód w tego rodzaju uprawach z 12,1% w sezonie 2003/2004 do 1,1% ogólnej liczby szkód w sezonie 2008/2009. Z kolei na terenie obwodu łowieckiego nr 137, w okresie badań wystąpił gwałtowny wzrost zarówno liczby jak i powierzchni szkód w uprawach ziemniaków. W okresie tym liczba szkód zwiększyła się ponad 3.krotnie, zaś ich powierzchnia blisko 5- krotnie. Łącznie w okresie oceny na terenie obwodu łowieckiego nr 136, zarówno liczba jak i wielkość uszkodzeń była zbliżona, a na terenie obwodu łowieckiego nr 137 nastąpił blisko 6.krotny wzrost ilości szkód oraz 3,5-krotny wzrost powierzchni uszkodzeń. W okresie objętym oceną na terenie obwodu łowieckiego nr 136, średni areał pojedynczej szkody w obydwu okresach oceny, kształtował się na poziomie 0,3 ha, Z kolei na terenie obwodu łowieckiego nr 137, średnia wielkość pojedynczej szkody w pierwszym okresie oceny wynosiła 0,25 ha, zaś po okresie 5 lat, wielkość ta zmniejszyła się i wynosiła 0,14 ha. Wykazany wzrost wielkości szkód wyrządzanych przez dziki w uprawach rolniczych, potwierdza ogólnopolską tendencję tego zjawiska. Najwięcej szkód wyrządzanych przez dziki występuje w rejonach największego ich zagęszczenia, tj. w rejonach zachodniej Polski (Flis, 2009 a). Niemniej jednak wzrost liczebności dzików, a tym samym i kwot wypłacanych odszkodowań dotyczy terenu całego kraju. W warunkach obwodu łowieckiego położonego na Lubelszczyźnie w ciągu ostatnich dziesięciu lat, wystąpił ponad 2,5-krotny wzrost wielkości powierzchni uszkodzeń, przy zbliżonej liczbie szkód, zaś w uprawach kukurydzy w ciągu ostatnich dziesięciu lat w tym samym terenie, wystąpił 30-krotny wzrost wielkości szkód (Flis, 2009 b). Uzyskane wyniki wskazują, że pomimo zróżnicowania w poszczególnych rejonach badań, w ostatnich latach zwiększeniu uległa średnia wielkość pojedynczej szkody w ujęciu powierzchni zredukowanej. Według Drozda (1988) w rejonie Lubelszczyzny w latach 1979–1983, wielkość ta kształtowała się na średnim poziomie 0,1 ha. Z kolei Flis (2009 b) podał, że w okresie 10 lat, w tym samym rejonie nastąpiło zwiększenie średniej wielkości pojedynczej szkody z 0,1 ha do 0,2 ha. Ocena powstawania szkód w ciągu sezonu wegetacyjnego wskazuje, że nasilenie ich występowania ściśle powiązane jest z sezonem wegetacyjnym oraz dostępnością żeru w poszczególnych okresach. WYNIKI I DYSKUSJA Tabela 1 Liczba i wielkość szkód (ha* i %) w uprawach rolniczych w ocenianych okresach Number and extent of damages (ha* and %) in agricultural cultivations in the evaluated periods Rodzaj uprawy Type of cultivation Nr obwodu łowieckiego Number of hunter district 136 137 2003/04 2008/09 2003/04 2008/09 Pszenica Wheat n 1 1 — 9 ha 0,03 0,16 — 1,81 % 0,4 2,1 — 11,8 Pszenżyto Triticale n 2 1 — — ha 0,07 0,05 — — % 1,0 0,6 — — Żyto Rye n — — — 3 ha — — — 0,22 % — — — 1,4 Mieszanka zbożowa Cereal mixture n — — — 1 ha — — — 0,05 % — — — 0,3 Owies Oats n — — 6 ha — — 0,96 % — — 6,2 Kukurydza Corn n 9 6 — 15 ha 5,39 3,54 — 2,78 % 76,5 45,2 — 18,1 Użytki zielone Green lands n 8 11 11 42 ha 0,71 3,56 4,04 7,65 % 10,0 45,5 92,7 49,8 Ziemniak Potato n 5 4 7 25 ha 0,85 0,09 0,32 1,47 % 12,1 1,1 7,3 9,6 Inne uprawy** Other cultivations n — 2 — 2 ha — 0,43 — 0,43 % — 5,5 — 2,8 Ogółem Total n 25 25 18 103 ha 7,05 7,83 4,36 15,37 % 100 100 100 100 * Wielkość szkód w hektarach podana dla powierzchni zredukowanej * Extent of damages in hectares for the reduced area Gryka, fasolka szparagowa Buckwheat, string bean Tabela Liczba i wielkość szkód (ha* i %) w uprawach rolniczych w ocenianych okresach Number and extent of damages (ha* and %) in agricultural cultivations in the evaluated periods Na terenie obwodu łowieckiego nr 137 w sezonie łowieckim 2003/2004 szkód w uprawach kukurydzy nie odnotowano, a sześć lat później stwierdzono 15 szkód, a ich powierzchnia zredukowana stanowiła 2,78 ha. Na użytkach zielonych szkody występowały we wszystkich okresach oceny, a ich wielkość w obydwu obwodach w tym okresie uległa zwiększeniu. W obwodzie łowieckim nr 136 nastąpił wzrost udziału tego typu szkód z 10% do 45,5% ogólnej powierzchni szkód, zaś na terenie obwodu łowieckiego nr 137 nastąpił spadek udziału szkód z 92,7% w sezonie 2003/2004 do poziomu 49,8% ogólnej 196 Marian Flis powierzchni uszkodzeń upraw rolniczych w sezonie 2008/2009. Jednak spadek szkód na użytkach zielonych, powiązany był ze wzrostem szkód w innych rodzajach upraw w tym terenie. WYNIKI I DYSKUSJA Pomimo zróżnicowania w poszczególnych rejonach badań, w uprawach zbóż okres ten ściśle związany jest z siewem zbóż jarych, które jako pierwsze pojawiają się na polach, jak również wzmożone nasilenie szkód występuje na przełomie lipca i sierpnia, co powiązać można z okresem dojrzałości ziarniaków u roślin zbożowych (rys. 1). Nasilenie szkód w uprawach kukurydzy występowało w maju tj. w okresie siewu tej rośliny oraz w sierpniu, wrześniu i październiku kiedy ziarniaki kukurydzy uzyskują poszczególne fazy dojrzałości (rys. 2). W uprawach ziemniaków największe nasilenie szkód występowało w maju i czerwcu, co powiązać można z terminem sadzenia tej rośliny, a tym samym dostępnością bulw w zależności od formy użytkowania i zmniejszało się w kolejnych miesiącach, aż do okresu zejścia tej rośliny z pól (rys. 3). Szkody wyrządzane 197 Marian Flis przez dziki na trwałych użytkach zielonych powstawały głównie w miesiącu marcu i kwietniu oraz począwszy od września do listopada (rys. 4). Fakt ten można powiązać w sposób bezpośredni z niewielką dostępnością innego rodzaju pożywienia na polach w okresie przedwiośnia jak również sukcesywnym ubywaniem pożywienia w okresie późno jesiennym. Rys. 1. Rozkład szkód w poszczególnych miesiącach w zbożach w latach 2003/2004-2008/2009 Fig. 1. Monthly distribution of damages in cereal plantations in 2003/2004-2008/2009 0 10 20 30 40 50 60 70 80 obwód 136, hunter district 136 obwód 137, hunter district 137 % Rys. 1. Rozkład szkód w poszczególnych miesiącach w zbożach w latach 2003/2004-2008/2009 Fig. 1. Monthly distribution of damages in cereal plantations in 2003/2004-2008/2009 Podobne wyniki w zakresie największego nasilenia powstawania szkód w uprawach rolniczych, uzyskał Drozd (1988), prowadząc badania w makroregionie środkowo wschodniej Polski w latach 1979–1983, jak również Flis (2009b) prowadząc badania w warunkach obwodu łowieckiego polnego położonego na Wyżynie Lubelskiej. Z kolei w rejonie Polski północno-wschodniej, według Dubasa (1996) występowały dwa okresy nasilenia szkód. Pierwszy z nich obejmował okres maja i czerwca, zaś drugi okres przypadał na sierpień i wrzesień. Dodatkowo autor ten wykazał, że w specyfice żerowej dzikich zwierząt, istnieje pewien specyficzny łańcuch żeru polowego, który uwarunkowany jest sezonem wegetacyjnym poszczególnych upraw jak również terminem ich sprzętu z pól. 198 Marian Flis 199 Rys. 2. Rozkład szkód w poszczególnych miesiącach w kukurydzy w latach 2003/2004-2008/2009 Fig. 2. Monthly distribution of damages in corns in 2003/2004-2008/2009 Rys. 3. Rozkład szkód w poszczególnych miesiącach w ziemniakach w latach 2003/2004-2008/2009 Fig. 3. WYNIKI I DYSKUSJA 200 Marian Flis Najmniej atrakcyjnymi roślinami żerowymi, były uprawy roślin zbożowych, dla których niezależnie od terenu badań, wskaźnik atrakcyjności przyjmował wartości ujemne. yj p yj j Uzyskane wyniki potwierdzają tezę o preferowaniu przez dziki upraw kukurydzy, które w zależności od regionu kraju, z reguły charakteryzują się większą jednostkową powierzchnią, a tym samym stwarzają optymalne warunki żerowe jak również osłonowe dla zwierzyny. Flis (2009 b) prowadząc badania w rejonie Wyżyny Lubelskiej wykazał, że najbardziej preferowaną rośliną przez dziki była również kukurydza. Z kolei Drozd (1988) prowadząc badania w tym samym rejonie w latach 1979–1983, wskazał, że pod względem atrakcyjności żerowej w tym okresie dominowały uprawy ziemniaków oraz owsa. W rejonie Polski północno-wschodniej pod względem atrakcyjności żerowej, w uprawach polowych dominowała również kukurydza oraz rzepak i ziemniaki (Dubas, 1996). Z kolei Mackin (1970) prowadząc badania w trzech rejonach Polski podała, że najbardziej preferowanymi roślinami żerowymi były uprawy owsa i ziemniaków. Jednak w okresie tym w Polsce nie występowały uprawy kukurydzy. Przesunięcie preferencji żerowych dzików w ostatnich kilkudziesięciu latach wynika, z faktu, zmniejszania udziału w strukturze upraw zarówno owsa jak i ziemniaków na korzyść upraw kukurydzy, wraz ze zwiększaniem jednostkowych powierzchni areału jej uprawy (GUS, 2009; Chotkowski i Rembeza, 2006; Flis, 2007). W okresie oceny liczebność oraz poziom łowieckiej eksploatacji populacji dzików uległ zwiększeniu w obydwu terenach badań. Na terenie obwodu łowieckiego nr 136 liczebność tego gatunku w ciągu 6 sezonów łowieckich zwiększyła się ponad 1,5-raza (rys. 5). W tym samym okresie poziom łowieckiej eksploatacji populacji tego gatunku wzrósł 4,5-krotnie i w ostatnim roku oceny wskaźnik ten kształtował się na poziomie 100% wiosennego stanu populacji tych zwierząt (dane na słupkach wykresu). Z kolei w obwodzie łowieckim nr 137, liczebność dzików w okresie oceny wykazywała cechy stabilności, z niewielką tendencją wzrostową (rys. 6). Stan taki wynikał z dość intensywnej presji łowieckiej na ten gatunek począwszy od sezonu łowieckiego 2005/06. W ostatnim sezonie łowieckim poziom łowieckiej eksploatacji populacji w tym terenie osiągnął wielkość 150% jej wiosennego stanu liczebnego. Uzyskane wyniki potwierdzają ogólnopolską tendencję wzrostu liczebności dzików, na przełomie ostatnich lat. Wielkość tego wzrostu jest zróżnicowana na terenie kraju i wynika z warunków środowiskowych jak również łowieckiej presji na ten gatunek (Kamieniarz i Panek, 2008; Flis, 2009 b, Budny i in., 2010). Wzrost liczebności uwarunkowany jest głównie poprawą warunków żerowych dla tego gatunku, w postaci intensyfikacji rolnictwa i wzrostu areału upraw kukurydzy. WYNIKI I DYSKUSJA Monthly distribution of damages in potatoes in 2003/2004-2008/2009 0 10 20 30 40 50 60 70 80 obwód 136, hunter district 136 obwód 137, hunter district 137 % 0 10 20 30 40 50 60 obwód 136, hunter district 136 obwód 137, hunter district 137 % 2 R kł d kód ól h i i h k k d l t h 2003/2004 2008/2009 0 10 20 30 40 50 60 70 80 obwód 136, hunter district 136 obwód 137, hunter district 137 % Rys. 2. Rozkład szkód w poszczególnych miesiącach w kukurydzy w latach 2003/2004-2008/2009 Fig. 2. Monthly distribution of damages in corns in 2003/2004-2008/2009 0 10 20 30 40 50 60 obwód 136, hunter district 136 obwód 137, hunter district 137 % Rys. 3. Rozkład szkód w poszczególnych miesiącach w ziemniakach w latach 2003/2004-2008/2009 Fig. 3. Monthly distribution of damages in potatoes in 2003/2004-2008/2009 199 Marian Flis Rys. 4. Rozkład szkód w poszczególnych miesiącach na trwałych użytkach zielonych w latach 2003/2004-2008/2009 Fig. 4. Monthly distribution of damages in sustainable green lands in 2003/2004-2008/2009 0 5 10 15 20 25 30 35 40 obwód 136, hunter district 136 obwód 137, hunter district 137 % 0 5 10 15 20 25 30 35 40 obwód 136, hunter district 136 obwód 137, hunter district 137 % obwód 136, hunter district 136 obwód 137, hunter district 137 Rys. 4. Rozkład szkód w poszczególnych miesiącach na trwałych użytkach zielonych w latach 2003/2004-2008/2009 Fig. 4. Monthly distribution of damages in sustainable green lands in 2003/2004-2008/2009 Tabela 2 Wskaźniki atrakcyjności żerowej roślin uprawnych w okresie 2003/2004–2008/2009 Prey attractiveness indicators for crops in 2003/2004–2008/2009 Rodzaj uprawy Type of cultivation Numer obwodu łowieckiego Number of hunter district 136 137 Zboża (razem) Cereal (total) -0,92 -0,63 Kukurydza Corn 2,13 0,13 Ziemniak Potato 1,52 1,80 Trwałe użytki zielone Sustainable green lands 0,57 0,65 Wskaźniki atrakcyjności żerowej roślin uprawnych w okresie 2003/2004–2008/2009 Prey attractiveness indicators for crops in 2003/2004–2008/2009 Obliczone wskaźniki atrakcyjności żerowej poszczególnych rodzajów roślin, wskazują, że w rejonie badań występowało zróżnicowanie preferencji żerowych (tab. 2). W obwodzie łowieckim nr 136 najbardziej preferowanym rodzajem upraw była kukurydza, dla której wskaźnik preferencji żerowej osiągnął wartość 2,13. Z kolei w obwodzie łowieckim nr 137, najbardziej preferowane były uprawy ziemniaków, dla których wskaźnik ten osiągnął wielkość 1,8. Również wysoką atrakcyjnością żerową cieszyły się uprawy ziemniaków na terenie obwodu łowieckiego nr 136, gdzie wskaźnik ich preferencji żerowej wynosił 1,52. WYNIKI I DYSKUSJA Dostępność wysokoenergetycznego żeru, w strukturach rozległych agrocenoz, wpływa dodatnio na wskaźniki rozrodu u tego gatunku (Kozdrowski i Dubiel, 2004; Flis, 2009 a). 201 Marian Flis Rys. 5. Liczebność i pozyskanie dzików w obwodzie 136 Fig. 5. Population and gaining of wild boars in hunter district 136 Rys. 6. Liczebność i pozyskanie dzików w obwodzie 137 Fig. 6. Population and gaining of wild boars in hunter district 137 22,2 93,3 53,3 40,0 52,0 100,0 0 5 10 15 20 25 30 35 2003/2004 2004/2005 2005/2006 2006/2007 2007/2008 2008/2009 liczba osobników number of individuals pozyskanie, gaining liczebność, number 53,3 12,5 32,5 54,2 66,6 150,0 0 10 20 30 40 50 60 70 80 2003/2004 2004/2005 2005/2006 2006/2007 2007/2008 2008/2009 liczba osobników number of individuals pozyskanie, gaining liczebność, number 22,2 93,3 53,3 40,0 52,0 100,0 0 5 10 15 20 25 30 35 2003/2004 2004/2005 2005/2006 2006/2007 2007/2008 2008/2009 liczba osobników number of individuals pozyskanie, gaining liczebność, number Rys. 5. Liczebność i pozyskanie dzików w obwodzie 136 Fig. 5. Population and gaining of wild boars in hunter district 136 Rys. 5. Liczebność i pozyskanie dzików w obwodzie 136 Fig. 5. Population and gaining of wild boars in hunter district 136 Fig. 5. Population and gaining of wild boars in hunter district 136 Rys. 6. Liczebność i pozyskanie dzików w obwodzie 137 Fig. 6. Population and gaining of wild boars in hunter district 137 53,3 12,5 32,5 54,2 66,6 150,0 0 10 20 30 40 50 60 70 80 2003/2004 2004/2005 2005/2006 2006/2007 2007/2008 2008/2009 liczba osobników number of individuals pozyskanie, gaining liczebność, number Rys. 6. Liczebność i pozyskanie dzików w obwodzie 137 Fig. 6. Population and gaining of wild boars in hunter district 137 202 Marian Flis WNIOSKI 1. W okresie oceny, wystąpiło zróżnicowanie ilości i areału szkód w zależności od struktury terenu badań. W obwodzie łowieckim polnym, poziom uszkodzeń upraw w ciągu sześcioletniego okresu oceny utrzymywał się na zbliżonym poziomie, zaś w obwodzie łowieckim o charakterze leśnym, w ciągu tego samego okresu liczba szkód zwiększyła się prawie 6-krotnie, a ich powierzchnia 3,5-krotnie. Najbardziej narażonymi uprawami są uprawy położone w bezpośrednim sąsiedztwie lasu. y p ą p y p p ą 2. Rozkład szkód w ciągu sezonu wegetacyjnego, ściśle powiązany jest z terminem siewu lub sadzenia poszczególnych roślin uprawnych oraz terminem wydania plonu przez te rośliny i ich zbiorem. W uprawach roślin zbożowych jest to okres wiosenny oraz miesiąc lipiec i sierpień, a w uprawach kukurydzy nasilenie szkód występuje w okresie siewu tej rośliny oraz w okresie jej dojrzewania. W ziemniakach największe nasilenie szkód występuje w maju i w czerwcu, zaś na trwałych użytkach zielonych w okresie wczesnowiosennym i późnojesiennym. 3. Obliczony wskaźnik preferencji żerowych wskazuje, że preferencje te są zróżnicowane i zależne od rejonu oceny. Niemniej jednak najbardziej preferowaną rośliną jest kukurydza i ziemniaki. Najmniej preferowanymi rodzajami upraw są rośliny zbożowe. y j j p y j p ą y 4. W okresie objętym oceną wystąpiło zwiększenie liczebności dzików jak również poziomu łowieckiej eksploatacji tego gatunku. W rejonie badań w ostatnim roku oceny łowiecka eksploatacja populacji zawierała się w przedziale od 100 do 150% zinwentaryzowanego jej wiosennego stanu i w porównaniu z początkowym okresem oceny była ona średnio czterokrotnie większa. Wzrost poziomu łowieckiej eksploatacji populacji, warunkował umiarkowany jej przyrost, co w sposób bezpośredni przedkładało się również na poziom wyrządzanych szkód. 4. W okresie objętym oceną wystąpiło zwiększenie liczebności dzików jak również poziomu łowieckiej eksploatacji tego gatunku. W rejonie badań w ostatnim roku oceny łowiecka eksploatacja populacji zawierała się w przedziale od 100 do 150% zinwentaryzowanego jej wiosennego stanu i w porównaniu z początkowym okresem oceny była ona średnio czterokrotnie większa. Wzrost poziomu łowieckiej eksploatacji populacji, warunkował umiarkowany jej przyrost, co w sposób bezpośredni przedkładało się również na poziom wyrządzanych szkód. Rozporządzenie Ministra Środowiska z dnia 15 lipca 2002 roku, w sprawie postępowania przy szacowaniu szkód oraz wypłat odszkodowań za szkody w uprawach i płodach rolnych (Dz. U. z 2002 r. Nr 126, poz. 1081). Mackin R. 1970. Dynamics of damage caused by wild boar to different agricultural crops. Acta Theriologica, Vol. 15: 447 — 458. 1081). Ustawa z dnia 13 października 1995 roku – Prawo łowieckie (Dz. U. 05.175.1462). Vol. 15: 447 458. Rocznik Statystyczny Rolnictwa i Obszarów Wiejskich. 2009. GUS Warszawa: 147 — 158. Ś Marian Flis LITERATURA Budny M., Kamieniarz R., Kolanoś B., Mąka H., Panek M. 2010. Sytuacja zwierząt łownych w Polsce w latach 2008–2009. Biuletyn Stacji Badawczej w Czempiniu Nr 6: 23 — 25. Chotkowski J., Rembeza J. 2006. Tendencje zmian na rynku ziemniaków w Polsce. [w:] Produkcja ziemniaków. Chotkowski J. (red.) Wydawnictwo Wieś Jutra. Warszawa: 7 — 15. ( ) y Drozd L. 1988. Wpływ rozdrobnienia kompleksów leśnych na szkody wyrządzane przez dziki w uprawach polowych w makroregionie środkowowschodniej Polski. Sylwan, Nr 11–12: 79 — 84. Dubas W. J. 1996. Szkody łowieckie w przyleśnych uprawach rolnych w północno-wschodniej Polsce. Sylwan, Nr 10: 45 — 56. Flis M. 2007. Szkody w ziemniakach. Łowiec Polski, Nr 9: 50 — 53. 2007. Szkody w ziemniakach. Łowiec Polski, Nr 9: 50 — Flis M. 2008. Procedura szacowania szkód wyrządzonych przez zwierzęta w uprawach rolniczych. Biul. IHAR 248: 117 — 123. Flis M. 2009 a. Szkody w uprawach rolniczych w świetle szkodliwego oddziaływania rolnictwa na ekosystemy. Biotop. Zagrożenia biotopów leśnych. Uniwersytet Opolski, Opole: 123 — 132. Flis M. 2009 b. Wielkość szkód wyrządzanych przez dziki w uprawach rolniczych w obwodzie łowieckim polnym w latach 1999–2000 i 2008–2009. Biuletyn Instytutu Hodowli i Aklimatyzacji Roślin, Nr 254: 179 — 187. Kamieniarz R., Panek M. 2008. Zwierzęta łowne w Polsce na przełomie XX i XXI wieku. Stacja Badawcza – OHZ PZŁ w Czempiniu: 46 — 49. p Kozdrowski R., Dubiel A. 2004. Biologia rozrodu dzika. Medycyna Weterynaryjna, 60: 1251 — 1253. 203 Marian Flis 204
https://openalex.org/W3167940477	https://bmcbiol.biomedcentral.com/counter/pdf/10.1186/s12915-021-01051-y	English	null	Divergent camptothecin biosynthetic pathway in Ophiorrhiza pumila	BMC biology	2,021	cc-by	12,770	© The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. * Correspondence: guoyinkai1@126.com †Mengquan Yang and Qiang Wang contributed equally to this work. ˆYouli Xiao is deceased. 1Laboratory of Medicinal Plant Biotechnology, College of Pharmacy, Zhejiang Chinese Medical University, Hangzhou 310053, Zhejiang, China Full list of author information is available at the end of the article Abstract Background: The anticancer drug camptothecin (CPT), first isolated from Camptotheca acuminata, was subsequently discovered in unrelated plants, including Ophiorrhiza pumila. Unlike known monoterpene indole alkaloids, CPT in C. acuminata is biosynthesized via the key intermediate strictosidinic acid, but how O. pumila synthesizes CPT has not been determined. Results: In this study, we used nontargeted metabolite profiling to show that 3α-(S)-strictosidine and 3-(S), 21-(S)- strictosidinic acid coexist in O. pumila. After identifying the enzymes OpLAMT, OpSLS, and OpSTR as participants in CPT biosynthesis, we compared these enzymes to their homologues from two other representative CPT-producing plants, C. acuminata and Nothapodytes nimmoniana, to elucidate their phylogenetic relationship. Finally, using labelled intermediates to resolve the CPT biosynthesis pathway in O. pumila, we showed that 3α-(S)-strictosidine, not 3-(S), 21- (S)-strictosidinic acid, is the exclusive intermediate in CPT biosynthesis. Conclusions: In our study, we found that O. pumila, another representative CPT-producing plant, exhibits metabolite diversity in its central intermediates consisting of both 3-(S), 21-(S)-strictosidinic acid and 3α-(S)-strictosidine and utilizes 3α-(S)-strictosidine as the exclusive intermediate in the CPT biosynthetic pathway, which differs from C. acuminata. Our results show that enzymes likely to be involved in CPT biosynthesis in O. pumila, C. acuminata, and N. nimmoniana have evolved divergently. Overall, our new data regarding CPT biosynthesis in O. pumila suggest evolutionary divergence in CPT-producing plants. These results shed new light on CPT biosynthesis and pave the way towards its industrial production through enzymatic or metabolic engineering approaches. Keywords: Biosynthesis, Camptothecin, In vivo labelling, Ophiorrhiza pumila, Strictosidine (TCM) [1]. CPT chemotype also appears sporadically in multiple taxa within the superasterids, with a total of 43 plant species [2]. Of those, C. acuminata, Nothapodytes nimmoniana, and Ophiorrhiza pumila are the three main representative CPT-producing plants (Fig. 1a). In 1994, the US Food and Drug Administration (FDA) ap- proved the therapeutic use of two well-known antitu- mour CPT derivatives, irinotecan and topotecan, which inhibit the replication, growth, and reproduction of can- cer cells by inhibiting DNA topoisomerase I [3]. The clinical applications of these two CPT-derived drugs to the treatment of cancer have greatly increased demand, raising issues about the sustainable production of CPT Divergent camptothecin biosynthetic pathway in Ophiorrhiza pumila Mengquan Yang2†, Qiang Wang1,3†, Yining Liu2, Xiaolong Hao1, Can Wang1, Yuchen Liang2, Jianbo Chen3, Youli Xiao2ˆ and Guoyin Kai1* Background The alkaloid camptothecin (CPT) was first isolated in 1966 from the bark of the tree Camptotheca acuminata (“Xi-Shu” in Chinese, which translates to happy tree). C. acuminata is a deciduous tree native to southern China that is extensively used in traditional Chinese medicine Yang et al. BMC Biology (2021) 19:122 https://doi.org/10.1186/s12915-021-01051-y Yang et al. BMC Biology (2021) 19:122 https://doi.org/10.1186/s12915-021-01051-y The biosynthesis of secologanin involves the successive action of the en- zymes geranyl diphosphate synthase (GPS), geraniol syn- thase (GE), geraniol 10-hydroxylase (G10H), 10- hydroxygeraniol oxidoreductase (10-HGO), iridoid syn- thase (IS), iridoid oxidase (IO), 7-deoxyloganetic acid UDP-glucosyltransferase (7-DLGT), 7-deoxyloganic acid hydroxylase (7-DLH, catalyzed by CYP72A224), secolo- ganin synthase (SLS), and cytochrome P450 reductase (CPR), finally forming secologanin [14]. In the shi- kimate pathway, several enzymes participate in multi- step reactions from chorismic acid to tryptamine, which is another precursor for CPT biosynthesis. These enzymes include anthranilate synthase (ASA), phosphoribosyl diphosphate anthranilate transferase (PRT), tryptophan synthase α (TSA), tryptophan syn- thase β (TSB), and tryptophan decarboxylase (TDC). TDC is considered to be a rate-limiting enzyme in CPT biosynthetic pathway [18]. Subsequently, trypta- mine and secologanin are condensed into strictosidine by strictosidine synthase (STR). However, multiple en- zymatic steps are still missing from our understanding of the poststrictosidine pathway, and only a few of the metabolite intermediates in the CPT biosynthetic [4]. Because chemical synthesis of CPT on an industrial scale is hindered by the complexity of its unique penta- cyclic pyrroloquinoline scaffold, the sourcing of CPT still depends heavily on extraction from its resource plants [5, 6]. However, the few plants that naturally produce CPT grow slowly and will not meet this increasing mar- ket demand, necessitating an alternative approach to raise CPT production [6, 7]. One such promising method involves the introduction of key biosynthetic genes or regulators through metabolic engineering or the reconstruction of the CPT biosynthetic pathway in heterologous microbial systems by synthetic biology. This method will require the dissection and thorough understanding of CPT biosynthesis, which is currently lacking [7–11]. [4]. Because chemical synthesis of CPT on an industrial scale is hindered by the complexity of its unique penta- cyclic pyrroloquinoline scaffold, the sourcing of CPT still depends heavily on extraction from its resource plants [5, 6]. However, the few plants that naturally produce CPT grow slowly and will not meet this increasing mar- ket demand, necessitating an alternative approach to raise CPT production [6, 7]. One such promising method involves the introduction of key biosynthetic genes or regulators through metabolic engineering or the reconstruction of the CPT biosynthetic pathway in heterologous microbial systems by synthetic biology. This method will require the dissection and thorough understanding of CPT biosynthesis, which is currently lacking [7–11]. Yang et al. BMC Biology (2021) 19:122 Page 2 of 16 Yang et al. BMC Biology A CAMPANULIDS LAMIIDS ASTERIDS SUPERASTERIDS A CAMPANULIDS LAMIIDS ASTERIDS SUPERASTERIDS Ophiorrhizapumila Nothapodytes nimmoniana Camptotheca acuminata B Fig. 1 Distribution of CPT in the plant kingdom and possible biosynthesis of alkaloids through strictosidine or strictosidinic acid. a Phylogenetic tree of the major lineages of land plants summarizing the known taxonomic distribution of CPT-producing species. Orders that contain at least one CPT- producing species are indicated with red diamonds. The three CPT-producing plants used in this study are shown on the right and belong to the Cornales (C. acuminata), Icacinales (N. nimmoniana), and Gentianales (O. pumila) orders. b The biosynthesis of monoterpene indole alkaloids (MIAs) through strictosidine or strictosidinic acid derived from tryptamine and secoiridoids. STR, strictosidine synthase; STRAS, strictosidinic acid synthase h t i t idi t i t idi i id Ph l ti t f A B Ophiorrhizapumila p y Ophiorrhizapumila Ophiorrhizapumila Fig. 1 Distribution of CPT in the plant kingdom and possible biosynthesis of alkaloids through strictosidine or strictosidinic acid. a Phylogenetic tree of the major lineages of land plants summarizing the known taxonomic distribution of CPT-producing species. Orders that contain at least one CPT- producing species are indicated with red diamonds. The three CPT-producing plants used in this study are shown on the right and belong to the Cornales (C. acuminata), Icacinales (N. nimmoniana), and Gentianales (O. pumila) orders. b The biosynthesis of monoterpene indole alkaloids (MIAs) through strictosidine or strictosidinic acid derived from tryptamine and secoiridoids. STR, strictosidine synthase; STRAS, strictosidinic acid synthase Fig. 1 Distribution of CPT in the plant kingdom and possible biosynthesis of alkaloids through strictosidine or strictosidinic acid. a Phylogenetic tree of the major lineages of land plants summarizing the known taxonomic distribution of CPT-producing species. Orders that contain at least one CPT- producing species are indicated with red diamonds. The three CPT-producing plants used in this study are shown on the right and belong to the Cornales (C. acuminata), Icacinales (N. nimmoniana), and Gentianales (O. pumila) orders. b The biosynthesis of monoterpene indole alkaloids (MIAs) through strictosidine or strictosidinic acid derived from tryptamine and secoiridoids. STR, strictosidine synthase; STRAS, strictosidinic acid synthase secologanin via the iridoid pathway [6]. Results We annotated 15 metabolites in the CPT biosynthetic pathway based on accurate mass measurements of positive ions and fragments ob- served in MS and MS/MS mass spectra (Fig. 2a and Additional file 1: Table S1) and confirmed them against the metabolite profiles detected in C. acumi- nata [15]. In this study, we detected both strictosidine and strictosidinic acid in O. pumila plant tissue and hairy root extracts. In addition, O. pumila plant tis- sues and hairy roots accumulated iridoids, loganic acid, loganin, and secologanic acid as well as secolo- ganin. Therefore, in contrast to C. acuminata, both carboxylic acid derivatives (loganic acid, secologanic acid, and strictosidinic acid) and methyl ester deriva- tives (loganin, secologanin, and strictosidine) coexist in O. pumila. These results suggest that O. pumila might harbour two parallel pathways that produce CPT, in sharp contrast to C. acuminata, which uses carboxylic acid intermediates [15], and to the best- studied MIA-producing plants (e.g. the Madagascar periwinkle, Catharanthus roseus), which use methyl esters as intermediates [25, 26]. We also successfully detected additional metabolites in the CPT biosynthetic pathway previously reported in O. pumila, such as stricto- samide, pumiloside, and deoxypumiloside [23]. To analyse CPT biosynthesis in O. pumila, we carried out nontargeted metabolite profiling and feeding experi- ments with deuterium-labelled tryptophan. Our results demonstrated the detection of both strictosidine and strictosidinic acid. Functional gene analyses and in vitro biochemical characterization further indicated that the O. pumila enzymes OpLAMT, OpSLS, and OpSTR par- ticipated in the biosynthesis of strictosidine and strictosi- dinic acid. Feeding experiments of O. pumila with d4- strictosidine and d4-strictosidinic acid suggested that strictosidine, rather than strictosidinic acid, was the ex- clusive intermediate involved in CPT biosynthesis. Our results demonstrate that the biosynthesis of CPT in O. pumila mainly recruits strictosidine and not strictosidi- nic acid, which is quite different from CPT biosynthesis in C. acuminata, suggesting divergence in their respect- ive biosynthetic pathways. p yp To evaluate the possibility that two parallel CPT bio- synthetic pathways might exist in O. pumila, we per- formed metabolite profiling and quantified the relative levels of all detectable metabolites as well as their iso- mers in extracts from four tissues (leaves, stems, roots, and hairy roots) (Fig. 2b). We identified several isomers with the same exact molecular masses and fragment ions as some of the metabolites we detected in O. pumila. Results Two parallel pathways of CPT biosynthesis were proposed by metabolite profiling of intermediates in O. Two parallel pathways of CPT biosynthesis were proposed by metabolite profiling of intermediates in O. pumila Based on our current understanding of CPT biosyn- thesis, strictosidine is commonly considered the key intermediate in the biosynthesis of MIAs such as vin- blastine and vincristine. However, although CPT belongs to the MIA family, no strictosidine was detected in the CPT-producing plant C. acuminata [15]. Furthermore, loganic acid, secologanic acid, and strictosidinic acid were all detected by metabolite profiling, whereas loga- nin, secologanin, and strictosidine were all undetectable in C. acuminata, leading to the conclusion that strictosi- dinic acid was the sole intermediate for CPT biosyn- thesis (Fig. 1b) [15]. In contrast, in N. nimmoniana, only secologanin was detected [21], indicating that strictosi- dine, and not strictosidinic acid, might be a key biosyn- thetic intermediate. In the case of O. pumila, independent metabolic profiling studies identified a strictosidinic backbone as a precursor but did not agree on the exact form, with one reporting strictosidinic acid and the other strictosidine [22, 23]. A bifunctional seco- loganin synthase (SLS) that catalyses the reaction of loganin (or loganic acid) to form secologanin (or secolo- ganic acid) was characterized in C. acuminata [16]. However, the enzyme that converts secologanic acid and tryptamine into strictosidinic acid has not been identi- fied or characterized in C. acuminata, N. nimmoniana, or O. pumila. Compared to the woody plants C. acumi- nata and N. nimmoniana, the herbaceous plant O. pumila (from the genus Ophiorrhiza) offers a number of advantages, including a shorter generation time and eas- ier genetic transformation. O. pumila would therefore be an excellent system for metabolic engineering research into CPT biosynthesis [24]. However, it remains unclear which intermediate (strictosidine or strictosidinic acid) is most likely to take part in the CPT biosynthesis pathway in O. pumila. The distribution of the bioactive compound CPT var- ies across O. pumila tissues [23]. To assess the diver- sity and abundance of putative intermediates in the CPT biosynthetic pathway in O. pumila, we collected different tissues (leaves, stems, roots, and hairy roots) for metabolite profiling (Additional file 1: Fig. S1). We subjected total methanolic extracts to ultra-high- performance liquid chromatography (UHPLC) followed by mass spectrometry (MS) for untargeted metabolic analysis. More than 2000 monoterpene indole alkaloids (MIAs) are thought to originate from the common intermediate strictosidine, which separates the MIA biosynthetic pathway into prestrictosidine and poststrictosidine path- ways or steps (Fig. 1b) [12–14]. In plants, the biosyn- thesis of strictosidine has been well studied [6, 15–17]. The methylerythritol 4-phosphate (MEP) and mevalonic acid (MVA) pathways provide basic terpene precursors such as isopentenyl pyrophosphate (IPP) to form Yang et al. BMC Biology (2021) 19:122 Page 3 of 16 Page 3 of 16 Page 3 of 16 pathway, such as pumiloside and deoxypumiloside, have been identified [19, 20]. Results We numbered these isomers according to their relative elution orders under the same liquid chromatography (LC) conditions (Additional file 1: Table S1). Strictosi- dine and strictosidinic acid coexisted in all tissues as a single isomer (Fig. 2b). This species differs from C. acu- minata, which contains strictosidinic acid as three iso- mers and lacks any detectable strictosidine [15]. In O. pumila, the strictosidine content was highest in stems, Page 4 of 16 Yang et al. BMC Biology (2021) 19:122 Yang et al. BMC Biology Fig. 2 Two parallel pathways of CPT biosynthesis were proposed by metabolite profiling of intermediates in O. pumila. a Metabolites and identified enzymes involved in CPT biosynthesis in O. pumila. LAMT, loganic acid methyltransferase; SLAS, secologanic acid synthase; SLS, secologanin synthase; STR, strictosidine synthase; STRAS, strictosidinic acid synthase. Steps shaded in blue are part of the prestrictosidine pathway, while orange denotespoststrictosidine is the poststrictosidine pathway. b Tissue distribution profiles of proposed CPT pathway metabolites in O. pumila plants and hairy roots (4 replicates). Tissues were collected from wild-type plants grown on Gamborg B5 medium for 3 months, and methanol extracts were analysed using a 38.5-min gradient elution method for mass spectrometry (MS) detection. Multiple isomers were detected for deoxypumiloside, pumiloside, and strictosidine epoxide. Data are shown as the mean ± SD (n = 4) for the most abundant and quantifiable isomers. Hr, hairy root; Rt, root; St, stem; Lf, leaf Fig. 2 Two parallel pathways of CPT biosynthesis were proposed by metabolite profiling of intermediates in O. pumila. a Metabolites and identified enzymes involved in CPT biosynthesis in O. pumila. LAMT, loganic acid methyltransferase; SLAS, secologanic acid synthase; SLS, secologanin synthase; STR, strictosidine synthase; STRAS, strictosidinic acid synthase. Steps shaded in blue are part of the prestrictosidine pathway, while orange denotespoststrictosidine is the poststrictosidine pathway. b Tissue distribution profiles of proposed CPT pathway metabolites in O. pumila plants and hairy roots (4 replicates). Tissues were collected from wild-type plants grown on Gamborg B5 medium for 3 months, and methanol extracts were analysed using a 38.5-min gradient elution method for mass spectrometry (MS) detection. Multiple isomers were detected for deoxypumiloside, pumiloside, and strictosidine epoxide. Data are shown as the mean ± SD (n = 4) for the most abundant and quantifiable isomers. Hr, hairy root; Rt, root; St, stem; Lf, leaf while the strictosidinic acid content was highest in hairy roots (Fig. 2b). Results We detected two isomers of pumiloside (isomer 1 at very low levels) in hairy roots but only isomer 2 in other plant tissues (leaves, stems, roots) (Fig. 2b). We also detected two isomers of deoxypumiloside in all four tissues, with the high- est levels in stems and roots (Fig. 2b). In addition, we identified three isomers for a compound we annotated as strictosamide epoxide, in accordance with C. acu- minata [15] (Fig. 2b). Finally, we resolved a single CPT isomer, which we detected in all tissues, with the highest levels in roots (Fig. 2b). The presence of comparable amounts of both strictosidine and stricto- sidinic acid in all O. pumila tissues implies the possi- bility that parallel biosynthetic pathways for CPT might indeed exist, at least during the prestrictosidine stage. This raised the question of how these two key precursors might become incorporated into the post- strictosidine biosynthetic steps to generate CPT as the single and final product. Page 5 of 16 Yang et al. BMC Biology (2021) 19:122 Yang et al. BMC Biology (2021) 19:122 determined their expression levels from our RNA-seq dataset across our different tissues in O. pumila. We then clustered the genes involved in prestrictosidine to compare the resulting pattern with that of metabolite contents in the same tissues. The resulting heatmap re- vealed that genes from the later iridoid stage with high similarity to strictosidine biosynthetic genes, including five genes involved in iridoid biosynthesis (IO, 7DLGT, 7DLH, LAMT, and SLS) and TDC, were highly expressed in stems (Additional file 1: Fig. S2a). They displayed the highest expression in stems and lower expression in roots, followed by leaves, with hairy roots showing the lowest expression of these genes, which is consistent with the pattern of strictosidine content obtained by me- tabolite profiling (Fig. 2b and Additional file 1: Fig. S2a). Genes encoding proteins involved in the MEP pathway and in the early stage of iridoid biosynthesis were highly expressed in leaves and roots, respectively (Additional file 1: Fig. S2a). Based on the gene expression pattern, we speculate that genes related to the poststrictosidine pathway would be highly expressed in stems. However, biosynthetic genes involved in the prestrictosidine stage might exhibit different expression patterns from those involved in the poststrictosidine stage. The quantifica- tion of metabolites indicated that strictosidine accumu- lated to high levels in stems (Fig. Results 2b), whereas CPT accumulated to high levels in the root (Fig. 2b). Interest- ingly, it was found that the key intermediates (strictosi- dine, pumiloside, and deoxypumiloside) involved in the CPT biosynthetic pathway, except strictosidinic acid, accumulated to the highest levels in the stem, which in- dicates that the precursors of CPT are synthesized in the stem. There are three hypotheses to explain this contra- diction. (1) Deoxypumiloside is transported into the roots and then converted into CPT in the roots by a series of enzymes. (2) CPT is also synthesized in the stems, which is consistent with other key intermediates (strictosidine, pumiloside, and deoxypumiloside), and then probably migrates from the stem to the root via transporters. (3) It is also possible that the very last steps for CPT biosynthesis are very active in the roots which leads to the high accumulation of CPT and low accumu- lation of intermediates, while these very last steps for CPT biosynthesis might be not very active in stems which results in high accumulation of intermediates and low accumulation of CPT. The identification of the rele- vant genes in O. pumila by comparable transcriptomic analyses opens the door to a more detailed exploration related to strictosidine biosynthesis. Transcriptomic analysis and candidate gene identification in CPT biosynthesis Transcriptomic analysis and candidate gene identification in CPT biosynthesis To further dissect the molecular basis of CPT biosyn- thesis, we performed deep sequencing and analysis of the transcriptome (RNA-seq) in the same O. pumila tis- sues used for extraction of metabolite intermediates. We followed the steps for transcriptome assembly, gene ex- pression qualification, and gene annotation as described in our previous study [27]. Strictosidine is a common precursor in MIA biosyn- thesis (such as vinblastine in C. roseus). We thus hypothe- sized that CPT biosynthesis in O. pumila would also prefer strictosidine as a key intermediate. We therefore ex- amined metabolite diversity in O. pumila in the prestricto- sidine stage and performed bioinformatic analyses to identify the genes in O. pumila that might be involved in strictosidine biosynthesis by looking for putative O. pumila orthologues to the corresponding genes in C. roseus [26, 28, 29]. This analysis revealed that most of the C. roseus genes encoding enzymes in the MEP pathway for biosynthesis of strictosidine exhibited very high simi- larity (76–90% identity) to O. pumila genes, including DXS, DXR, CMS, CMK, MCS, HDS, HDR, IPI, GPPS, G8H, GOR, ISY, IO, 7DLGT, 7DLH, LAMT, and SLS (Additional file 1: Table S2). However, poststrictosidine genes encoding the enzymes responsible for catharanthine and tabersonine biosynthesis showed comparatively lower similarity (44–60%, with the exception of Redox1 at 71%, Additional file 1: Table S2), for example, STR, strictosidine β-D-glucosidase (SGD), geissoschizine synthase (GS), geis- soschizine oxidase (GO), Redox1, Redox2, stemmadenine O-acetyltransferase (SAT), precondylocarpine acetate syn- thase (PAS), dehydroprecondylocarpine acetate synthase (DPAS), tabersonine synthase (TS), and catharanthine syn- thase (CS). In addition, these prestrictosidine genes from C. acuminata shared between 65 and 91% identity with C. roseus genes, with the exception of LAMT (54%), while poststrictosidine genes shared 38–60% identity (with the exception of Redox1 (66%), Additional file 1: Table S2). Even though O. pumila and C. roseus belong to the same order (Gentianales), their poststrictosidine genes share low sequence similarity. This observation indicated that O. pumila and C. roseus diverged in the Gentianales order, at least in the context of their CPT biosynthetic pathway. The low sequence identity noted here between poststric- tosidine genes in C. roseus and CPT-producing plants O. pumila and C. acuminata thus suggests a profound diver- gence in their respective biosynthetic pathways, adding confusion to our understanding of the poststrictosidine portion of their biosynthetic pathways. OpLAMT shows loganic acid methyltransferase activity in vitro OpLAMT shows loganic acid methyltransferase activity in vitro OpLAMT shows loganic acid methyltransferase activity in vitro acuminata, N. nimmoniana, and O. pumila, we searched published transcriptomic databases for these three species. Indeed, the three species each code for one LAMT enzyme. Among them, CaLAMT shared 53% identity with the C. roseus orthologue CrLAMT (ABW38009.1), while OpLAMT shared 78% identity with CrLAMT. Unfortunately, the se- quence available for NnLAMT (c51527_g1_i3) did not cover the full length of the gene [21]. However, N. nimmoniana ac- cumulated the precursor secologanin [30], implying that NnLAMT is a functional enzyme in vitro. We also character- ized the cytochrome P450 gene SLS in C. acuminata and C. roseus and their encoded proteins. The bifunctional CaSLS enzyme shared 65% identity with CrSLS (AAA33106.1). NnSLS (c54487_g1_i1) showed 76% identity with CrSLS. OpSLS shared 83% identity with CrSLS. Unlike C. roseus, the intermediates loganin, secologa- nin, and strictosidine were not detected in C. acumi- nata during a previous metabolic study [15]. Their absence indicates that the relevant methyltransferases catalyzing the methylation of loganic acid, secologanic acid, and strictosidinic acid are either missing or have lost their function due to mutations in C. acuminata. Since we observed methyl ester intermediates in O. pumila, we suspected that a functional methyltrans- ferase should exist to methylate the carboxylic acid intermediates. CrLAMT demonstrated catalytic activ- ity in C. roseus in a previous report [31]. Thus, we searched the transcriptomic database of O. pumila for CrLAMT-like sequences and identified a single pre- dicted OpLAMT with 78% identity to CrLAMT (Add- itional file 1: Table S2). To evaluate its biochemical function, we cloned OpLAMT into the bacterial ex- pression vector pET-30a and heterologously produced the protein in Escherichia coli BL21 (DE3) cells. The purified recombinant protein converted loganic acid and S-adenosyl methionine (SAM) into loganin (Fig. 3a). In addition, a microsome assay with secolo- ganic acid as a substrate for recombinant OpLAMT revealed methylation of secologanic acid (Additional file 1: Fig. S4a). These results indicated that OpLAMT is a methyltransferase catalyzing both loganic acid and secologanic acid methylation into loganin and secologanin, respectively. y Is the sequence divergence between STR proteins re- stricted to a small domain of the protein? Comparing the STR sequences from these three species with that of CrSTR showed that CaSTR1 shared 38% identity with CrSTR, while OpSTR showed 55% identity with CrSTR, and NnSTR shared 37% identity with CrSTR. OpLAMT shows loganic acid methyltransferase activity in vitro We then aligned the pro- tein sequences of LAMT, SLS, and STR (Additional file 1: Fig. S3), revealing many differences between STRs from the three species (Additional file 1: Table S3). This left unproven whether the observed sequence divergence might correlate with enzymatic activity. Phylogenetic analysis suggests greater evolutionary distance in CPT-producing plants To further understand the evolutionary relationship be- tween these proteins and their encoding genes, we next performed a molecular phylogenetic analysis of STRs from these three plant species, which revealed that they clustered in different clades (Additional file 1: Fig. S2b). This finding was in agreement with their relative posi- tions in the general phylogenetic tree, which also indi- cated that they belong to different clades of flowering plants (Fig. 1a). In addition, we performed phylogenetic analyses of SLS and LAMT protein sequences deduced from available transcriptomic data. The six OpSLSs were divided into three clades and showed high similarity to NnSLS, DcSLS, and CrSLSs (CYP72A1 and CrCYP72C). In contrast, the CaSLS proteins CaCYP72A565 and CaCYP72A610 formed a fourth clade (Additional file 1: Fig. S2c). The putative OpLAMT from O. pumila clus- tered away from other LAMTs, such as CaLAMT and OeLAMT, suggesting greater evolutionary distance (Additional file 1: Fig. S2d). OpSTR shows promiscuous Pictet-Spengler reaction activity in vitro Based on the results of metabolite profiling, we postu- lated that another STR-like enzyme might be involved in strictosidinic acid biosynthesis. However, a search of the O. pumila transcriptome database identified only one candidate, OpSTR. To validate the phenomenon of the coexistence of strictosidine and strictosidinic acid in O. pumila, the activities of OpSTR towards secologanin and secologanic acid were determined in recombinant enzyme assays. We purified recombinant OpSTR to test its activity against secologanin and secologanic acid. Interestingly, LC-MS analysis indicated that OpSTR can convert both secologanin and secologanic acid into 3α-(S)- strictosidine and 3-(S), 21-(S)-strictosidinic acid in vitro, respectively (Fig. 3b, compared with CrSTR). To compare the substrate specificity of OpSTR, we performed a time-course assay (Fig. 3c). OpSTR showed greater activity towards secologanin than to- wards secologanic acid as substrates. We quantified the products of the reactions against a standard curve for strictosidine and strictosidinic acid based on LC- LAMTs, SLSs, and STRs from three genera of CPT-producing plants provide clues regarding CPT biosynthesis LAMTs, SLSs, and STRs from three genera of CPT-producing plants provide clues regarding CPT biosynthesis Expression patterns of prestrictosidine genes in O. pumila Armed with the putative O. pumila orthologues for genes involved in strictosidine biosynthesis, we To determine whether the gene encoding LAMT is present in the three representative CPT-producing species C. Page 6 of 16 Yang et al. BMC Biology (2021) 19:122 Yang et al. BMC Biology (2021) 19:122 Page 6 of 16 In vitro biochemical characterization of OpLAMT, OpSLS, and OpSTR Following the identification of O. pumila genes encoding putative enzymes participating in CPT biosynthesis, the next step was to investigate their biocatalytic functions. Yang et al. BMC Biology (2021) 19:122 (2021) 19:122 Yang et al. BMC Biology Page 7 of 16 A B C min 9 9.5 10 10.5 11 L SL L SL min 9 9.5 10 10.5 11 min 8 9 10 LA SLA ii iv i iii v vi vii ii iv i iii v vii i vi D E F ii iv min 8 10 12 14 16 18 20 mAU 0 200 400 600 800 i iii v vi SAMLA L x105 0 1 2 x105 0 1 2 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 iii strictosidinic acid x105 0 1 2 x105 1 2 i ii strictosidine min iv Fig. 3 (See legend on next page.) C A D E F Page 8 of 16 Yang et al. BMC Biology (2021) 19:122 (See figure on previous page.) Fig. 3 In vitro biochemical characterization of successive functional enzymes (OpLAMT, OpSLS, OpSTR) involved in CPT biosynthesis in O. pumila. a Loganic acid methyltransferase HPLC assay with purified recombinant OpLAMT enzyme. (i) OpLAMT assay with loganic acid and SAM, (ii) boiled OpLAMT assay with loganic acid and SAM, (iii) OpLAMT assay with loganic acid, (iv) loganin (L) standard, (v) loganic acid (LA) standard, and (vi) S- adenosyl methionine (SAM) standard. b OpSTR LC-MS assay with recombinant OpSTR enzyme. Purified recombinant protein was assayed for strictosidine activity in reaction mixtures with secologanin/secologanic acid and tryptamine. (i) boiled STR assay with secologanin, (ii) STR assay with secologanin, (iii) boiled STR assay with secologanic acid, and (iv) STR assay with secologanic acid. c Time-course assay of STR activity. OpSTR assay with secologanin (producing strictosidine) and secologanic acid (producing strictosidinic acid) (three repeats). The assays were quenched at 2 min, 4 min, 6 min, 8 min, and 10 min and then measured by LC-MS. Curve fitting was performed by GraphPad Prism 8. d Six OpSLS assays with loganic acid (LA). The solid lines in black indicate samples from the microsome assay and secologanic acid standard (SLA). The dashed lines in grey indicate samples from the boiled microsome assay (control) and loganic acid standard. OpSLS shows secologanin synthase activity only To test the catalytic ability of our candidates in convert- ing loganin and loganic acid, we cloned the six OpSLS genes (OpSLS1, CYP72A865; OpSLS2, CYP72A866; OpSLS3, CYP72A867; OpSLS4, CYP72A868; OpSLS5, CYP72A869; OpSLS6, CYP72A870) identified from the transcriptomic analysis into the yeast expression vector pESC-Leu and transformed the resulting constructs into yeast (strain WAT11). We extracted microsomes for ac- tivity assays: five OpSLS proteins (OpSLS1, OpSLS3, OpSLS4, OpSLS5, OpSLS6) exhibited secologanin syn- thase activity but not secologanic acid activity. However, OpSLS2 showed no detectable activity towards either secologanin or secologanic acid (Fig. 3d and e). These results demonstrate that the activities of OpSLS pro- teins are distinct from those of CaSLSs and reflect their position within the phylogenetic tree (Additional file 1: Fig. S2c). Critically, these OpSLS assays also support a role for loganin and secologanin in CPT biosynthesis in O. pumila. STR assays from CPT-producing plants prove the validity of two parallel pathways in the plant kingdom STR assays from CPT-producing plants prove the validity of two parallel pathways in the plant kingdom MS peak integrations (Additional file 1: Fig. S5). We then fitted the data in GraphPad Prism 8 and calcu- lated the resulting velocity (slope) ratio of assays to- wards secologanin and secologanic acid: OpSTR displayed an activity towards secologanin 150 times higher than that for secologanic acid in the 10 min of the assay (Fig. 3c). In addition, we furtherly character- ized the kinetic parameters of OpSTR using LCMS by monitoring the production of strictosidine and stricto- sidinic acid. The results of the kinetic analysis are as follows: Kcat/Km = 8.23 min−1 mM−1 for secologanin and Kcat/Km = 0.00995 min−1 mM−1for secologanic acid (Additional file 1: Table S4). Here, we character- ized OpSTR showing promiscuous Pictet-Spengler re- action activity in vitro and supposed OpSTR preferred secologanin as the substrate. MS peak integrations (Additional file 1: Fig. S5). We then fitted the data in GraphPad Prism 8 and calcu- lated the resulting velocity (slope) ratio of assays to- wards secologanin and secologanic acid: OpSTR displayed an activity towards secologanin 150 times higher than that for secologanic acid in the 10 min of the assay (Fig. 3c). In addition, we furtherly character- ized the kinetic parameters of OpSTR using LCMS by monitoring the production of strictosidine and stricto- sidinic acid. The results of the kinetic analysis are as follows: Kcat/Km = 8.23 min−1 mM−1 for secologanin and Kcat/Km = 0.00995 min−1 mM−1for secologanic acid (Additional file 1: Table S4). Here, we character- ized OpSTR showing promiscuous Pictet-Spengler re- action activity in vitro and supposed OpSTR preferred secologanin as the substrate. y p g p p y of two parallel pathways in the plant kingdom To better understand the differences in their CPT biosynthetic pathways, we cloned the STR genes from the three CPT-producing plants C. acuminata, O. pumila, and N. nimmoniana into the bacterial expres- sion vector pET-30a and introduced the resulting constructs into E. coli BL21 (DE3). We then evaluated the enzymatic activities of OpSTR, CaSTRs, and NnSTR towards secologanic acid and secologanin by LC-MS (Fig. 4). OpSTR and CaSTR2 used both seco- loganin and secologanic acid activity as their sub- strates. In contrast, NnSTR, CaSTR1, and CaSTR3 displayed substrate specificity towards secologanin only. STR assays from CPT-producing plants prove the validity of two parallel pathways in the plant kingdom The observed specificities of STR enzymes indi- cated that strictosidine may play a major role in CPT biosynthesis, at least in O. pumila and N. nimmoni- ana. The activity exhibited by NnSTR is consistent with the metabolite profile of N. nimmoniana, as these plants contain only secologanin and no secolo- ganic acid [30]. It remains unclear why the three CaSTRs each showed distinct enzymatic activity, even though their activity towards strictosidinic acid con- firms the detection and isolation of strictosidinic acid in C. acuminata [15]. In vitro biochemical characterization of OpLAMT, OpSLS, and OpSTR (i) Loganic acid and secologanic acid standards, (ii) OpSLS6, (iii) OpSLS5, (iv) OpSLS4, (v) OpSLS3, vi) OpSLS2, and (vii) OpSLS1. Wavelength, 254 nm. e Six OpSLSs assay with loganin (L). The solid lines in black indicate samples from the microsome assay and secologanin standard (SL). The dashed lines in grey indicate samples from the boiled microsome assay (control) and loganin standard. (i) Loganic acid and secologanic acid standards, (ii) OpSLS6, (iii) OpSLS5, (iv) OpSLS4, (v) OpSLS3, (vi) OpSLS2, and (vii) OpSLS1. Wavelength, 254 nm. f Summary of the CPT biosynthesis pathway in O. pumila. Loganic acid is converted into loganin in a reaction catalyzed by OpLAMT, and then loganin is converted into secologanin by OpSLSs. Secologanin and tryptamine are then condensed into strictosidine carried out by OpSTR, which is involved in CPT biosynthesis in O. pumila In vivo labelling studies demonstrate that strictosidine is a key intermediate involved in CPT biosynthesis in O. pumila The detection of both the acid and methyl ester forms of the intermediates in the biosynthesis of strictosidinic acid and strictosidine in O. pumila lends support to our hypothesis that parallel biosynthetic pathways act in the prestrictosidine stage in this species. To further dissect metabolite flux in the poststrictosidine stage of CPT bio- synthesis, key isotopically labelled metabolic intermedi- ates are necessary for in vivo feeding studies. Starting from commercially available deuterated d5-L-tryptophan Page 9 of 16 Yang et al. BMC Biology (2021) 19:122 Yang et al. BMC Biology Fig. 4 STR assays from CPT-producing plants prove the validity of two parallel pathways in the plant kingdom. a OpSTR, CaSTR, and NnSTR activities towards secologanin. (i) CaSTR1, (ii) CaSTR2, (iii) CaSTR3, (iv) NnSTR, and (v) OpSTR. The dashed box indicates the expected elution time of the STR product strictosidine. b OpSTR, CaSTR, and NnSTR activities towards secologanic acid. (i) CaSTR1, (ii) CaSTR2, (iii) CaSTR3, (iv) NnSTR, and (v) OpSTR. The dashed box indicates the expected elution time of the STR product strictosidinic acid Fig. 4 STR assays from CPT-producing plants prove the validity of two parallel pathways in the plant kingdom. a OpSTR, CaSTR, and NnSTR activities towards secologanin. (i) CaSTR1, (ii) CaSTR2, (iii) CaSTR3, (iv) NnSTR, and (v) OpSTR. The dashed box indicates the expected elution time of the STR product strictosidine. b OpSTR, CaSTR, and NnSTR activities towards secologanic acid. (i) CaSTR1, (ii) CaSTR2, (iii) CaSTR3, (iv) NnSTR, and (v) OpSTR. The dashed box indicates the expected elution time of the STR product strictosidinic acid feeding experiment. Meanwhile, no d4-strictosidinic acid was detected in the d4-strictosidine feeding experiment, and no d4-strictosidine was detected in the d4-strictosi- dinic acid feeding experiment (Additional file 1: Fig. S9). It indicates that d4-strictosidine and d4-strictosidinic acid will not be converted into each other in vivo. Inter- estingly, we detected the poststrictosidine compounds pumiloside and deoxypumiloside, with a pentacyclic pyr- roloquinoline scaffold [19], as well as CPT, in the ex- tracts of plants incubated with d4-strictosidine but not with d4-strictosidinic acid compared to the extracts of plants incubated with d5-tryptophan (Fig. 6). (d5-Trp), we chemoenzymatically synthesized three deuterium-labelled metabolites by using purified recom- binant proteins (OpTDC, OpSTR) (Fig. 5a, b and Add- itional file 1: Fig. S6). In vivo labelling studies demonstrate that strictosidine is a key intermediate involved in CPT biosynthesis in O. pumila We purified the deuterium- labelled products and characterized them by LC-MS to confirm that they harboured the correct number of deu- terium atoms due to labelling (Additional file 1: Fig. S7 and S8). With these synthesized deuterated intermedi- ates, d4-strictosidine and d4-strictosidinic acid, as well as d5-L-tryptophan (d5-Trp), we conducted in vivo labelling studies by feeding O. pumila apical cuttings with the above-deuterated metabolites (Fig. 5c). To trace the CPT biosynthetic pathway, we performed feeding experiments with each deuterium-labelled key intermediate (d5-tryptophan, d4-strictosidine, and d4- strictosidinic acid) provided individually at a concentra- tion of 250 μM (Fig. 6). We incubated apical cuttings from wild-type plants in an aqueous solution with d5- tryptophan, d4-strictosidine, or d4-strictosidinic acid. After 45 days, we collected the stems and leaves for me- tabolite analysis via LC-MS. As expected, d4-strictosidine and d4-strictosidinic acid were detected in d5-tryptophan O. pumila generates both carboxylic acids and methyl esters as proposed precursors in CPT biosynthesis CPT was first identified in C. acuminata and later found in species belonging to unrelated angiosperm orders that were successively discovered [2], revealing an apparently random phylogenetic distribution of CPT production. Among known CPT-producing plants, the three repre- sentative species N. nimmoniana, C. acuminata, and O. Page 10 of 16 Yang et al. BMC Biology (2021) 19:122 Yang et al. BMC Biology Fig. 5 In vivo labelling to study the biosynthesis of O. pumila apical cuttings by feeding with deuterated metabolites. a, b Selected deuterium intermediates in the CPT biosynthesis pathway and their chemoenzymatic synthesis. c O. pumila apical cuttings were given the deuterated metabolites d4-strictosidine and d4-strictosidinic acid as feeding precursors. d Labelled poststrictosidine metabolite products with pentacyclic pyrroloquinoline scaffolds detected by in vivo feeding experiments Fig. 5 In vivo labelling to study the biosynthesis of O. pumila apical cuttings by feeding with deuterated metabolites. a, b Selected deuterium intermediates in the CPT biosynthesis pathway and their chemoenzymatic synthesis. c O. pumila apical cuttings were given the deuterated metabolites d4-strictosidine and d4-strictosidinic acid as feeding precursors. d Labelled poststrictosidine metabolite products with pentacyclic pyrroloquinoline scaffolds detected by in vivo feeding experiments pumila (Fig. 1b) exhibit chemical diversity in their CPT biosynthesis pathways. In many MIA-producing plants [14], such as Apocynaceae [32], Rubiaceae [7, 33], and Icacinaceae [30], strictosidine accumulates as a key intermediate in CPT biosynthesis and is then activated by conversion via the enzyme strictosidine β-D-glucosi- dase (SGD), whose unstable product strictosidine agly- cone is rapidly converted into thousands of MIAs, such as vindoline, vinblastine, and vincristine, in C. roseus [25, 26, 34]. However, although C. acuminata produces MIAs, it was recently reported to accumulate only carboxylic acid precursors (loganic acid, secologanic acid, and strictosidinic acid). In agreement with this, strictosidinic acid was isolated from C. acuminata ex- tracts [2, 15, 16, 35]. It is thought that strictosidine can also act as a key intermediate in CPT biosynthesis [22, 33]. However, C. acuminata appears to favour strictosi- dinic acid instead of strictosidine for CPT biosynthesis [15]. The coexistence of both carboxylic acids (loganic acid, secologanic acid, and strictosidinic acid) and me- thyl esters (loganin, secologanin, and strictosidine) in O. pumila was explained by global untargeted metabolite Fig. 6 In vivo labelling studies demonstrate that strictosidine is a key intermediate involved in CPT biosynthesis in O. pumila. O. pumila generates both carboxylic acids and methyl esters as proposed precursors in CPT biosynthesis a–c Metabolites (d5-tryptophan [i], d4-strictosidine [ii], d4-strictosidinic acid [iii]) detected by LC-MS in extracts from feeding experiments with three different deuterium-labelled substrates: d4- pumiloside (a), d4-deoxypumiloside (b), and d4-camptothecin (c). Extracted ion chromatograms (EICs) of deuterium-labelled intermediates were compared studies demonstrate that strictosidine is a key intermediate involved in CPT biosynthesis Fig. 6 In vivo labelling studies demonstrate that strictosidine is a key intermediate involved in CPT biosynthesis in O. pumila. a–c Metabolites (d5-tryptophan [i], d4-strictosidine [ii], d4-strictosidinic acid [iii]) detected by LC-MS in extracts from feeding experiments with three different deuterium-labelled substrates: d4- pumiloside (a), d4-deoxypumiloside (b), and d4-camptothecin (c). Extracted ion chromatograms (EICs) of deuterium-labelled intermediates were compared Page 11 of 16 Page 11 of 16 Yang et al. BMC Biology (2021) 19:122 proteins showed activity towards loganin, although no OpSLS exhibited any activity towards loganic acid. These results indicate that either OpSLS activity towards loganic acid is too low to be detectable or that another OpSLS plays a role in converting loganic acid into seco- loganic acid. In an earlier study, the activities of OpSTR were determined in recombinant enzyme assays [23]. They showed that OpSTR converted tryptamine and secologanin into strictosidine. Here, we first determined that OpSTR was a promiscuous enzyme capable of con- verting secologanic acid or secologanin into strictosidi- nic acid or strictosidine, respectively (Fig. 3b and c). The results from competition (Additional file 1: Fig. S4b) and time-course experiments (Fig. 3c) indicated that OpSTR converted secologanin and tryptamine into strictosidine as its main product rather than catalyzing the formation of strictosidinic acid from secologanic acid and trypta- mine (Additional file 1: Fig. S4b). This observation also implies that strictosidine may play a major role in CPT biosynthesis. Collectively, methyl ester intermediates (loganin, secologanin, strictosidine) and successive func- tional enzymes (OpLAMT, OpSLS, OpSTR) are indeed involved in CPT biosynthesis in O. pumila (Fig. 3f). Based on their biochemical characterization, we postu- late that strictosidine, not strictosidinic acid, is the main intermediate involved in CPT biosynthesis. To validate our hypothesis, we performed feeding experiments with proposed labelled precursors in vivo to determine their biotransformation profile. profiling in our study, which confirmed the results of previous studies that detected either strictosidinic acid or strictosidine in O. pumila [22, 23]. Interestingly, it was reported that only secologanin, the precursor of strictosidine, was detected in N. nimmoniana [21]. Even though both C. acuminata and N. O. pumila generates both carboxylic acids and methyl esters as proposed precursors in CPT biosynthesis nimmoniana are woody plants, they exhibit substantial differences in their key intermediates for CPT biosynthesis. In our study, we detected both strictosidine and strictosidinic acid in the leaves and roots of N. nimmoniana (Additional file 1: Fig. S10), indicating that N. nimmoniana and O. pumila accumulate both carboxylic acids and methyl esters as their proposed precursors during CPT biosynthesis. In addition, we identified loganic acid, loganin, secologanic acid, and secologanin in O. pumila. Collectively, these results strongly imply the evolution of at least two routes for CPT biosynthesis in CPT-producing plants. Carbox- ylic acid intermediates may be considered markers for the biosynthesis pathway seen in C. acuminata, while the coexistence of carboxylic acids and methyl esters in O. pumila and N. nimmoniana identifies another path to CPT. Here, we observed that strictosidinic acid accumu- lated to slightly higher levels than strictosidine in plants based on the quantification of metabolites. We hypothesize that 3α-(S)-strictosidine, but not 3-(S), 21- (S)-strictosidinic acid, is the key intermediate incorpo- rated into CPT biosynthesis. Thus, 3-(S), 21-(S)-strictosi- dinic acid is probably the byproduct in the CPT biosynthetic pathway in O. pumila, resulting in high accumulation. Evolution resulted in large differences in three representative CPT-producing plants Most MIA-producing plants use strictosidine rather than strictosidinic acid as their central intermediate. We com- pared the genes involved in the CPT biosynthesis path- way with those of C. roseus, the best-studied MIA- producing plant. By comparing the enzymes involved in vincristine biosynthesis in C. roseus with those of C. acu- minata and O. pumila, the enzymes involved in prestric- tosidine all shared high identity. However, O. pumila appears to diverge in the poststrictosidine pathway according to low identity with the genes involved in the biosynthesis of specific MIAs such as vincristine (Add- itional file 1: Table S2), indicating that CPT-producing plants diverged from C. roseus. We hypothesize that the three CPT-producing plants probably utilize different precursors and show different enzymatic activities, indi- cative of independent evolution. Our in vivo deuterium- labelled metabolite feeding studies confirmed that stric- tosidine is the key intermediate in the poststrictosidine CPT biosynthetic pathway in O. pumila. The phylogen- etic tree also supported the independent evolution of N. nimmoniana, C. acuminata, and O. pumila (Fig. 1b). Resistance to CPT treatment is a hallmark of CPT- producing plants [17, 36, 37]. We therefore phylogenet- ically analysed DNA topoisomerase I sequences from several flowering plants. In CPT-producing and nonpro- ducing species, we uncovered three key amino acid mu- tation sites related to CPT resistance (Additional file 1: Fig. S11). The endogenous biological function of CPT as a chemical defence molecule in host plants against biotic or abiotic insults is currently unknown. DNA topoisom- erase I enzymes in these plants are resistant to the en- dogenous CPT they accumulate, presumably due to a mutation in CPT binding site (Additional file 1: Fig. S11) [17, 36, 37]. O. pumila, Ophiorrhiza liukiuensis, and C. acuminata share the N-to-S mutation. N. nimmoniana and C. acuminata share a specific N-to-K mutation. O. pumila and O. liukiuensis share a specific G-to-S muta- tion. Although the three plant species belong to three different orders (Gentianales (O. pumila), Icacinales (N. nimmoniana), and Nyssaceae (C. acuminata)), they dis- play both divergence and some similarities regarding the genetic basis of their CPT resistance mechanism. p ( g ) Why might C. acuminata produce strictosidinic acid as its intermediate for CPT biosynthesis? The enzymes involved in CPT biosynthesis in C. acuminata may pro- vide some clues. Enzymatic evidence for the coexistence of carboxylic acids and methyl esters in O. pumila y p Since both strictosidinic acid and strictosidine accumu- lated in all O. pumila tissues, we were unsure which might be involved in CPT biosynthesis in O. pumila. Satisfyingly, however, detectable amounts of labelled products with a pentacyclic pyrroloquinoline scaffold ac- cumulated in tissues incubated with d4-strictosidine and d5-L-tryptophan (Fig. 6). However, we did not detect any labelled poststrictosidine compounds in extracts of tis- sues incubated with d4-strictosidinic acid (Fig. 6). These results are in sharp contrast with the observation that strictosidinic acid plays the role of a major precursor in CPT biosynthesis in C. acuminata but not strictosidine [15]. In addition, our metabolite analysis first showed that N. nimmoniana contained both strictosidine and strictosidinic acid (Additional file 1: Fig. S10), in contrast to only strictosidinic acid in C. acuminata [15]. These results indicate that both methyl ester derivatives and carboxylic acid derivatives coexist in N. nimmoniana, as in O. pumila. Collectively, our results indicate that stric- tosidine may play the same key role in O. pumila and N. nimmoniana and that strictosidinic acid fills in C. acu- minata. Based on these observations, strictosidine, and To better understand the enzymatic basis of the chem- ical diversity underlying CPT biosynthesis, we performed transcriptome sequencing and analysis. In most MIA- producing species, such as C. roseus, secologanin and tryptamine are condensed into strictosidine; therefore, we searched and analysed the gene candidates for the prestrictosidine steps in CPT biosynthesis in O. pumila. As visualized by coexpression analysis, genes closely re- lated to OpSTR (OpIO, Op7DLGT, Op7DLH, OpLAMT, OpSLS, OpTDC) were highly expressed in stems and roots. Loganic acid O-methyltransferase (LAMT), seco- loganin synthase (SLS), and strictosidine synthase (STR) are involved in the formation of carboxylic acids and methyl esters, resulting in strictosidine and strictosidinic acid. We functionally characterized these enzymes in vitro (Fig. 3). We first demonstrated OpLAMT to be a promiscuous enzyme converting loganic acid and secolo- ganic acid into loganin and secologanin, respectively (Fig. 3a and Additional file 1: Fig. S4a). By searching the O. pumila transcriptome database, we identified six OpSLS genes. Surprisingly, five out of the six OpSLS Page 12 of 16 Page 12 of 16 Yang et al. BMC Biology (2021) 19:122 Yang et al. BMC Biology (2021) 19:122 secologanic acid and secologanin show divergences in enzymatic activity (Fig. 4) and clustered into different clades in the phylogenetic tree (Additional file 1: Fig. Enzymatic evidence for the coexistence of carboxylic acids and methyl esters in O. pumila S2b), which is consistent with the species tree (Fig. 1a). These observations indicate that the CPT biosynthesis pathway may have evolved divergently in flowering plants based on a comparison of the enzymes involved and the metabolite profiles in the three plant species. Thus, CPT biosynthesis in different CPT-producing plants likely utilizes two different routes. One is the traditional iridoid pathway, whereby loganin is converted into secologanin and then strictosidine by CrSLS and CrSTR and later incorporated into MIA biosynthesis. The second route is the carboxylic acid pathway, in which loganin acid is converted into secologanic acid and then strictosidinic acid by CaSLSs and CaSTRs, fi- nally producing CPT through a series of bioconversion reactions in C. acuminata [15, 16]. In addition, NnSTR showed secologanin activity, which indicates strictosi- dine as the key intermediate incorporated into the CPT biosynthetic pathway in O. pumila and N. nimmoniana. not strictosidinic acid, is very likely a central intermedi- ate in CPT biosynthesis in O. pumila, especially in the poststrictosidine stage. We further suggest that the CPT biosynthetic pathway in O. pumila is similar to most previously characterized MIA pathways, such as vin- blastine biosynthesis in C. roseus and that C. acuminata differs from the more common CPT biosynthesis route. To further investigate the divergence among CPT- producing plants, we compared homologues across CPT-producing plants by biochemical assay. not strictosidinic acid, is very likely a central intermedi- ate in CPT biosynthesis in O. pumila, especially in the poststrictosidine stage. We further suggest that the CPT biosynthetic pathway in O. pumila is similar to most previously characterized MIA pathways, such as vin- blastine biosynthesis in C. roseus and that C. acuminata differs from the more common CPT biosynthesis route. To further investigate the divergence among CPT- producing plants, we compared homologues across CPT-producing plants by biochemical assay. Evolution resulted in large differences in three representative CPT-producing plants Due to the absence of loganin, secolo- ganin, and strictosidine, we postulate that CaLAMT should not be a functional loganic acid methyltransfer- ase. The recently characterized bifunctional CaSLS [16] can convert both loganin and loganic acid with similar catalytic efficiency. According to the previous study [15], we postulate that CaSTRs probably show secologanic acid activity. However, we first discovered that strictosi- dine synthases (STRs) in C. acuminata mainly exhibited secologanin activity, and only one CaSTR showed de- tectable activity towards both secologanin and secolo- ganic acid in our study (Fig. 4). The lack of methyl ester intermediates, combined with environmental pressures (biotic and abiotic stress), may have pushed C. acumi- nata to evolve a strictosidinic acid-dependent branch of the CPT biosynthetic pathway. At the same time, STRs from the three CPT-producing plants towards Gene expression analysis We determined the expression levels of most genes from the MEP pathway to the biosynthesis of strictosidine. The complete list of genes analysed here is as follows (further in- formation and accession numbers are provided in Additional file 1: Table S2): 1-deoxy-D-xylulose-5-phosphate synthase (DXS), 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR), 4-diphosphocytidylmethylerythritol 2-phosphate syn- thase (CMS), 4-diphosphocytidyl-2C-methyl-D-erythritol kin- ase (CMK), 2C-methyl-D-erythritol-2,4-cyclodiphosphate synthase (MCS), 1-hydroxy-2-methylbutenyl-4-diphosphate synthase (HDS), 1-hydroxy-2-methylbutenyl 4-diphosphate reductase (HDR), isopentenyl diphosphate isomerase (IPI), geraniol 8-hydroxylase (G8H), 8-hydroxygeraniol oxidore- ductase (GOR), iridodial synthase (ISY), iridoid oxidase (IO), 7-deoxyloganetic acid glucosyltransferase (7DLGT), 7- deoxyloganic acid hydroxylase (7DLH), loganic acid O- Application in CPT production by metabolic engineering approaches Medicinal plants accumulate very low levels of natural products, including various drugs with clinical applica- tions, such as vinblastine and vincristine in C. roseus or CPT in C. acuminata and O. pumila [8, 11]. Here, our study provides a path towards improving CPT produc- tion in Ophiorrhiza species. OpLAMT is the key enzyme that controls the methylation of carboxylic acid interme- diates. Together with OpSTR, OpLAMT produces both strictosidinic acid and strictosidine. Strictosidine and Page 13 of 16 Page 13 of 16 Yang et al. BMC Biology (2021) 19:122 powder); the methanol solution also contained 50 μM telmisartan as an internal standard. We vortexed the so- lution for 1 min, followed by extraction by ultrasonica- tion at 4 °C for 30 min in an ice bath for 1 h. We then centrifuged all samples at 4 °C at 12,000g for 10 min, fil- tered the supernatants through a 0.22-μm filter mem- brane, and injected 1 μL of each sample into an Agilent 1290 UHPLC system coupled to an Agilent 6545 Q-TOF ESI high-resolution mass spectrometer (HRMS) for ana- lysis. The column used for separation was an Agilent 300 Extend-C18 (4.6 × 150 mm, 3.5 μm) with the temperature set to 40 °C. Mobile phases A (H2O + 0.1% formic acid) and B (acetonitrile + 0.1% formic acid) were run in the following gradient programme at 0.3 mL/min: 0–1 min, 5% B; 1–3 min, 5–15% B; 3–11 min, 15–24% B; 11–18 min, 24–26% B; 18–30 min, 26–50% B; 30–33 min, 50–70% B; 33–34.5 min, 70–98% B; 34.5–38 min, 98% B; and 38–38.5 min, 98–5% B. The mass spectrom- eter was set to positive mode with a mass range between 70 and 1000 m/z. Other parameters were as follows: ac- quisition rate, 1.2 spectra/s; acquisition time, 833.3 ms/ spectrum; gas temperature, 300 °C; drying gas, 6 L/min; nebulizer, 35 psig; vcap, 4000 V; fragmentor, 135 V; skimmer, 65 V; and oct 1 RF Vpp, 750 V. The collision energies used for MS fragmentation analysis were 20 V, 40 V, and 65 V. strictosidinic acid coexist in O. pumila, and strictosidine production is a key factor in enhancing the production of CPT. Therefore, remodelling the pathway with a secologanin-specific STR and overexpression of LAMT might prove helpful to increase CPT production. In addition, our results provide some clues for the meta- bolic engineering of different CPT-producing plants in a microbe chassis. Transcriptome sequencing and bioinformatic analysis Transcriptome sequencing and bioinformatic analysis We extracted total RNA from four different O. pumila tissues: leaves, stems, roots, and hairy roots. We pre- pared RNA sequencing libraries using the TIANGEN RNAprep Pure Plant Kit. We sequenced the resulting li- braries on a NovaSeq 6000 platform according to the manufacturer’s instructions. Transcriptome assembly, gene quantification, and annotation were carried out as previously reported [27]. Plant materials used in this study O. pumila plants and hairy roots were obtained as re- ported previously [7]. Different tissues (leaves, stems, and roots) of 6-month-old O. pumila sterile seedlings and 3-month-old hairy roots grown on Gamborg’s B5 solid medium plates were collected for RNA-seq and metabolite profiling, respectively. In addition, apical cut- tings of 6-month-old O. pumila plantlets were used in feeding experiments and cultured in Gamborg’s B5 li- quid medium with deuterium-labelled substrates in 15 mL polypropylene round-bottom tubes (under 16 light, 8 h dark, 25 °C). After 45 days, the plant materials were used for metabolite extraction and LC-MS analysis. Conclusions Most MIA-producing plants are thought to use strictosi- dine as the central intermediate in the biosynthesis of end-products, but the full CPT biosynthetic pathway in CPT-producing plants remains unclear. Surprisingly, it was found that C. acuminata uses strictosidinic acid in- stead of strictosidine as an intermediate for CPT biosyn- thesis. However, O. pumila, another representative CPT- producing plant, exhibits metabolite diversity in the cen- tral intermediates consisting of both 3-(S), 21-(S)-stricto- sidinic acid and 3α-(S)-strictosidine. We characterized a series of candidate genes in the prestrictosidine branch of the CPT pathway and compared them across the three representative CPT-producing plants O. pumila, C. acuminata, and N. nimmoniana. Our results show that enzymes likely to be involved in CPT biosynthesis in O. pumila, C. acuminata, and N. nimmoniana have evolved divergently. Overall, our new data about CPT biosyn- thesis in O. pumila suggest evolutionary divergence in CPT-producing plants. In addition, the promiscuity of LAMT and STR enzymes may pave the way towards the industrial production of CPT through enzymatic or metabolic engineering approaches. Plasmid construction and enzymes preparation Plasmid construction and enzymes preparation We extracted the total RNA from plant tissues with the SPARKeasy RNA extraction kit (Sparkjade Science Co., Ltd.). Genes of interest were amplified by PCR from cDNA using the primers listed in Additional file 1: Table S5. Escherichia coli strain Top10 was used as the cloning host for plasmid construction, and E. coli BL21 (DE3) was used as the host for re- combinant protein production. We introduced the plasmids pET30a-OpTDC, pET30a-OpLAMT, pET30a- OpSTR, pET30a-CaSTR1, pET30a-CaSTR2, pET30a- CaSTR3, and pET30a-NnSTR individually into E. coli BL21(DE3). We inoculated 10 mL LB medium with single colonies for each construct, followed by cultiva- tion at 37 °C for 12 h. We then transferred the cul- ture into 1 L of fresh LB medium with kanamycin (50 mg/L) until the OD600 reached 0.6. For protein expression, we added 200 μM isopropyl-β-D-thiogalac- toside (IPTG) to the cultures to induce protein pro- duction over 18 h at 16 °C. After collection by centrifugation, we suspended the cell pellets in 30 mL of lysis buffer (Sangon Biotech, B548117, consisting of 50 mM potassium phosphate buffer, pH 7.5, 100 mM NaCl and 5% glycerol) and lysed them via a Union- Biotech high-pressure homogenizer. After centrifuga- tion at 20,000g for 40 min, we loaded the supernatant onto a column with Ni2+ resin. We used lysis buffer containing increasing concentrations of imidazole (25 mM, 50 mM, 100 mM, and 500 mM) to wash the column. Each fraction was sampled by SDS-PAGE analysis. We concentrated and desalted the target proteins on a PD-10 column and determined the pro- tein concentration by Bradford assay using BSA to generate a standard curve. For OpSTR, a typical enzymatic assay was carried out in 100 μL aliquots of a reaction mixture consisting of 50 mM PBS buffer (pH 7.5), 1 mM tryptamine, and 1 mM secologanin or secologanic acid in the presence of stric- tosidine synthase (STR) (1 mg/mL). We incubated the reaction mixtures at 30 °C for 2 h and quenched the reactions with the addition of 100 μL of methanol and vortexing for 5 min. After centrifugation at 12,000g for 5 min and filtration, we used a 10-μL sample for LC-MS analysis. The column applied for analysis was an Agilent Eclipse plus C18 column (4.6 × 150 mm, 3.5 μm) on an Agilent 1260-6125+ LC-MS system with the temperature set at 35 °C. Phylogenetic analyses y g y We downloaded the protein sequences for DNA topo- isomerase I, STRs, SLSs, and LAMTs from the National Center for Biotechnology Information (NCBI) and the predicted protein sequences from the O. pumila tran- scriptome database for phylogenetic analyses. We aligned sequences with the help of ClustalW and gener- ated the corresponding trees by the neighbour-joining method with the JTT model and bootstrap values set to 1,000 [38]. Enzymatic assays of OpLAMT and OpSTR and HPLC-MS analysis For OpLAMT, we performed a typical enzymatic assay in 100 μL aliquots of a reaction mixture containing 50 mM phosphate-buffered saline (PBS) buffer (pH 7.5), 1 mM loganic acid, and 1 mM S-adenosyl methionine (SAM) in the presence of loganic acid methyltransferase (LAMT) (1 mg/mL). Plasmid construction and enzymes preparation Mobile phases A (H2O + 0.1% formic acid) and B (acetonitrile) were run in the following gradient programme at 0.8 mL/min: 0–3 min, 5% B; 3–12 min, 5–30% B; 12–15 min, 30–95% B; 15–18 min, 95% B; 18–21 min, 95–5% B; and 21–24 min, 5% B. A 10-μL sam- ple was injected for analysis. OpLAMT assays were moni- tored at 254 nm, and OpSTR assays were monitored by the extracted ion chromatogram of the products. Nontargeted metabolites analysis To analyse the metabolites of different plant tissues and hairy roots, we ground the above samples (including leaves, stems, roots, and hairy roots; Additional file 1: Fig. S1) to a fine powder under liquid nitrogen. We then added 500 μL of methanol to each sample (50 mg of Page 14 of 16 Page 14 of 16 Yang et al. BMC Biology (2021) 19:122 methyltransferase (LAMT), secologanin synthase (SLS), and strictosidine synthase (STR). We visualized the gene expres- sion levels and their hierarchical clustering as a heatmap (Additional file 1: Fig. S2a). glucose as the carbon source. Yeast transformants were grown in 200 mL of synthetic dropout medium lacking leucine with glucose until they reached the logarithmic phase, at which point we harvested cells by centrifuga- tion at 6000g for 5 min. We resuspended the cells in synthetic dropout medium lacking leucine with galactose as a carbon source to induce protein production for 36 h before collection. We prepared microsomes as previ- ously reported [39]. Acknowledgements h k We thank Dr. Wenjuan Yuan and Mr. Shizheng Bu in the Core Facility Centre of the Shanghai Institute of Plant Physiology and Ecology for the mass spectrometry assistance. The authors also thank Prof. Shuai Zhan and Dr. Qun Liu for the helpful discussion. Availability of data and materials Feeding experiments and metabolite detection by LC-MS We used deuterium-labelled substrates (d5-tryptophan, d4-strictosidine, and d4-strictosidinic acid) in the feeding experiment. We incubated the apical cuttings from plants grown on Gamborg’s B5 medium in an aqueous solution containing 250 μM d5-tryptophan, d4-strictosi- dine, and d4-strictosidinic acid. After 30 days, we col- lected the stems and leaves for metabolite analysis via LC-MS with the method mentioned in the “Nontargeted metabolite analysis” section. The raw sequence data reported in this paper have been deposited in the Genome Sequence Archive in BIG Data Center, Beijing Institute of Genomics (BIG), Chinese Academy of Sciences, under accession number: CRA003143 [40]. The GenBank accession numbers for OpLAMT, OpSLS1, OpSLS2, OpSLS3, OpSLS4, OpSLS5, OpSLS6, CaSTR1, CaSTR2, and CaSTR3 are MT942677, MT942678, MT942679, MT942680, MT942681, MT942682, MT942683, MT942684, MT942685, and MT942686, respectively. Other data supporting the results in this study were shown in the Additional file. Funding g This work was financially supported by the National Natural Science Fund of China (31571735, 82073963, and 81522049), the National Key R&D Program of China (2018YFC1706200, 2019YFA0905100, and 2019YFA0905700), Zhejiang Provincial Ten Thousands Program for Leading Talents of Science and Technology Innovation (2018R520), and Zhejiang Provincial Program for the Cultivation of High-level In- novative Health Talents and the Chinese Academy of Sciences (XDB27020203, GJHZ2074, 153D31KYSB20170121, and 153D31KYSB20160074). To check the purity of the deuterium-labelled prod- uct, we characterized the products by LC-MS to con- firm the correct number of deuterium atoms incorporated (Additional file 1: Fig. S7 and S8). Authors’ contributions GK and YX conceived the study. MY, QW, YL, CW, and YL performed the experiments. MY performed the bioinformatics analyses. GK, MY, QW, XH, and JC wrote the paper. However, we are deeply saddened by the passing of Prof. Youli Xiao, who did not have the opportunity to approve the final manuscript. Prof. Youli Xiao provided great help, provided scientific research ideas, scientific instruments and lots of article writing opinions. In memory of his immense contribution to this project, good scientific research practice and reporting and humanity, we dedicate this work to him. All other authors read and approved the final manuscript. OpSLS microsome assay and HPLC analysis OpSLS microsome assay and HPLC analysis We performed OpSLS microsome assays in 100 μL of the above-prepared microsomes containing 1 mM nico- tinamide adenine dinucleotide phosphate (NADPH) and 1 mM specific substrate (loganin or loganic acid). We initiated the catalytic reaction through the addition of NADPH and incubated the reaction mixture at 30 °C. We quenched the reaction mixtures after 2 h with the addition of 100 μL of methanol. After the removal of the denatured proteins by centrifugation at 12,000g for 5 min, we analysed the supernatants by HPLC. The column applied for analysis was a Phenomenex Luna C18(2) (4.6 × 250 mm, 5 μm) on an Agilent 1260 Infinity II system with the temperature set at 35 °C. Mobile phases A (H2O + 0.1% formic acid) and B To assess the activity of OpSLSs, we transformed the yeast expression vector pESC-Leu-SLSs into the WAT11 yeast strain. We selected transformants on a solid syn- thetic dropout medium lacking leucine and containing Page 15 of 16 Yang et al. BMC Biology (2021) 19:122 (acetonitrile + 0.1% formic acid) were run in the follow- ing gradient programme at 0.8 mL/min: 0–3 min, 5% B; 3–12 min, 5–60% B; 12–15 min, 60–95% B; and 15–19 min, 95% B. A 10-μL sample was injected for analysis. OpSLS assays were monitored at 254 nm. STRs from C. acuminata, N. nimmoniana, and O. pumila used in this work. Table S4. Kinetic parameters of OpSTR towards secologanin and secolo- ganic acid. Table S5. Primers list used in this study. STRs from C. acuminata, N. nimmoniana, and O. pumila used in this work. Table S4. Kinetic parameters of OpSTR towards secologanin and secolo- ganic acid. Table S5. Primers list used in this study. Additional file 2:. The original, uncropped SDS-PAGE of Fig. S6. Additional file 2:. The original, uncropped SDS-PAGE of Fig. S6. Consent for publication Not applicable. Consent for publication Not applicable. Consent for publication Not applicable. The online version contains supplementary material available at https://doi. org/10.1186/s12915-021-01051-y. Competing interests Th h d l h The authors declare that they have no competing interests. Additional file 1: Fig. S1. O. pumila plant materials were used for metabolite profiling. Fig. S2. Analysis of gene expression patterns and phylogenic analysis of STRs, SLSs, LAMTs enzymes in CPT biosynthesis. Fig. S3. Protein sequence alignments mentioned in this article. Fig. S4. OpLAMT assay with secologanic acid and OpSTR competion expriments. Fig. S5. The standard curve of strictosidine and strictosidinic acid. Fig. S6. The SDS-PAGE gel of purified recombinant proteins used in the chemo-enzymatic synthesis of deuterium-labeled metabolites and bio- chemical assay. Fig. S7. Scheme of labeled substrates synthesis. Fig. S8. Chemoenzymatic synthesis of labeled substrates. Fig. S9. Detection of d4-strictosidinic acid and d4-strictosidine in the feeding experiments in O. pumila. Fig. S10. Metabolites detection of N. nimmoniana by LC-MS. Fig. S11. Phylogenetic relationship of DNA topoisomerase I in CPT-producing and non-producing species. Table S1. Relevant Compounds Detected in O. pumila plant and hairy root. Table S2. Identification of candidate CPT biosynthetic pathway genes in O. pumila as revealed by sequence iden- tity with characterized genes from the pre-strictosidine biosynthetic path- ways in Catharanthus roseus. Table S3. Identities and similarities among Chemo-enzymatic synthesis of deuterium-labelled substrates To trace the biosynthetic pathway of CPT, we performed a large-scale enzymatic reaction for deuterium-labelled product production. For d5-tryptamine production, we mixed 2 mM d5-tryptophan and 5 mM pyridoxal 5′- phosphate (PLP) in 30 mL OpTDC (2 mg/mL) solution at 30 °C for 10 h and concentrated the isolated product (d5-tryptamine) to dryness. For d4-strictosidine and d4- strictosidinic acid production, we mixed 2 mM purified d5-tryptamine and 5 mM secologanin or secologanic acid, respectively, in 10 mL at 30 °C until d5-tryptamine was completely consumed. d4-strictosidine and d4-stric- tosidinic acid were concentrated to dryness. Additional file 1: Fig. S1. O. pumila plant materials were used for metabolite profiling. Fig. S2. Analysis of gene expression patterns and phylogenic analysis of STRs, SLSs, LAMTs enzymes in CPT biosynthesis. Fig. S3. Protein sequence alignments mentioned in this article. Fig. S4. OpLAMT assay with secologanic acid and OpSTR competion expriments. Fig. S5. The standard curve of strictosidine and strictosidinic acid. Fig. S6. The SDS-PAGE gel of purified recombinant proteins used in the chemo-enzymatic synthesis of deuterium-labeled metabolites and bio- chemical assay. Fig. S7. Scheme of labeled substrates synthesis. Fig. S8. Chemoenzymatic synthesis of labeled substrates. Fig. S9. Detection of d4-strictosidinic acid and d4-strictosidine in the feeding experiments in O. pumila. Fig. S10. Metabolites detection of N. nimmoniana by LC-MS. Fig. S11. Phylogenetic relationship of DNA topoisomerase I in CPT-producing and non-producing species. Table S1. Relevant Compounds Detected in O. pumila plant and hairy root. Table S2. Identification of candidate CPT biosynthetic pathway genes in O. pumila as revealed by sequence iden- tity with characterized genes from the pre-strictosidine biosynthetic path- ways in Catharanthus roseus. Table S3. Identities and similarities among Author details 1 1Laboratory of Medicinal Plant Biotechnology, College of Pharmacy, Zhejiang Chinese Medical University, Hangzhou 310053, Zhejiang, China. 2CAS Key Laboratory of Synthetic Biology, CAS Center for Excellence in Molecular Plant Sciences, Core Facility Centre, Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai 200032, China. 3Institute of Plant Biotechnology, School of Life Sciences, Shanghai Normal University, Shanghai 200234, China. Received: 22 December 2020 Accepted: 13 May 2021 Declarations Ethics approval and consent to participate Not applicable. References ll BMC Genomics. 2017;18(1):245. https://doi. org/10.1186/s12864-017-3615-8. 8. Suttipanta N, Pattanaik S, Kulshrestha M, Patra B, Singh SK, Yuan L. The transcription factor CrWRKY1 positively regulates the terpenoid indole alkaloid biosynthesis in Catharanthus roseus. Plant Physiol. 2011;157(4): 2081–93. https://doi.org/10.1104/pp.111.181834. 28. Kellner F, Kim J, Clavijo BJ, Hamilton JP, Childs KL, Vaillancourt B, et al. Genome-guided investigation of plant natural product biosynthesis. Plant J. 2015;82(4):680–92. https://doi.org/10.1111/tpj.12827. 9. Patra B, Pattanaik S, Schluttenhofer C, Yuan L. A network of jasmonate- responsive bHLH factors modulate monoterpenoid indole alkaloid biosynthesis in Catharanthus roseus. New Phytol. 2018;217(4):1566–81. https://doi.org/10.1111/nph.14910. 29. Tatsis EC, Carqueijeiro I, BErnonville DD, et al. A three enzyme system to generate the strychnos alkaloid scaffold from a central biosynthetic intermediate. Nat Commun. 2017;8:1–10. 30. Rather GA, Sharma A, Jeelani SM, Misra P, Kaul V, Lattoo SK. Metabolic and transcriptional analyses in response to potent inhibitors establish mep pathway as major route for camptothecin biosynthesis in Nothapodytes nimmoniana (graham) mabb. BMC Plant Biol. 2019;19(1):301. https://doi. org/10.1186/s12870-019-1912-x. 10. Jin Z, Cong Y, Zhu S, Xing R, Zhang D, Yao X, et al. Two classes of cytochrome P450 reductase genes and their divergent functions in Camptotheca acuminata Decne. Int J Biol Macromol. 2019;138:1098–108. https://doi.org/10.1016/j.ijbiomac.2019.07.141. 11. Chang C, Liu Z, Wang Y, Tang Z, Yu F. A bZIP transcription factor, CaLMF, mediated light-regulated camptothecin biosynthesis in Camptotheca acuminata. Tree Physiol. 2019;39(3):372–80. https://doi.org/10.1093/treephys/tpy106. 31. Petronikolou N, Hollatz AJ, Schuler MA, Nair SK. Loganic acid methyltransferase: insights into the specificity of methylation on an iridoid glycoside. Chembiochem. 2018;19(8):784–8. https://doi.org/10.1002/cbic.201700679. 12. O’Connor SE, Maresh JJ. Chemistry and biology of monoterpene indole alkaloid biosynthesis. Nat Prod Rep. 2006;23(4):532–47. https://doi.org/10.1039/b512615k. 32. Duge BT, Papon N, Clastre M, et al. Identifying missing biosynthesis enzymes of plant natural products. Trends Pharmacol Sci. 2020;41(3):142–6. https://doi.org/10.1016/j.tips.2019.12.006. 13. Luca VD, Salim V, Thamm A, et al. Making iridoids/secoiridoids and monoterpenoid indole alkaloids: progress on pathway elucidation. Curr Opin Plant Biol. 2014;19:35–42. https://doi.org/10.1016/j.pbi.2014.03.006. 33. Yamazaki Y, Sudo H, Yamazaki M, Aimi N, Saito K. Camptothecin biosynthetic genes in hairy roots of Ophiorrhiza pumila: cloning, characterization and differential expression in tissues and by stress compounds. Plant Cell Physiol. 2003;44(4):395–403. https://doi.org/10.1093/pcp/pcg051. 14. Wu S, Yang M, Xiao Y. Synthetic biology studies of monoterpene indole alkaloids. Chin J Org Chem. 2018;38(9):2243–58. https://doi.org/10.6023/ cjoc201806001. 34. Qu Y, Easson M, Froese J, et al. Completion of the seven-step pathway from tabersonine to the anticancer drug precursor vindoline and its assembly in yeast. References ll 1. Wall ME, Wani MC, Cook CE, Palmer KH, McPhail AT, Sim GA. Plant antitumor agents. I. The isolation and structure of camptothecin, a novel alkaloidal leukemia and tumor inhibitor from Camptotheca acuminata. J Am Chem Soc. 1966;88(16):3888–90. https://doi.org/10.1021/ja00968a057. Page 16 of 16 Page 16 of 16 Yang et al. BMC Biology (2021) 19:122 Yang et al. BMC Biology (2021) 19:122 2. Pu X, Zhang C, Zhu L, et al. Possible clues for camptothecin biosynthesis from the metabolites in camptothecin-producing plants. Fitoterapia. 2019; 134:113–28. https://doi.org/10.1016/j.fitote.2019.02.014. biosynthesis of the anti-cancer alkaloid camptothecin and anthraquinones. Plant Cell Physiol. 2013;54(5):686–96. https://doi.org/10.1093/pcp/pct040. 23. 23. Yamazaki Y, Urano A, Sudo H, Kitajima M, Takayama H, Yamazaki M, et al. Metabolite profiling of alkaloids and strictosidine synthase activity in camptothecin producing plants. Phytochemistry. 2003;62(3):461–70. https:// doi.org/10.1016/S0031-9422(02)00543-5. 3. Kai G, Dai L, Mei X, et al. In vitro plant regeneration from leaf explants of Ophiorrhiza japonica. Biol Plant. 2008;52(3):557–60. https://doi.org/10.1007/s1 0535-008-0110-8. 24. Watase I, Sudo H, Yamazaki M, Saito K. Regeneration of transformed Ophiorrhiza pumila plants producing camptothecin. Plant Biotech. 2004; 21(5):337–42. https://doi.org/10.5511/plantbiotechnology.21.337. 4. Ulukan H, Swaan PW. Camptothecins - a review of their chemotherapeutic potential. Drugs. 2002;62(14):2039–57. https://doi.org/10.2165/00003495-2 00262140-00004. 5. Lorence A, Nessler CL. Camptothecin, over four decades of surprising findings. Phytochemistry. 2004;65(20):2735–49. https://doi.org/10.1016/j. phytochem.2004.09.001. 25. Qu Y, Easson ME, Simionescu R, et al. Solution of the multistep pathway for assembly of corynanthean, strychnos, iboga, and aspidosperma monoterpenoid indole alkaloids from 19E-geissoschizine. Proc Natl Acad Sci U S A. 2018;115(12):3180–5. https://doi.org/10.1073/pnas.1719979115. 6. Kai G, Wu C, Gen L, Zhang L, Cui L, Ni X. Biosynthesis and biotechnological production of anti-cancer drug camptothecin. Phytochem. Rev. 2015;14(3): 525–39. https://doi.org/10.1007/s11101-015-9405-5. 6. Kai G, Wu C, Gen L, Zhang L, Cui L, Ni X. Biosynthesis and biotechnological production of anti-cancer drug camptothecin. Phytochem. Rev. 2015;14(3): 525–39. https://doi.org/10.1007/s11101-015-9405-5. 26. Caputi L, Franke J, Farrow SC, Chung K, Payne RME, Nguyen TD, et al. Missing enzymes in the biosynthesis of the anticancer drug vinblastine in Madagascar periwinkle. Science. 2018;360(6394):1235–9. https://doi.org/1 0.1126/science.aat4100. 7. Shi M, Gong H, Cui L, Wang Q, Wang C, Wang Y, et al. Targeted metabolic engineering of committed steps improves anti-cancer drug camptothecin production in Ophiorrhiza pumila hairy roots. Ind Crop Prod. 2020;148: 112277. https://doi.org/10.1016/j.indcrop.2020.112277. 27. Yang M, You W, Wu S, Fan Z, Xu B, Zhu M, et al. Global transcriptome analysis of Huperzia serrata and identification of critical genes involved in the biosynthesis of huperzine A. References ll Proc Natl Acad Sci U S A. 2015;112(19):6224–9. https://doi.org/10.1 073/pnas.1501821112. 15. Sadre R, Lundback MM, Pradhan S, et al. Metabolite diversity in alkaloid biosynthesis: a multilane (diastereomer) highway for camptothecin synthesis in camptotheca acuminata. Plant Cell. 2016;28(8):1926–44. https://doi.org/1 0.1105/tpc.16.00193. 35. Jin Z, Wan R, Yan R, Su Y, Huang H, Zi L, et al. Microwave-assisted extraction of multiple trace levels of intermediate metabolites for camptothecin biosynthesis in Camptotheca acuminata and their simultaneous determination by HPLC-LTQ-Orbitrap-MS/MS and HPLC-TSQ-MS. Molecules. 2019;24(4):815. https://doi.org/10.3390/molecules24040815. 16. Yang Y, Wei L, Jiang L, et al. Bifunctional cytochrome P450 enzymes involved in camptothecin biosynthesis. ACS Chem Biol. 2019;14(6):1091–6. https://doi.org/10.1021/acschembio.8b01124. 17. Zhao D, Hamilton JP, Pham GM, et al. De novo genome assembly of Camptotheca acuminata, a natural source of the anti-cancer compound camptothecin. GigaSci. 2017;6:gix065. 17. Zhao D, Hamilton JP, Pham GM, et al. De novo genome assembly of Camptotheca acuminata, a natural source of the anti-cancer compound camptothecin. GigaSci. 2017;6:gix065. 36. Sirikantaramas S, Yamazaki M, Saito K. Mutations in topoisomerase I as a self-resistance mechanism coevolved with the production of the anticancer alkaloid camptothecin in plants. Proc Natl Acad Sci U S A. 2008;105(18): 6782–6. https://doi.org/10.1073/pnas.0801038105. 18. Asano T, Sudo H, Yamazaki M, et al. Camptothecin production by in vitro cultures and plant regeneration in Ophiorrhiza species. Methods Mol Biol. 2009;547:337–45. https://doi.org/10.1007/978-1-60327-287-2_27. 37. Viraporn V, Yamazaki M, Mami S, et al. Correlation of camptothecin- producing ability and phylogenetic relationship in the genus Ophiorrhiza. Planta Medica. 2011;77(07):759–64. https://doi.org/10.1055/s-0030-1250568. 19. Aimi N, Nishimura M, Miwa A, Hoshino H, Sakai SI, Haginiwa J. Pumiloside and deoxypumiloside - plausible intermediates of camptothecin biosynthesis. Tetrahedron Lett. 1989;30(37):4991–4. https://doi.org/10.1016/ S0040-4039(01)80563-3. 38. Kumar S, Stecher G, Li M, Knyaz C, Tamura K. MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol. 2018;35(6):1547–9. https://doi.org/10.1093/molbev/msy096. 20. Carte BK, Brosse CD, Eggleston D, et al. Isolation and characterization of a presumed biosynthetic precursor of camptothecin from extracts of Camptotheca acuminata. Tetrahedron. 1990;46(8):2747–60. https://doi.org/1 0.1016/S0040-4020(01)88369-1. 39. Pompon D, Louerat B, Bronine A, et al. Yeast expression of animal and plant P450s in optimized redox environments. Methods Enzymol. 1996;272:51–64. https://doi.org/10.1016/S0076-6879(96)72008-6. 21. Rather GA, Sharma A, Pandith SA, Kaul V, Nandi U, Misra P, et al. De novo transcriptome analyses reveals putative pathway genes involved in biosynthesis and regulation of camptothecin in Nothapodytes nimmoniana (Graham) Mabb. Plant Mol Biol. 2018;96(1-2):197–215. https://doi.org/10.1 007/s11103-017-0690-9. 40. Yang, M. Divergent camptothecin biosynthetic pathway in Ophiorrhiza pumila. Genome Sequence Archive (GSA). References ll 2021.https://bigd.big.ac.cn/gsa/ browse/CRA003143. Accessed 3 May 2021. 40. Yang, M. Divergent camptothecin biosynthetic pathway in Ophiorrhiza pumila. Genome Sequence Archive (GSA). 2021.https://bigd.big.ac.cn/gsa/ browse/CRA003143. Accessed 3 May 2021. Publisher’s Note 22. Yamazaki M, Mochida K, Asano T, Nakabayashi R, Chiba M, Udomson N, et al. Coupling deep transcriptome analysis with untargeted metabolic profiling in Ophiorrhiza pumila to further the understanding of the 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. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
https://openalex.org/W3173826441	https://www.nature.com/articles/srep44966.pdf	English	null	Correction: Corrigendum: Universal stress protein Rv2624c alters abundance of arginine and enhances intracellular survival by ATP binding in mycobacteria	Scientific reports	2,017	cc-by	101	www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Corrigendum: Universal stress protein Rv2624c alters abundance of arginine and enhances intracellular survival by ATP binding in mycobacteria Figure 1. This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ Scientific ReportS \| 7:44966 \| DOI: 10.1038/srep44966
https://openalex.org/W4306764950	https://www.jnmp.ru/jour/article/download/1445/1200	Russian	null	The Effectiveness of Compression Therapy in the Prevention of Venous Thromboembolic Complications in Patients With COVID-19	Neotložnaâ medicinskaâ pomoŝʹ	2,022	cc-by	7,367	ОРИГИНАЛЬНЫЕ СТАТЬИ ОРИГИНАЛЬНЫЕ СТАТЬИ Научная статья https://doi.org/10.23934/2223-9022-2022-11-3-436-443 Эффективность компрессионной терапии в профилактике венозных тромбоэмболических осложнений у пациентов с COVID-19 Р.Е. Калинин, И.А. Сучков , А.Б. Агапов, Н.Д. Мжаванадзе, Д.А. Максаев, А.А. Чобанян Кафедра сердечно-сосудистой, рентгенэндоваскулярной хирургии и лучевой диагностики ФГБОУ ВО «Рязанский государственный медицинский университет им. акад. И.П. Павлова» МЗ РФ Российская Федерация, 390026, Рязань, ул. Высоковольтная, д. 9 Контактная информация: Сучков Игорь Александрович, доктор медицинских наук, профессор, профессор кафедры сердечно-сосудис- той, рентгенэндоваскулярной хирургии и лучевой диагностики ФГБОУ ВО РязГМУ. Еmail: suchkov_med@mail.ru © Калинин Р.Е., Сучков И.А., Агапов А.Б., Мжаванадзе Н.Д., Максаев Д.А., Чобанян А.А. М., 2022 Russian Sklifosovsky Journal of Emergency Medical Care. 2022;11(3):436–443. https://doi.org/10.23934/2223-9022-2022-11-3-436-443 Цель работы Оценить эффективность применения ЭК нижних конечностей в профилактике венозных тромбо- эмболических осложнений (ВТЭО) у пациентов с новой коронавирусной инфекцией. Исследование одобрено локальным этическим комитетом ФГБОУ ВО РязГМУ Минздрава России и зарегистрировано на платформе ClinicalTrials.gov (идентификатор NCT05143567). В исследова- ние включены 69 пациентов без применения трикотажа (1-я группа) и 65 пациентов, которым применялся профилактический компрессионный трикотаж (2-я группа). Больные проходили ле- чение в ковидном госпитале с июля по ноябрь 2021 г. Все пациенты были с верифицированной коронавирусной инфекцией, им назначали АКТ и проводили ультразвуковое дуплексное ска- нирование вен нижних конечностей при поступлении, в отделении реанимации и при выписке. Оценивали частоту венозных тромбоэмболических осложнений (ВТЭО), геморрагические ослож- нения, летальность. Для оценки тяжести кровотечении использовали классификацию Комите- та Международного общества по тромбозу и гемостазу (International Society on Thrombosis and Haemostasis, ISTH). У пациентов без трикотажа (1-я группа) ВТЭО составили 7 случаев (10,14%); тромбоз глубоких вен (ТГВ) — 4 (5,8%), тромбоэмболия легочной артерии (ТЭЛА) — 3 (4,3%). Все 6 пациентов (8,7%) с ТЭЛА — со смертельным исходом. Отмечено, что 2 случая ТЭЛА (2,8%) были верифицированы на аутопсии, а не клинически. У пациентов 2-й группы с применением эластической компрессии общая частота ВТЭО составила всего один случай (1,6%). При анализе частоты кровотечений в 1-й группе было одно легочное кровотечение (1,4%), во 2-й группе также один случай напряженной межмышечной гематомы (1,6%). Значимые кровотечения в 1-й группе наблюдались у одного больного (1,4%), а во 2-й группе — у 3 (4,8%). Малые кровотечения в 1-й группе отмечены у 11 пациентов (15,9%), во 2-й группе — у 8 (12,7%). Летальность во время госпитализации у пациентов 1-й группы (без ЭК) со- ставила 11 больных (15,9%) , во 2-й группе (с ЭК) — 7 (11,1%) (р=0,419). Летальность у пациентов с коронавирусной инфекцией без компрессионной терапии выше, чем у пациентов с применением компрессионного трикотажа (р=0,419). У пациентов, носивших комп- рессионные чулки в стационаре, обнаружена низкая частота развития ВТЭО (1,6% случаев во 2-й группе против 10,14% случаев в 1-й группе, р=0,039). В исследуемых группах была одинаковая частота больших кровотечений (1-я группа — один случай (1,4%), 2-я группа — один (1,6%) слу- чай). Применение профилактического трикотажа на стационарном этапе лечения новой коро- навирусной инфекции позволяет выполнять профилактику ВТЭО у пациентов с кровотечениями при невозможности проведения антикоагулянтной терапии. новая коронавирусная инфекция, тромбоз, кровотечения, эластическая компрессия, венозные тромбоэмболические осложнения, COVID-19 Калинин Р.Е., Сучков И.А., Агапов А.Б., Мжаванадзе Н.Д., Максаев Д.А., Чобанян А.А. Эффек- тивность компрессионной терапии в профилактике венозных тромбоэмболических осложне- ний у пациентов с COVID-19. Актуальность Основным методом профилактики тромботических осложнений у больных коронавирусной ин- фекцией является антикоагулянтная терапия (АКТ). Однако и ее применение не всегда возможно, в частности, у пациентов с кровотечениями. Единственным способом профилактики в данном случае является эластическая компрессия (ЭК) нижних конечностей. Цель работы Материал и методы Цель работы Журнал им. Н.В. Склифосовского Неотложная медицинская помощь. 2022;11(3):436–443. https://doi.org/10.23934/2223-9022-2022-11-3-436-443 Калинин Р.Е., Сучков И.А., Агапов А.Б., Мжаванадзе Н.Д., Максаев Д.А., Чобанян А.А. Эффек- тивность компрессионной терапии в профилактике венозных тромбоэмболических осложне- ний у пациентов с COVID-19. Журнал им. Н.В. Склифосовского Неотложная медицинская помощь. 2022;11(3):436–443. https://doi.org/10.23934/2223-9022-2022-11-3-436-443 Авторы заявляют об отсутствии конфликта интересов Авторы заявляют об отсутствии конфликта интересов Благодарность, финансирование Компрессионный трикотаж для проведения исследования предоставлен фирмой ООО «ЭЛАСТ МЕДИКАЛ» ирование Компрессионный трикотаж для проведения исследования предоставлен фирмой ООО «ЭЛАСТ МЕДИКАЛ» © Калинин Р.Е., Сучков И.А., Агапов А.Б., Мжаванадзе Н.Д., Максаев Д.А., Чобанян А.А. М., 2022 436 ОРИГИНАЛЬНЫЕ СТАТЬИ АКТ — антикоагулянтная терапия АЧТВ — активированное частичное тромбопластиновое время БА — бронхиальная астма ВПОТ — высокопоточная оксигенотерапия ВТЭО — венозные тромбоэмболические осложнения ДВС — диссеминированное внутрисосудистое свертывание ИБС — ишемическая болезнь сердца ИВЛ — искусственная вентиляция легких КТ — компьютерная томография НИВЛ — неинвазивная вентиляция легких НФГ — нефракционированный гепарин ОРИТ — отделение реанимации и интенсивной терапии ПВ — протромбиновое время ПИКС — постинфарктный кардиосклероз ПКТ — прокальцитонин ПЦР — полимеразная цепная реакция РКТ — рентгеновская компьютерная томография СОЭ — скорость оседания эритроцитов ТГВ — тромбоз глубоких вен ТЭЛА — тромбоэмболия легочной артерии ХЗВ — хронические заболевания вен ХОБЛ — хроническая обструктивная болезнь легких ЭК — эластическая компрессия СРБ — С-реактивный белок Актуальность Критерии исключения из исследования: мужчины или женщины младше 18 лет с декомпенсированной соматической патологией, беременностью или в пери- од кормления грудью (женщины), а также пациенты, которым противопоказано применение компрессион- ной терапии. Противопоказания к эластической ком- прессии: сердечная недостаточность высокого функ- ционального класса, состояние после шунтирующих операций на артериях нижних конечностей, систо- лическое давление на уровне лодыжки менее 70 мм рт.ст., тяжелая периферическая нейропатия, дерматит, аллергические реакции на компоненты компрессион- ных изделий, истонченная кожа над костными дефор- мациями. COVID-19-ассоциированная коагулопатия, которая лежит в основе патогенеза пандемии последних трех лет, является следствием воспалительного ответа на вирусную нагрузку, что приводит к активации иммун- ных комплексов [1]. В начале заболевания срабатывает адаптивный механизм, происходит активация гемос- таза и отложение фибрина. Дальнейшее воздействие вируса приводит к формированию гипервоспалитель- ного ответа благодаря цитокиновому шторму и синд- рому активации макрофагов [2]. Данные патогенетические механизмы приводят к тромбозам на микроциркуляторном уровне, высокой частоте венозных тромбоэмболических осложнений (ВТЭО) и смертности [1, 2]. Лечение пациентов проводилось соглас- но Временным методическим рекомендациям Министерства здравоохранения РФ «Профилактика, диагностика и лечение новой коронавирусной инфек- ции» [4]. Пациентам осуществляли комплексную тера- пию, направленную на лечение основного и сопутс- твующих заболеваний, а также коррекцию состояний, обусловленных тяжестью заболевания и проводимым лечением. Основным методом профилактики ВТЭО является антикоагулянтная терапия (АКТ). Однако ее примене- ние не всегда возможно. Например, у пациентов с боль- шими и значимыми кровотечениями. Единственным способом профилактики в данном случае является эластическая компрессия (ЭК) [3]. Данный метод недо- оценивается в реальной клинической практике, но ее применение обосновано тем, что пациенты находятся на длительном постельном режиме для проведения кислородотерапии. Назначали препараты из различных фармаколо- гических групп: антикоагулянтная, противовирусная, антибактериальная, гормональная терапия, препара- ты из группы моноклональных антител, блокаторы протонной помпы, инсулинотерапия по показаниям, противокашлевые средства, инфузионная терапия до 1500 мл в сутки, инсуффляция кислорода в зависи- мости от уровня сатурации и тяжести основного забо- левания. Всем пациентам проводили ультразвуковое дуплексное сканирование (УЗДС) согласно общепри- нятым протоколам осмотра вен нижних конечностей при поступлении, при переводе в реанимацию (если он происходил) и при выписке [5]. Оценивали частоту ВТЭО, геморрагических осложнений, летальность на стационарном этапе лечения. Для оценки тяжести кро- вотечения использовалась классификация Комитета Международного общества по тромбозу и гемостазу (International Society on Thrombosis and Haemostasis, ISTH) [6]. Цель работы: оценить эффективность примене- ния ЭК нижних конечностей в профилактике ВТЭО у пациентов с новой коронавирусной инфекцией. Russian Sklifosovsky Journal of Emergency Medical Care. 2022;11(3):436–443. https://doi.org/10.23934/2223-9022-2022-11-3-436-443 Результаты Таблица 1 Клинико-анамнестическая характеристика пациентов Table 1 Clinical and anamnestic characteristics of patients Показатель 1-я группа, n=69 2-я группа, n=63 р Возраст 56,13±14,4 года 57,5±11,9 года 0,548 Мужчины 23 (33,3%) 2 (3,2%) 0,001 Женщины 46 (66,7%) 61 (96,8%) Положительный ПЦР- тест при поступлении 55 (79,7%) 55 (87,3%) 0,217 Тяжесть COVID-19 — Легкая 9 (13,04%) 1 (1,6%) 0,013 — Средняя 30 (43,5%) 23 (36,5%) 0,415 — Тяжелая 21 (30,4%) 31 (49,2%) 0,028 — Крайне тяжелая 9 (13,04%) 8 (12,7%) 0,841 КТ-1 17 (24,6%) 6 (9,6%) 0,022 КТ-2 33 (47,8%) 28 (44,4%) 0,697 КТ-3 16 (23,2%) 26 (41,2%) 0,026 КТ-4 3 (4,3%) 3 (4,8%) 0,909 Потребность в кислороде 57 (83%) 55 (87%) 0,453 ОРИТ 14 (20,3%) 8 (12,7%) 0,242 Переведены из ОРИТ 3 (4,3%) 1 (1,6%) 0,602 ВПОТ 3 (4,3%) 1 (1,6%) 0,602 НИВЛ 3 (4,3%) 1 (1,6%) 0,602 ИВЛ 8 (11,6%) 6 (9,5%) 0,699 Койко-день 16,6±10,14 20,9±13,52 0,015 Летальность 11 (15,9%) 7 (11,1%) 0,419 Примечания: ПЦР — полимеразная цепная реакция; КТ — компьютерная томография; ОРИТ — отделение реанимации и интенсивной терапии; ВПОТ — высокопоточная оксигенотерапия; НИВЛ — неинвазивная вентиляция легких; ИВЛ — искусственная вентиляция легких Notes: ПЦР — polymerase chain reaction; КТ — computed tomography; ОРИТ — re- suscitation and intensive care unit; ВПОТ — high-flow oxygen therapy; НИВЛ — non- invasive ventilation of the lungs; ИВЛ — artificial lung ventilation Клинико-анамнестическая характеристика паци- ентов, включенных в исследование, представлена в табл. 1. Средний возраст пациентов без трикотажа (1-я группа) составил 56,1±14,4 года, с трикотажем (2-я группа) — 57,5±11,9 года. По полу: в группе с исполь- зованием трикотажа была 61 женщина (96,8%) и двое мужчин (3,2%). В группе без ЭК (1-я группа) женщин также было больше — 46 (66,7%); мужчин — 23 (33,3%) (табл. 1). По тяжести коронавирусной инфекции в обеих группах наиболее часто встречались пациенты со сред- ней и тяжелой формами заболевания, причем тяжелая форма встречалась больше у пациентов 2-й группы (1-я группа — 21 (30,4%), 2-я — 31 (49,2%), р=0,028). Тяжесть заболевания напрямую связана с объемом поражения легочной ткани по данным РКТ органов грудной клетки: 1-я группа — КТ-3 у 16 пациентов (23,2%), 2-я группа — у 26 (41,2%), р=0,026. Респираторную поддержку всем пациентам прово- дили согласно рекомендациям Федерации анестезио- логов-реаниматологов России [7]. Инсуффляция кис- лорода применялась в 83% случаев в 1-й группе и в 87% случаев во 2-й группе. Материал и методы Клиническое исследование проведено на базе «ковидных госпиталей» ГБУ РО «Областная клини- ческая больница» и ГБУ РО «Больница скорой меди- цинской помощи г. Рязани» с июля по ноябрь 2021 г. В исследование включены 69 пациентов без ЭК (1-я группа) и 65 пациентов, которым применялся про- филактический компрессионный трикотаж (чулки) ˝Tonus Elast˝ (2-я группа). Исследование одобрено локальным этическим комитетом ФГБОУ ВОРязГМУ Минздрава России и зарегистрировано на платформе ClinicalTrials.gov (идентификатор NCT05143567). Критерии включения в исследование: мужчины или женщины старше 18 лет, находившиеся на лечении в ковидном госпитале с двусторонней вирусной пневмо- нией по данным рентгеновской компьютерной томог- рафии (РКТ) органов грудной клетки; с подозрением на COVID-19 или подтвержденной новой коронавирус- ной инфекцией по данным ПЦР-теста (полимеразная цепная реакция); подписавшие добровольное согласие на использование профилактического компрессион- ного трикотажа. Подбор трикотажа (чулки) проводили индивидуально по замерам окружности верхней трети бедра и голени, нижней трети голени, показателям роста и согласно инструкции производителя. Статистический анализ данных проведен с использованием программы Statistica 10 for Windows. Количественные переменные описывались следую- щими величинами: количество случаев, абсолютные и относительные величины (проценты). Принятый уро- вень статистической значимости — р<0,05. Числовые данные представлены как среднее арифметическое и стандартное отклонение. 437 Russian Sklifosovsky Journal of Emergency Medical Care. 2022;11(3):436–443. https://doi.org/10.23934/2223-9022-2022-11-3-436-443 ОРИГИНАЛЬНЫЕ СТАТЬИ Результаты В отделение реанимации без применения ЭК (1-я группа) переведены 14 пациентов (20,3%), а в группе с использованием ЭК пациентов, переведенных в ОРИТ, было в 2 раза меньше: их число составило 8 больных (12,7%) . В отделении реанимации применялись методы, помогающие отсрочить перевод больного на искусст- венную вентиляцию легких (ИВЛ). Так, высокопоточ- ная оксигенотерапия (ВПОТ) использовалась у всех пациентов в ОРИТ, но из-за тяжести состояния остава- лись на ней не все, возникала необходимость в неинва- зивной вентиляции легких (НИВЛ). Остались на ВПОТ 3 пациента (4,3%) 1-й группы и один (1,6%) пациент 2- й группы; данные больные были переведены из ОРИТ в палаты. При неэффективности НИВЛ, нару- шении гемодинамики и сознания проводилось ИВЛ. Все пациенты в обеих группах на НИВЛ и ИВЛ умер- ли. Таким образом, перевод больных на ИВЛ и НИВЛ сопровождался высокой летальностью в ОРИТ. Russian Sklifosovsky Journal of Emergency Medical Care. 2022;11(3):436–443. https://doi.org/10.23934/2223-9022-2022-11-3-436-443 ц Сопутствующая патология у пациентов 1-й и 2-й группы Table 2 Table 2 Concomitant disorders in patients of the 1st and 2nd groups Сопутствующие заболевания 1-я группа (n=69) 2-я группа (n=63) р Гипертоническая болезнь 47 (68) 46 (73%) 0,336 ИБС 10 (14%) 7 (11%) 0,376 ПИКС 3 (4%) 4 (6%) 0,449 Нарушение ритма 4 (6%) 2 (3%) 0,384 Сахарный диабет 24 (35%) 21 (33%) 0,504 Заболевания легких (ХОБЛ, БА) 4 (6%) 5 (8%) 0,443 Ревматологические заболевания 4 (6%) 3 (5%) 0,551 Онкологические заболевания 4 (6%) 5 (8%) 0,443 Гастроэнтерологические заболевания 3 (4%) 4 (6%) 0,449 Ожирение 19 (28%) 15 (24%) 0,387 ХЗВ 7 (10%) 6 (10%) 0,569 ВТЭО в анамнезе 2 (3%) 3 (5%) 0,457 Примечания: ИБС — ишемическая болезнь сердца; ПИКС — постинфарктный кардиосклероз; ХОБЛ — хроническая обструктивная болезнь легких; БА — бронхиальная астма; ХЗВ — хронические заболевания вен; ВТЭО — венозные тромбоэмболические осложнения Notes: ИБС — coronary artery disease; ПИКС — postinfarction cardiosclerosis; ХОБЛ — chronic obstructive pulmonary disease; БА — bronchial asthma; ХЗВ — chronic ve- nous disease; ВТЭО — venous thromboembolic complications При анализе сопутствующей патологии отмечено, что все пациенты статистически сопоставимы между собой (р>0,05). Наиболее часто у госпитализированных пациентов встречаются: кардиологическая патология (гипертоническая болезнь), сахарный диабет 2-го типа и ожирение (табл. 2). Сахарный диабет у пациентов с коронавирусной инфекцией усугубляется применением гормональной терапии. А такой фактор, как ожирение, способствует увеличению дыхательной недостаточности. По динамике лабораторных показателей в обоих группах были отмечены высокие значения СРБ и фер- ритина, что указывает на наличие активного воспа- ления в организме (табл. 3). Также при поступлении у всех пациентов наблюдалась коагулопатия с повыше- нием уровня фибриногена и Д-димера. При поступ- лении больных в реанимацию наблюдалось увеличе- ние концентрации маркеров воспаления (лейкоцитоз, СРБ, ферритин, прокальцитонин). В коагулограмме наблюдалось удлинение активированного частичного тромбопластинового времени (АЧТВ), протромбино- вого времени, что может быть обусловлено переводом пациентов на внутривенную инфузию гепарина через инфузомат. Также у всех пациентов отмечена гипер- 438 Russian Sklifosovsky Journal of Emergency Medical Care. 2022;11(3):436–443. ц Сопутствующая патология у пациентов 1-й и 2-й группы Table 2 https://doi.org/10.23934/2223-9022-2022-11-3-436-443 ОРИГИНАЛЬНЫЕ СТАТЬИ Та бл и ц а 3 Динамика лабораторных показателей у пациентов Ta b l e 3 The dynamics of laboratory parameters in patients Показатель (норма) При поступлении р В ОРИТ р При выписке/смерти р 1-я группа 2-я группа 1-я группа 2-я группа 1-я группа 2-я группа Эритроциты 3,5–5,5х1012/л 4,6 (3,4–6) 4,6 (3,1–5,5) 0,973 4,7 (4,2–5,8) 4,4 (2,2–5,3) 0,781 4,4 (2,8–5,9) 4,3 (3,2–5,9) 0,476 Гемоглобин 110–160 г/л 139 (85–188) 135,4 (90–170) 0,429 143 (118–176) 126,1 (72–158) 0,074 133,9 (78–181) 126,4 (90–185) 0,699 Тромбоциты 100–400х109/л 216,1 (34–520) 244,5 (55–690) 0,149 246,7 (57–426) 231,8 (34–401) 0,358 244,2 (34–568) 249,5 (39–474) 0,567 Лейкоциты 4,0–10,0х109/л 7,2 (2–32,6) 8,3 (2–23,5) 0,206 11,9 (6,2–20,1) 17,8 (1,4–41,8) 0,004 12,5 (3,5–69) 12,9 (2–43,8) 0,437 СОЭ 2–18 мм/час 24,3 (1–63) 22,3 (1–60) 0,479 14,1 (1–30) 34,7 (14–55) 0,001 12,9 (1–60) 19,3 (1–77,1) 0,042 Глюкоза 3,5—6,3 ммоль/л 8,8 (2–38,2) 8,63 (3,6–24,6) 0,361 19,1 (4,1–33) 7,4 (1,9–16,3) 0,109 11,3 (2–31,4) 10,7 (2,8–40,4) 0,422 СРБ ≤5 мг/л 78,7 (6,6–356) 75,7 (3,2–205,5) 0,531 73,4 (10–197) 105,2 (16–200) 0,372 28,3 (0,3–197,9) 20,1 (1–207) 0,031 Ферритин 10–200 мкг/л 843,2 (310–1064)720,9 (312–1040) 0,013 1128 (562–1467)1089 (428–1367) 0,458 903,7 (570–1059) 484,9 (292–945) 0,024 ПКТ ≤0,5 нг/мл 0,104 (0,02–0,4) 0,101 (0,002–0,4) 0,793 1,56 (0,036–14,7) 1,1 (0,068–3,9) 0,493 0,25 (0,02–4,6) 0,09 (0,04–0,48) 0,124 АЧТВ 12,6–28,7 сек 39,6 (19,9–155,9) 33,9 (21,1–70,2) 0,012 74,4 (26,6–155,9) 40,8 (27,8–75) 0,110 42,9 (17,4–158) 35,4 (11,8–105,5) 0,271 ПВ 9,8–12,2 сек 12,1 (9,4–20,1) 12,5 (10–21,5) 0,138 15,2 (11,2–25,2) 17,3 (11,8–45,6) 0,329 15,7 (9,4–36,8) 18,9 (9,8-39) 0,457 Фибриноген 1,8–3,5 г/л 3,6 (1,1–7,4) 4,4 (1,1–7,8) 0,003 3,3 (1,1–8) 4,6 (1,2–8) 0,224 2,5 (1,1–7,2) 3,12 (1,01–7,3) 0,009 Д-димер ≤0,5 мг/л 1,2 (0,9–5,5) 0,56 (0,12–2,1) 0,008 3,23 (0,6–5) 1,2 (0,6–2,4) 0,033 1,4 (0,2–5,6) 0,6 (0,12–4,4) 0,001 Примечания: СРБ — С-реактивный белок; АЧТВ — активированное частичное тромбопластиновое время; ПВ — протромбиновое время; ПКТ — прокальцитонин; СОЭ — скорость оседания эритроцитов Notes: СОЭ — erythrocyte sedimentation rate; СРБ — C-reactive protein; ПКТ — procalcitonin; ПВ — prothrombin time; АЧТВ — activated partial thromboplastin time Рисунок. Антикоагулянтная терапия у пациентов исследуемых групп Figure. Anticoagulant therapy in patients of studied groups 30 % 20 10 0 70 60 50 40 1-я степень 2-я степень Лечебная доза Повышенная доза гликемия, что обусловлено высокой встречаемостью сахарного диабета 2-го типа либо является следствием приема глюкокортикостероидов. При выписке из ста- ционара наблюдалась динамика снижения маркеров воспаления и гемостаза. Russian Sklifosovsky Journal of Emergency Medical Care. 2022;11(3):436–443. https://doi.org/10.23934/2223-9022-2022-11-3-436-443 ц Сопутствующая патология у пациентов 1-й и 2-й группы Table 2 Таким образом, наиболее чувствительными рутинными маркерами воспаления и коагуляции, по нашим данным, являются концент- рация СРБ, лейкоцитов, прокальцитонин, ферритин, фибриноген, Д-димер. При поступлении всем пациентам вводили лечеб- ную дозу гепарина. «Стартовую» повышенную дозу антикоагулянтов назначали пациентам с ожирением, высоким исходным уровнем Д-димера и фибриноге- на, наличием ВТЭО в анамнезе. У пациентов без ЭК (1-я группа) лечебная доза гепаринов была назначена 46 пациентам (67%), повышенная — у 23 (33%), а у больных с применением ЭК (2-я группа) лечебная доза была у 43 больных (63%), повышенная — у 20 (37%), р=0,993 (рисунок). Рисунок. Антикоагулянтная терапия у пациентов исследуемых групп Figure. Anticoagulant therapy in patients of studied groups По результатам стационарного этапа лечения сле- дует отметить, что в основном тромботические собы- тия возникли у пациентов без использования про- филактического компрессионного трикотажа. ВТЭО у пациентов без трикотажа (1-я группа) произошло у 7 больных (10,14%), а у пациентов 2-й группы с приме- нением ЭК общая частота ВТЭО составила всего один случай (1,6%), который был представлен ТЭЛА мелких ветвей, обнаруженных на аутопсии (р=0,039). низкая частота развития ВТЭО. Наличие ВТЭО прямо пропорционально тяжести заболевания и способство- вало высокой смертности. Смертность пациентов 1-й группы (без трикотажа) составила 11 больных (15,9%), во 2-й группе (с трикотажем) — 7 (11,1%). При анализе частоты геморрагических осложне- ний отмечено, что большие и значимые кровотечения наблюдали в отделении реанимации. В 1-й группе было одно легочное кровотечение (1,4%), во 2-й — также один (1,6%) случай напряженной межмышечной гематомы, потребовавший проведения гемотрансфу- зии (табл. 4). Тромбоз глубоких вен (ТГВ) — 4 случая (5,8%, р=0,052), ТЭЛА — 3 (4,3%). Все 6 пациентов (8,7%) с ТЭЛА — со смертельным исходом. Проксимальная гра- ница тромба у пациентов с ТГВ без ТЭЛА встречалась у 2 пациентов (2,8%) в илеофеморальном сегменте, еще у 2 (2,8%) — в бедренно-подколенном. У одного пациента с ТЭЛА тромботические массы были лока- лизованы в поверхностных венах и в бедренной вене. Отмечено, что 2 случая ТЭЛА (2,8%) были верифициро- ваны на аутопсии, и не клинически. Значимое геморрагическое осложнение у больных без эластической компрессии (1-я группа) наблюда- лось в одном случае (1,4%); представлено носовым кровотечением. Данный пациент получал лечебную дозу гепарина в палате по поводу ТГВ. Во 2-й группе у 3 больных (4,8%) наблюдались кровотечения, кото- рые потребовали отмены АКТ и были представле- ны межмышечной напряженной гематомой голени, геморроидальным кровотечением, носовым крово- Таким образом, у пациентов с использованием профилактических компрессионных чулок доказана 439 Russian Sklifosovsky Journal of Emergency Medical Care. 2022;11(3):436–443. ц Сопутствующая патология у пациентов 1-й и 2-й группы Table 2 https://doi.org/10.23934/2223-9022-2022-11-3-436-443 ОРИГИНАЛЬНЫЕ СТАТЬИ Табли ц а 4 Тромботические и геморрагические осложнения Table 4 Thrombotic and hemorrhagic complications Группы пациентов ВТЭО Геморрагические осложнения ТГВ ТЭЛА Большие Значимые Малые Всего ТЭЛА+ ТГВ ТЭЛА без ТГВ 1-я группа (n=69) 4 (5,8%) 3 (4,3%) 1 (1,4%) 2 (3%) 1 (1,4%) 1 (1,4%) 11 (15,9%) 2-я группа (n=63) 0 (0%) 1 (1,6%) 0 (0%) 1 (1,6%) 1 (1,6%) 3 (4,8%) 8 (12,7%) р 0,052 0,355 0,338 0,614 0,729 0,268 0,390 Примечания: ВТЭО — венозные тромбоэмболические осложнения; ТГВ — тромбоз глубоких вен; ТЭЛА — тромбоэмболия легочной артерии Notes: ВТЭО — venous thromboembolic complications; ТГВ— deep vein thrombosis; ТЭЛА — pulmonary embolism результатам наиболее крупного метаанализа, вклю- чавшего 49 исследований, частота ВТЭО составила 17,0% (95% ДИ 13,4–20,9), из которых 12,1% (95% ДИ 8,4–16,4) приходилось на ТГВ и 7,1% (95% ДИ 5,3– 9,1) — на ТЭЛА. ВТЭО чаще регистрировались в отде- лениях реанимации (27,9 против 7,1%). Особенностью результатов данного метаанализа является то, что частота ВТЭО не зависела от характера фармакологи- ческой профилактики: 21% — без АКТ, 18,2% — при использовании профилактических доз антикоагулян- тов, 19,4% — при использовании повышенных доз антикоагулянтов [13]. течением. Следует отметить, что ВТЭО у пациентов с применением компрессионной терапии после отмены антикоагулянтов не наблюдалось. При анализе кровотечений не учитывали подкож- ные гематомы передней брюшной стенки, если они были не напряженными, потому что практически все пациенты получали нефракционированный гепарин (НФГ) в дозе 5 тыс. ЕД 4 раза в день и у всех наблюда- лось данное нежелательное явление. Малые кровоте- чения (носовые и десневые) в 1-й группе наблюдались у 11 пациентов (15,9%), во 2-й — у 8 (12,7%). После завершения курса АКТ на амбулаторном этапе малые кровотечения прекратились. По результатам нашего исследования ВТЭО выяв- лены у пациентов без ЭК (1-я группа) в 7 случаях (10,14%) и в одном случае (1,6%) с использованием ЭК, представленной ТЭЛА (р=0,039). Следует отметить, что данный единичный случай ТЭЛА во 2-й группе и 2 слу- чая ТЭЛА (3%) в 1-й группе были выявлены на аутоп- сии, при отсутствии источника в нижних конечностях. Распространенный легочный микротромбоз является уникальным признаком коронавирусной инфекции. В сравнительном исследовании аутопсийного мате- риала больных COVID-19 и больных гриппом H1N1 в альвеолярных капиллярах микротромбы в легочных артериях встречались в 9 раз чаще у умерших от коро- навирусной инфекции больных [14]. Обсуждение До пандемии при понимании патогенеза острого венозного тромбоза мы руководствовались триадой Вирхова и привычными факторами риска, на которые обращали внимание клиницисты. Были иммобилиза- ция, травма, обширные хирургические вмешательства, тромбофилии, заместительная гормональная терапия, онкология и другие [8, 9]. В настоящее время про- цесс гиперкоагуляции у больных новой коронавирус- ной инфекцией протекает совместно с «цитокиновым штормом», синдромом активации макрофагов, тром- боцитопатией, дисфункцией эндотелия и в литературе носит название «COVID-19-ассоциированная коагуло- патия» [10, 11]. В зоне тромбоза легочных артерий наблюдалось повреждение эндотелия и наличие внутриклеточных вирусных частиц, что указывает на взаимосвязь пов- реждения эндотелия и воспаления, зачастую являю- щихся причиной первичного тромбоза легочных арте- рий [15]. [ ] Новыми факторами, способствующими тромбооб- разованию, сегодня являются вирусная интоксикация, наличие дыхательной недостаточности, обусловлен- ной объемным поражением легких [12]. Однако обна- руживаются и ранее известные факторы риска, только теперь их можно встретить при лечении COVID-19 — это гиподинамия, вынужденный постельный режим для получения инсуффляции кислорода. По сути — эта та же иммобилизация пациента, только с наличи- ем вирусного заболевания. В нашем исследовании бóльшая часть пациентов получала кислородотерапию (83% — в 1-й группе и 87% — во 2-й группе, р=0,453). Все эти факторы делают пациентов с коронавирусной инфекцией уязвимыми перед сосудистыми тромботи- ческими осложнениями, поэтому применение средств профилактики ВТЭО наиболее оправдано у данных больных. При анализе лабораторных показателей исходно в обеих группах прослеживается высокая концентрация провоспалительных маркеров (СОЭ, СРБ, ферритина) и коагуляционных показателей (Д-димера, фибриноге- на), причем уровень фибриногена значительно боль- ше у пациентов с применением ЭК (1-я группа — 3,6 (1,1–7,4), 2-я группа — 4,4 (1,1–7,8), р=0,003). Уровень эритроцитов, гемоглобина, тромбоцитов находился у всех пациентов в пределах нормальных значений. В конце лечения наблюдалось закономерное снижение провоспалительных маркеров: СРБ, прокальцитони- на, СОЭ (табл. 3). Однако уровень Д-димера оставался высоким у всех больных, что указывает на высокие риски тромботических осложнений в стационаре и на амбулаторном этапе лечения. Исследуемые группы в нашем наблюдении были сопоставимы по клинико-анамнестическим данным, лечению, но, несмотря на это, ВТЭО наиболее часто наблюдались у пациентов без эластической компрес- сии. У пациентов, находившихся в реанимации, отмече- но резкое увеличение маркеров воспаления СРБ, фер- ритина, прокальцитонина. Уровень прокальцитонина имеет значение в диагностике и прогнозе течения сепсиса [16]. По нашим данным, у всех пациентов в В литературе уже достаточно данных о частоте встречаемости ВТЭО у пациентов с COVID-19. По 440 Russian Sklifosovsky Journal of Emergency Medical Care. 2022;11(3):436–443. https://doi.org/10.23934/2223-9022-2022-11-3-436-443 ОРИГИНАЛЬНЫЕ СТАТЬИ Полученные данные наглядно показывают, что использование профилактического компрессионного трикотажа предупреждает развитие тромботических осложнений. Список источников 1. Celovska D, Wawruch M, Stvrtinova V. COVID-19-associated coagulo pathy and immuno-thrombosis. Acta Phlebologica. 2021;22(2):55–60. https://doi.org/10.23736/S1593-232X.21.00510-5 1. Celovska D, Wawruch M, Stvrtinova V. COVID-19-associated coagulo pathy and immuno-thrombosis. Acta Phlebologica. 2021;22(2):55–60. https://doi.org/10.23736/S1593-232X.21.00510-5 после имплантации двухкамерных электрокардиостимуляторов. Российский медико-биологический вестник имени академика И.П. Павлова. 2021;29(4):497–504. https://doi.org/https://doi.org/10.17816/ PAVLOVJ79285 2. Shi S, Qin M, Shen B, Cai Y, Liu T, Yang F, et al. Association of cardiac injury with mortality in hospitalized patients with COVID-19 in Wuhan, China. JAMA Cardiol. 2020;5(7):802–810. PMID: 32211816 https://doi. org/10.1001/jamacardio.2020.0950 2. Shi S, Qin M, Shen B, Cai Y, Liu T, Yang F, et al. Association of cardiac injury with mortality in hospitalized patients with COVID-19 in Wuhan, China. JAMA Cardiol. 2020;5(7):802–810. PMID: 32211816 https://doi. org/10.1001/jamacardio.2020.0950 11. Верткин А.Л., Авдеев С.Н., Ройтман Е.В., Сучков И.А., Кузне- цова И.В., Замятин М.Н., и др. Вопросы лечения COVID-19 с позиции коррекции эндотелиопатии и профилактики тромботи- ческих осложнений. Согласованная позиция экспертов. Профи- лактическая медицина. 2021;24(4):45–51. https://doi.org/10.17116/ profmed20212404145 3. Бицадзе В.О., Бредихин Р.А., Булатов В.Л., Головина В.И., Дже- нина О.В., Золотухин И.А., и др. Флебит и тромбофлебит повер- хностных сосудов. Флебология. 2021;15(3):211–244. https://doi. org/10.17116/flebo202115031211 12. Bikdeli B, Madhavan MV, Jimenez D, Chuich T, Dreyfus I, Driggin E, et al. COVID-19 and Thrombotic or Thromboembolic Disease: Implications for Prevention, Antithrombotic Therapy, and Follow-up. J Am Coll Cardiol. 2020;75(23):2950–2973. PMID: 32311448 https://doi. org/10.1016/j.jacc.2020.04.031 4. Министерство здравоохранения РФ. Профилактика, диагностика и лечение новой коронавирусной инфекции (COVID-19): Временные методические рекомендации. Версия 14 (27.12.2021). URL: https:// стопкоронавирус.рф/ai/doc/1213/attach/vmr_COVID-19_V14_27-12- 2021.pdf [Дата обращения 7 июля 2022] 13. Karakike E, Giamarellos-Bourboulis EJ. Macrophage activation-like syndrome: a distinct entityleading to early death in sepsis. Front Immunol. 2019;10:55. PMID: 30766533 https://doi.org/10.3389/ fimmu.2019.00055 5. Лишов Д.Е., Бойко Л.В., Золотухин И.А., Илюхин Е.А., Каторкин С. Е., Березко М.П. и др. Ультразвуковое исследование вен нижних конечностей. Рекомендации экспертов Ассоциации флебологов России. Флебология. 2021;15(4):318–340. https://doi.org/10.17116/ flebo202115041318 14. Jimenez D, Garcia-Sanchez A, Rali P, MurielA, Bikdeli B, Ruiz-Artacho P, et al. Incidence of VTe and bleeding among Hospitalized Patients with Coronavirus Disease 2019: A Systematic Review and Meta- analysis. Chest. 2021;159(3):1182–1196. PMID: 33217420 https://doi. org/10.1016/ j.chest.2020.11.005 6. Schulman, S. Definition of major bleeding in clinical investigations of antihemostatic medicinal products in non-surgical patients. J Thromb Haemost. 2005;3(4):692–694. PMID: 15842354. http://doi.org/10.1111/ j.1538-7836.2005.01204.x 15. Ackermann M, Verleden SE, Kuehnel M, Haverich A, Welte T, Laenger F, et al. Pulmonary vascular endothelialitis, thrombosis, and angiogenesis in Covid-19. N Engl J Med. 2020;383(2):120–128. PMID: 32437596 https://doi.org/10.1016/10.1056/NEJMoa2015432 7. 1. Летальность у пациентов с коронавирусной инфекцией без компрессионной терапии выше, чем у пациентов с применением компрессионного трико- тажа. Особенно большие показатели протромбинового времени у пациентов 2-й группы (1-я группа — 15,2 (11,2–25,2) сек и 2-я группа — 17,3 (11,8–45,6) сек). Данный показатель также сигнализирует о высоком риске кровотечений. По результатам нашего исследо- вания в 1-й группе наблюдалось одно (1,4%) большое легочное кровотечение (табл. 4). Несмотря на консер- вативные и хирургические методы лечения крово- течения больной скончался. У пациентов 2-й группы зафиксирован один (1,6%) случай большого кровоте- чения (межмышечная гематома), которое потребовало гемотрансфузии (р=0,729). 2. У пациентов, носивших компрессионные чулки в стационаре, имеет место низкая частота развития венозных тромбоэмболических осложнений (1,6% слу- чаев во 2-й группе против 10,14% случаев в 1-й группе, р=0,039). 3. В исследуемых группах была одинаковая часто- та больших кровотечений: 1-я группа — один случай (1,4%), 2-я группа — один случай (1,6%). 4. Применение профилактического трикотажа на стационарном этапе лечения новой коронавирусной инфекции позволяет выполнять профилактику веноз- ных тромбоэмболических осложнений у пациентов с кровотечениями — при невозможности проведения антикоагулянтной терапии. Среди кровотечений один (1,4%) случай в 1-й груп- пе и 3 (4,8%) — во 2-й (р=0,268). Учитывая, что при коронавирусной инфекции всем больным показана АКТ, невозможность ее применения делает профилак- тический компрессионный трикотаж единственным средством профилактики ВТЭО. Среди кровотечений один (1,4%) случай в 1-й груп- пе и 3 (4,8%) — во 2-й (р=0,268). Учитывая, что при коронавирусной инфекции всем больным показана АКТ, невозможность ее применения делает профилак- тический компрессионный трикотаж единственным средством профилактики ВТЭО. Обсуждение При этом важным наблюдением стало то, что наличие ВТЭО было причиной смерти у 6 пациен- тов с ТЭЛА (8,7%). Такая связь демонстрирует наличие тяжелых микроциркуляторных нарушений в организ- ме с развитием фатальных осложнений у пациентов с новой коронавирусной инфекцией. ОРИТ наблюдаются большие значения прокальцито- нина (1-я группа — 1,56 (0,036–14,7) нг/мл и 2-я груп- па — 1,1 (0,063–3,9) нг/мл, р=0,493), что указывает на присоединение бактериальной ко-инфекции у паци- ентов с COVID-19. В коагулограмме наблюдается уве- личение уровня АЧТВ, протромбинового времени и Д-димера. Увеличение АЧТВ у всех пациентов может быть связано с ведением данных больных на внутри- венной инфузии гепарина. Уровень протромбинового времени отражает время свертывания крови и его увеличение наблюдается при 2-й фазе ДВС-синдрома (диссеменированное сосудистое свертывание), кото- рый является неотъемлемой частью тяжелых пациен- тов с COVID-19 [17, 18]. Russian Sklifosovsky Journal of Emergency Medical Care. 2022;11(3):436–443. https://doi.org/10.23934/2223-9022-2022-11-3-436-443 Выводы 1. Летальность у пациентов с коронавирусной инфекцией без компрессионной терапии выше, чем у пациентов с применением компрессионного трико- тажа. 1. Летальность у пациентов с коронавирусной инфекцией без компрессионной терапии выше, чем у пациентов с применением компрессионного трико- тажа. References of dual-chamber pacemakers. I.P. Pavlov Russian Medical Biological Herald. 2021;29(4):497–504. (In Russ.) https://doi.org/https://doi. org/10.17816/PAVLOVJ79285 1. Celovska D, Wawruch M, Stvrtinova V. COVID-19-associated coagulopathy and immuno-thrombosis. Acta Phlebologica. 2021;22(2):55–60. https:// doi.org/10.23736/S1593-232X.21.00510-5 2. Shi S, Qin M, Shen B, Cai Y, Liu T, Yang F, et al. Association of cardiac injury with mortality in hospitalized patients with COVID-19 in Wuhan, China. JAMA Cardiol. 2020;5(7):802–810. PMID: 32211816 https://doi. org/10.1001/jamacardio.2020.0950 11. Vertkin AL, Avdeev SN, Roitman EV, Suchkov IA, Kuznetsova IV, Zamyatin MN, et al. Treatment of COVID-19 from the perspective of endotheliopathy correction and prevention of thrombotic complications. The agreed position of the experts. Profilakticheskaya Meditsina. 2021;24(4):45–51. (In Russ.). https://doi.org/10.17116/ profmed20212404145 3. Bitsadze VO, Bredikhin RA, Bulatov VL, Golovina VI, Dzhenina OV, Zolotukhin IA, et al. Superficial phlebitis and thrombophlebitis. Flebologiya. 2021;15(3):211–244. (In Russ.). https://doi.org/10.17116/ flebo202115031211 12. Bikdeli B, Madhavan MV, Jimenez D, Chuich T, Dreyfus I, Driggin E, et al. COVID-19 and Thrombotic or Thromboembolic Disease: Implications for Prevention, Antithrombotic Therapy, and Follow-up. J Am Coll Cardiol. 2020;75(23):2950–2973. PMID: 32311448 https://doi. org/10.1016/j.jacc.2020.04.031 4. Ministerstvo zdravookhraneniya RF. Profilaktika, diagnostika i lechenie novoy koronavirusnoy infektsii (COVID-19): Vremennye metodicheskie rekomendatsii. Versiya 14 (27.12.2021) Available at: https://xn-- 80aesfpebagmfblc0a.xn--p1ai/ai/doc/1213/attach/vmr_COVID-19_V14_ 27-12-2021.pdf [Accessed Jul 7, 2022] 13. Karakike E, Giamarellos-Bourboulis EJ. Macrophage activation-like syndrome: a distinct entityleading to early death in sepsis. Front Immunol. 2019;10:55. PMID: 30766533 https://doi.org/10.3389/ fimmu.2019.00055 5. Lishov DE, Boyko LV, Zolotukhin IA, Ilyukhin EA, Katorkin SE, Berezko MP, et al. Duplex Ultrasound of Lower Limbs Venous System. Russian Phlebology Association Expert Panel Report. Flebologiya. 2021;15(4):318– 340. (In Russ.). https://doi.org/10.17116/flebo20211504131 14. Jimenez D, Garcia-Sanchez A, Rali P, MurielA, Bikdeli B, Ruiz-Artacho P, et al. Incidence of VTe and bleeding among Hospitalized Patients with Coronavirus Disease 2019: A Systematic Review and Meta- analysis. Chest. 2021;159(3):1182–1196. PMID: 33217420 https://doi. org/10.1016/ j.chest.2020.11.005 6. Schulman, S. Definition of major bleeding in clinical investigations of antihemostatic medicinal products in non-surgical patients. J Thromb Haemost. 2005;3(4):692–694. PMID: 15842354. http://doi.org/10.1111/ j.1538-7836.2005.01204.x 15. Ackermann M, Verleden SE, Kuehnel M, Haverich A, Welte T, Laenger F, et al. Pulmonary vascular endothelialitis, thrombosis, and angiogenesis in Covid-19. N Engl J Med. 2020;383(2):120–128. PMID: 32437596 https://doi.org/10.1016/10.1056/NEJMoa2015432 7. Anesteziologo-reanimatsionnoe obespechenie patsientov s novoy koronavirusnoy infektsiey COVID-19. Metodicheskie rekomendatsii Obshcherossiyskoy obshchestvennoy organizatsii ”Federatsiya anesteziologov i reanimatologov”, versiya 5. Accessed: https://www. arfpoint.ru/wp-content/uploads/2021/03/mr-ar-covid-19-v5-1.pdf [Accessed Jul 7, 2022] 16. Li Y, Li H, Zhu S, Xie Y, Wang B, He L, et al. References Prognostic value of right ventricular longitudinal strain in patients with COVID-19. JACC Cardiovasc Imaging. 2020;13(11):2287–2299. PMID: 32654963 https:// doi.org/10.1016/j.jcmg.2020.04.014 8. Bokeriya LA, Zatevakhin II, Kirienko AI. (project coordinators). Rossiyskie klinicheskie rekomendatsii po diagnostike, lecheniyu i profilaktike venoznykh tromboembolicheskikh oslozhneniy (VTEO). Flebologiya. 2015;9(4, 2):3–52. (In Russ.). 17. Borodina IA, Selezneva IA, Borisova OV, Baldina OA, Gil’miyarova FN, Gusyakova OA, et al. Blood groups and secretory state in COVID-19. Science of the Young (Eruditio Juvenium). 2021;9(4):589–596. (In Russ.) https://doi.org/10.23888/HMJ202194589-596 9. Kalinin RE, Suchkov IA, Agapov AB. The Effectiveness of the Various Options of Anticoagulant Therapy for the Treatment of the Patients with Thrombosis of the Deep Veins of the Lower Extremities in the Routine Clinical Practice. Flebologiya. 2017;11(1):21–27. (In Russ.). https://doi.org/10.17116/flebo201711121-27 18. Petrikov AS, Dudin DV, Popkova LN, Volodin IV, Shoikhet YaN. Clinical Course of Deep Venous Thrombosis of Lower Limbs in Acute Period With Underlying Anticoagulant Therapy Depending on Concentration of D- dimers and C-reactive Protein. Science of the Young (Eruditio Juvenium). 2018;6(2):233–241. (In Russ.) https://doi.org/10.23888/HMJ201862233- 241 10. Kalinin RE, Suchkov IA, Povarov VO, Mzhavanadze ND, Jurina ON. Hemostasis system in patients with bradycardias after the implantation 10. Kalinin RE, Suchkov IA, Povarov VO, Mzhavanadze ND, Jurina ON. Hemostasis system in patients with bradycardias after the implantation Список источников Анестезиолого-реанимационное обеспечение пациентов с новой коронавирусной инфекцией COVID-19. Методические рекомендации Общероссийской общественной организации «Федерация анесте- зиологов и реаниматологов», версия 5. URL: https://www.arfpoint. ru/wp-content/uploads/2021/03/mr-ar-covid-19-v5-1.pdf [Дата обра- щения 7 июля 2022] 16. Li Y, Li H, Zhu S, Xie Y, Wang B, He L, et al. Prognostic value of right ventricular longitudinal strain in patients with COVID-19. JACC Cardiovasc Imaging. 2020;13(11):2287–99. PMID: 32654963 https://doi. org/10.1016/j.jcmg.2020.04.014 8. Бокерия Л.А., Затевахин И.И., Кириенко А.И. (координаторы проек- та). Российские клинические рекомендации по диагностике, лече- нию и профилактике венозных тромбоэмболических осложнений (ВТЭО). Флебология. 2015;9(4, вып. 2):3–52. 17. Бородина И.А., Селезнева И.А., Борисова О.В., Балдина О.А., Гиль- миярова Ф.Н., Гусякова О.А., и др. Группы крови и секретор- ное состояние при COVID-19. Наука молодых (Eruditio Juvenium). 2021;9(4):589–596. https://doi.org/10.23888/HMJ202194589-596 9. Калинин Р.Е., Сучков И.А., Агапов А.Б. Эффективность различных вариантов антикоагулянтной терапии при тромбозе глубоких вен нижних конечностей в рутинной клинической практике. Флеболо- гия. 2017;11(1):21–27. https://doi.org/10.17116/flebo201711121-27 18. Петриков А.С., Дудин Д.В., Попкова Л.Н., Володин И.В., Шойхет Я. Н. Течение тромбоза глубоких вен нижних конечностей в остром периоде на фоне антикоагулянтной терапии в зависимости от содержания D-димеров и С-реактивного белка. Наука молодых (Eruditio Juvenium). 2018;6(2):233–241. 10. Калинин Р.Е., Сучков И.А., Поваров В.О., Мжаванадзе Н.Д., Жури- на О.Н. Состояние системы гемостаза пациентов с брадикардиями 441 Russian Sklifosovsky Journal of Emergency Medical Care. 2022;11(3):436–443. https://doi.org/10.23934/2223-9022-2022-11-3-436-443 ОРИГИНАЛЬНЫЕ СТАТЬИ информация об авторах информация об авторах Калинин Роман Евгеньевич доктор медицинских наук, профессор, заведующий кафедрой сердечно-сосудистой, рентгенэн- доваскулярной хирургии и лучевой диагностики ФГБОУ ВО РязГМУ Минздрава России; Калинин Роман Евгеньевич доктор медицинских наук, профессор, заведующий кафедрой сердечно-сосудистой, рентгенэн- доваскулярной хирургии и лучевой диагностики ФГБОУ ВО РязГМУ Минздрава России; доктор медицинских наук, профессор, заведующий кафедрой сердечно-сосудистой, рентгенэн- доваскулярной хирургии и лучевой диагностики ФГБОУ ВО РязГМУ Минздрава России; Калинин Роман Евгеньевич доктор медицинских наук, профессор, заведующий кафедрой сердечно-сосудистой, рентгенэн- доваскулярной хирургии и лучевой диагностики ФГБОУ ВО РязГМУ Минздрава России; https://orcid.org/0000-0002-0817-9573, kalinin-re@yandex.ru; 35%: концепция исследования, редактирование Сучков Игорь Александрович доктор медицинских наук, профессор, профессор кафедры сердечно-сосудистой, рентгенэндо- васкулярной хирургии и лучевой диагностики ФГБОУ ВО РязГМУ Минздрава России; https://orcid.org/0000-0002-1292-5452, suchkov_med@mail.ru; 25%: концепция исследования, редактирование Агапов Андрей Борисович кандидат медицинских наук, врач отделения сосудистой хирургии ГБУ Рязанской области «Об- ластная клиническая больница»; https://orcid.org/0000-0003-0178-1649, agapchik2008@yandex.ru; 20%: сбор материала, написание текста Мжаванадзе Нина Джансуговна кандидат медицинских наук, доцент кафедры сердечно-сосудистой, рентгенэндоваскулярной хирургии и лучевой диагностики ФГБОУ ВО РязГМУ Минздрава России; https://orcid.org/0000-0001-5437-1112, nina_mzhavanadze@mail.ru; 10%: сбор материала, редактирование Максаев Денис Алексеевич кандидат медицинских наук, аспирант кафедры сердечно-сосудистой, рентгенэндоваскулярной хирургии и лучевой диагностики ФГБОУ ВО РязГМУ Минздрава России; https://orcid.org/0000-0003-3299-8832, denma1804@yandex.ru; 5%: сбор материала Чобанян Артём Александрович аспирант кафедры сердечно-сосудистой, рентгенэндоваскулярной хирургии и лучевой диагнос- тики ФГБОУ ВО РязГМУ Минздрава России; https://orcid.org/0000-0002-8129-5976, artaleksandrovich@gmail.com; 5%: сбор материала Авторы заявляют об отсутствии конфликта интересов https://orcid.org/0000-0002-0817-9573, kalinin-re@yandex.ru; https://orcid.org/0000-0002-0817-9573, kalinin-re@yandex.ru; 35%: концепция исследования, редактирование доктор медицинских наук, профессор, профессор кафедры сердечно-сосудистой, рентгенэндо- васкулярной хирургии и лучевой диагностики ФГБОУ ВО РязГМУ Минздрава России; доктор медицинских наук, профессор, профессор кафедры сердечно-сосудистой, рентгенэндо- васкулярной хирургии и лучевой диагностики ФГБОУ ВО РязГМУ Минздрава России; tps://orcid.org/0000-0002-1292-5452, suchkov_med@mail.ru кандидат медицинских наук, врач отделения сосудистой хирургии ГБУ Рязанской области «Об- ластная клиническая больница»; Мжаванадзе Нина Джансуговна кандидат медицинских наук, доцент кафедры сердечно-сосудистой, рентгенэндоваскулярной хирургии и лучевой диагностики ФГБОУ ВО РязГМУ Минздрава России; аспирант кафедры сердечно-сосудистой, рентгенэндоваскулярной хирургии и лучевой диагнос- тики ФГБОУ ВО РязГМУ Минздрава России; 442 Russian Sklifosovsky Journal of Emergency Medical Care. 2022;11(3):436–443. https://doi.org/10.23934/2223-9022-2022-11-3-436-443 ОРИГИНАЛЬНЫЕ СТАТЬИ R.E. Kalinin , I.A. Suchkov, A.B. Agapov, N.D. Mzhavanadze, D.A. Maksayev, A.A. Chobanyan sults In patients without prophylactic compression (Group 1) there were 7 VTEC cases (10.14%); deep vein thrombosis (DVT) 4 (5.8%), pulm ) — 3 (4.3%). All 6 cases (8.7%) with PE were fatal. It was noted that 2 cases of PE (2.8%) were verified upon autopsy, and not clinically. In patients of the 2nd group with the use of elastic compression, the overall incidence of VTEC was only one case (1.6%). When analyzing the frequency of bleeding in the 1st group, there was one pulmonary bleeding (1.4%), in the 2nd group there was also one case of intense intermuscular hematoma (1.6%). Significant bleeding was observed in one patient (1.4%) of the 1st group, and in 3 (4.8%) cases of the 2nd group. The minor bleeding was observed in 11 patients (15.9%) of the 1st group, and in 8 (12.7%) patients of the 2nd group. The mortality during hospitalization was 11 (15.9%) cases in patients of the 1st group (without EC) and 7 (11.1%) in patients of the 2nd group (with EC) (p=0.419). Conclusions The mortality in patients with coronavirus infection without compression therapy is higher than in patients with compression stockings (p=0.419). Patients wearing compression stockings in the hospital had a low incidence of VTEC (1.6% of cases in group 2 versus 10.14% of cases in group 1, p=0.039). The study groups had the same frequency of bleeding (group 1 — one case (1.4%), group 2 — one case (1.6%). The use of preventive compression at the inpatient stage of treatment of a new coronavirus infection makes it possible to prevent VTEC in patients with bleeding when anticoagulant therapy is not possible. Keywords: new coronavirus infection, thrombosis, bleeding, elastic compression, venous thromboembolic complications, COVID-19 Keywords: new coronavirus infection, thrombosis, bleeding, elastic compression, venous thromboembolic complications, COVID-19 For citation Kalinin RE Suchkov IA Agapov AB Mzhavanadze ND Maksayev DA Chobanyan AA The Effectiveness of Compression Therapy in the Preve y , , g, p , p , For citation Kalinin RE, Suchkov IA, Agapov AB, Mzhavanadze ND, Maksayev DA, Chobanyan AA. The Effectiveness of Compression Therapy in the Prevention of Venous Thromboembolic Complications in Patients With COVID-19. Russian Sklifosovsky Journal of Emergency Medical Care. 2022;11(3):436–443. https://doi. R.E. Kalinin , I.A. Suchkov, A.B. Agapov, N.D. Mzhavanadze, D.A. Maksayev, A.A. Chobanyan Chobanyan Postgraduate Student of the Department of Cardiovascular, X-ray Endovascular Surgery and Radiation Diagnosis, Ryazan State Medical University; https://orcid.org/0000-0002-8129-5976, artaleksandrovich@gmail.com; 5%, material collection Поступила в редакцию 18.02.2022 Рецензирование завершено 12.04.2022 Принята к печати 29.06.2022 Received on 18.02.2022 Review completed on 12.04.2022 Accepted on 29.06.2022 Doctor of Medical Sciences, Professor, Head of the Department of Cardiovascular, X-ray Endovascular Surgery and Radiation Diagnosis, Ryazan State Medical University; https://orcid.org/0000-0002-0817-9573, kalinin-re@yandex.ru; 35%, concept of the study, editing Doctor of Medical Sciences, Professor, Professor of the Department of Cardiovascular, X-ray Endovascular Surgery and Radiation Diagnosis, Ryazan State Medical University; https://orcid.org/0000-0002-1292-5452, suchkov_med@mail.ru; 25%, concept of the study, editing Candidate of Medical Sciences, Physician of the Department of Vascular Surgery, Ryazan Territory Regional Clinical Hospital; https://orcid.org/0000-0003-0178-1649, agapchik2008@yandex.ru; 20%, material collection, text writing Candidate of Medical Sciences, Associate Professor of the Department of Cardiovascular, X-ray Endovascular Surgery and Radiation Diagnosis, Ryazan State Medical University; https://orcid.org/0000-0001-5437-1112, nina_mzhavanadze@mail.ru; 10%, collection of material, editing Candidate of Medical Sciences, postgraduate student of the Department of Cardiovascular, X-ray Endovascular Surgery and Radiation Diagnosis, Ryazan State Medical University; https://orcid.org/0000-0003-3299-8832, denma1804@yandex.ru; 5%, material collection Postgraduate Student of the Department of Cardiovascular, X-ray Endovascular Surgery and Radiation Diagnosis, Ryazan State Medical University; https://orcid.org/0000-0002-8129-5976, artaleksandrovich@gmail.com; 5%, material collection Поступила в редакцию 18.02.2022 Рецензирование завершено 12.04.2022 Принята к печати 29.06.2022 22 Roman E. Kalinin Doctor of Medical Sciences, Professor, Head of the Department of Cardiovascular, X-ray Endovascular Surgery and Radiation Diagnosis, Ryazan State Medical University; https://orcid.org/0000-0002-0817-9573, kalinin-re@yandex.ru; 35%, concept of the study, editing Igor A. Suchkov Doctor of Medical Sciences, Professor, Professor of the Department of Cardiovascular, X-ray Endovascular Surgery and Radiation Diagnosis, Ryazan State Medical University; https://orcid.org/0000-0002-1292-5452, suchkov_med@mail.ru; 25%, concept of the study, editing Andrey B. Agapov Candidate of Medical Sciences, Physician of the Department of Vascular Surgery, Ryazan Territory Regional Clinical Hospital; https://orcid.org/0000-0003-0178-1649, agapchik2008@yandex.ru; 20%, material collection, text writing Nina D. Mzhavanadze Candidate of Medical Sciences, Associate Professor of the Department of Cardiovascular, X-ray Endovascular Surgery and Radiation Diagnosis, Ryazan State Medical University; https://orcid.org/0000-0001-5437-1112, nina_mzhavanadze@mail.ru; 10%, collection of material, editing Denis A. Maksayev Candidate of Medical Sciences, postgraduate student of the Department of Cardiovascular, X-ray Endovascular Surgery and Radiation Diagnosis, Ryazan State Medical University; https://orcid.org/0000-0003-3299-8832, denma1804@yandex.ru; 5%, material collection Artyom A. R.E. Kalinin , I.A. Suchkov, A.B. Agapov, N.D. Mzhavanadze, D.A. Maksayev, A.A. Chobanyan org/10.23934/2223-9022-2022-11-3-436-443 (in Russ.) Conflict of interest Authors declare lack of the conflicts of interests Acknowledgments, sponsorship The compression stockings for the study were provided by ELAST MEDICAL LLC Affiliations Acknowledgments, sponsorship The compression stockings for the study were provided by ELAST MEDICAL LLC Roman E. Kalinin Doctor of Medical Sciences, Professor, Head of the Department of Cardiovascular, X-ray Endovascular Surgery and Radiation Diagnosis, Ryazan State Medical University; https://orcid.org/0000-0002-0817-9573, kalinin-re@yandex.ru; 35%, concept of the study, editing Igor A. Suchkov Doctor of Medical Sciences, Professor, Professor of the Department of Cardiovascular, X-ray Endovascular Surgery and Radiation Diagnosis, Ryazan State Medical University; https://orcid.org/0000-0002-1292-5452, suchkov_med@mail.ru; 25%, concept of the study, editing Andrey B. Agapov Candidate of Medical Sciences, Physician of the Department of Vascular Surgery, Ryazan Territory Regional Clinical Hospital; https://orcid.org/0000-0003-0178-1649, agapchik2008@yandex.ru; 20%, material collection, text writing Nina D. Mzhavanadze Candidate of Medical Sciences, Associate Professor of the Department of Cardiovascular, X-ray Endovascular Surgery and Radiation Diagnosis, Ryazan State Medical University; https://orcid.org/0000-0001-5437-1112, nina_mzhavanadze@mail.ru; 10%, collection of material, editing Denis A. Maksayev Candidate of Medical Sciences, postgraduate student of the Department of Cardiovascular, X-ray Endovascular Surgery and Radiation Diagnosis, Ryazan State Medical University; https://orcid.org/0000-0003-3299-8832, denma1804@yandex.ru; 5%, material collection Artyom A. R.E. Kalinin , I.A. Suchkov, A.B. Agapov, N.D. Mzhavanadze, D.A. Maksayev, A.A. Chobanyan R.E. Kalinin , I.A. Suchkov, A.B. Agapov, N.D. Mzhavanadze, D.A. Maksayev, A.A. Chobanyan Department of Cardiovascular, X-ray Endovascular Surgery and Radiation Diagnosis I.P. Pavlov Ryazan State Medical University 9, Vysokovoltnaya St., Ryazan, 390026, Russian Federation E. Kalinin, Doctor of Medical Sciences, Professor, Head of theDepartment of Cardiovascular, X-ray Endovascular Surgery and Radiation Diagnosis, Ryazan State Med alinin-re@yandex.ru Background The main method for preventing thrombotic complications in patients with coronavirus infection is anticoagulant therapy (ACT). However, its use is not always possible, in particular in patients with bleeding. The only method of prevention in this case is elastic compression (EC) of the lower extremities. Aim of study To evaluate the effectiveness of lower extremity EC for the prevention of venous thromboembolic complications (VTEC) in patients with a new coronavirus infection. Material and methods The study was approved by the local Ethics Committee of the Ryazan State Medical University of the Ministry of Health of Russian Federation and registered on the ClinicalTrials.gov platform (identifier NCT05143567). The study included 69 patients without prophylactic compression (Group 1) and 65 patients who used prophylactic compression stockings (Group 2). The patients were treated in the covid hospital from July to November 2021. All patients had verified coronavirus infection, they took ACT and underwent ultrasound duplex scanning of the veins of the lower extremities upon admission, in the intensive care unit and upon discharge. We assessed the frequency of venous thromboembolic complications (VTEC), hemorrhagic complications, and mortality. To assess the severity of bleeding, the classification of the Committee of the International Society on Thrombosis and Haemostasis (ISTH) was used. i sults In patients without prophylactic compression (Group 1) there were 7 VTEC cases (10.14%); deep vein thrombosis (DVT) — 4 (5.8%), p Results In patients without prophylactic compression (Group 1) there were 7 VTEC cases (10.14%); deep vein thrombosis (DVT) — 4 (5 (PE) 3 (4 3%) All 6 cases (8 7%) with PE were fatal It was noted that 2 cases of PE (2 8%) were verified upon autopsy and not clinica ents without prophylactic compression (Group 1) there were 7 VTEC cases (10.14%); deep vein thrombosis (DVT) — 4 (5.8%), pulmonary embol ll 6 cases (8.7%) with PE were fatal. It was noted that 2 cases of PE (2.8%) were verified upon autopsy, and not clinically. R.E. Kalinin , I.A. Suchkov, A.B. Agapov, N.D. Mzhavanadze, D.A. Maksayev, A.A. Chobanyan Chobanyan Postgraduate Student of the Department of Cardiovascular, X-ray Endovascular Surgery and Radiation Diagnosis, Ryazan State Medical University; https://orcid.org/0000-0002-8129-5976, artaleksandrovich@gmail.com; 5%, material collection Поступила в редакцию 18.02.2022 Рецензирование завершено 12.04.2022 Принята к печати 29.06.2022 Received on 18.02.2022 Review completed on 12.04.2022 Accepted on 29.06.2022 Doctor of Medical Sciences, Professor, Head of the Department of Cardiovascular, X-ray Endovascular Surgery and Radiation Diagnosis, Ryazan State Medical University; Roman E. Kalinin Doctor of Medical Sciences, Professor, Head of the Department of Cardiovascular, X-ray Endovascular Surgery and Radiation Diagnosis, Ryazan State Medical University; Radiation Diagnosis, Ryazan State Medical University https://orcid.org/0000-0002-0817-9573, kalinin-re@yandex.ru; Igor A. Suchkov Doctor of Medical Sciences, Professor, Professor of the Department of Cardiovascular, X-ray Endovascular Surgery and Radiation Diagnosis, Ryazan State Medical University; Doctor of Medical Sciences, Professor, Professor of the Department of Cardiovascular, X-ray Endovascular Surgery and Radiation Diagnosis, Ryazan State Medical University; Radiation Diagnosis, Ryazan State Medical University; https://orcid.org/0000-0002-1292-5452, suchkov_med@mail.ru; Andrey B. Agapov Candidate of Medical Sciences, Physician of the Department of Vascular Surgery, Ryazan Territory Regional Clinical Hospital; Candidate of Medical Sciences, Physician of the Department of Vascular Surgery, Ryazan Territory Regional Clinical Hospital; https://orcid.org/0000-0003-0178-1649, agapchik2008@yandex.ru; Denis A. Maksayev ostgraduate Student of the Department of Cardiovascular, X-ray Endovascular Surgery and Radiation Diagnosis, Ryazan tate Medical University; y https://orcid.org/0000-0002-8129-5976, artaleksandrovich@gmail.com; 5% i l ll i Received on 18.02.2022 Review completed on 12.04.2022 Accepted on 29.06.2022 Поступила в редакцию 18.02.2022 Рецензирование завершено 12.04.2022 Принята к печати 29.06.2022 443 Russian Sklifosovsky Journal of Emergency Medical Care. 2022;11(3):436–443. https://doi.org/10.23934/2223-9022-2022-11-3-436-443
https://openalex.org/W2911248329	https://link.springer.com/content/pdf/10.1007/s00010-019-00638-9.pdf	English	null	Extension problem for principles of equivalent utility	Aequationes mathematicae	2,019	cc-by	11,093	Mathematics Subject Classiﬁcation. 39B12, 39B82, 91B16. Keywords. Principle of equivalent utility, Extension, Insurance premium, General linear equa- tion. Aequat. Math. 93 (2019), 217–238 c⃝The Author(s) 2019 0001-9054/19/010217-22 published online February 14, 2019 https://doi.org/10.1007/s00010-019-00638-9 Aequat. Math. 93 (2019), 217–238 c⃝The Author(s) 2019 0001-9054/19/010217-22 published online February 14, 2019 https://doi.org/10.1007/s00010-019-00638-9 Aequat. Math. 93 (2019), 217–238 c⃝The Author(s) 2019 0001-9054/19/010217-22 published online February 14, 2019 https://doi.org/10.1007/s00010-019-00638-9 Aequationes Mathematicae Extension problem for principles of equivalent utility Jacek Chudziak Dedicated to Professor Karol Baron on the occasion of his 70-th birthday. Abstract. We prove that, under the Cumulative Prospect Theory, every principle of equivalent utility, deﬁned on a family of ternary risks, possesses a unique extension to the family of all risks. Mathematics Subject Classiﬁcation. 39B12, 39B82, 91B16. 1. Introduction In insurance mathematics, risks are usually represented by non-negative bounded random variables on a given probability space. A premium principle is a functional assigning to every risk a non-negative real number. There are several methods of deﬁning principles. In this paper we deal with the principle of equivalent utility, belonging to the so-called economic methods of insurance contracts pricing. The principle, introduced by B¨uhlmann [2], involves the no- tion of a utility function and postulates a fairness in terms of utility. In order to recall the principle, assume that w ∈[0, ∞) is an insurance company’s initial wealth level and ⪯is its preference relation over a family X+ of risks. Then the relation ⪯in a natural way induces on X+ the indiﬀerence relation ∼: X ∼Y ⇐⇒(X ⪯Y ) and (Y ⪯X) for X, Y ∈X+. The principle of equivalent utility for the risk X ∈X+ is a real number H⪯(X) such that for X, Y ∈X+. The principle of equivalent utility for the risk X ∈X+ is a real number H⪯(X) such that w + H⪯(X) −X ∼w. (1) (1) hudziak AEM 218 J. Chudziak AEM This condition has the following interpretation: the company is indiﬀerent between rejecting the contract and entering into it. Thus, H⪯(X) is a minimal This condition has the following interpretation: the company is indiﬀerent between rejecting the contract and entering into it. Thus, H⪯(X) is a minimal price for which the insurance company would be ready to insure the risk X. price for which the insurance company would be ready to insure the risk X. In general, the principle of equivalent utility need not exist and, even if it exists, it need not be uniquely determined. Under the Expected Utility theory ( ) p p y y In general, the principle of equivalent utility need not exist and, even if it exists, it need not be uniquely determined. Under the Expected Utility theory (1) becomes (1) becomes E[u(w + Hu(X) −X)] = u(w), (2) (2) According to [4, Theorem 3.1], in the case where w ∈(0, ∞), for every continuous and strictly increasing function u : R →R satisfying u(0) = 0 and every continuous probability distortion Extension problem for principles of equivalent utility Vol. 93 (2019) Extension problem for principles of equivalent utility 219 functions g and h, Eq. (5) uniquely determines H(u,g,h)(X) for X ∈X+. If w = 0, then H(u,g,h)(X) is uniquely determined by (5) for every X ∈X+ if and only if functions g and h, Eq. (5) uniquely determines H(u,g,h)(X) for X ∈X+. If w = 0, then H(u,g,h)(X) is uniquely determined by (5) for every X ∈X+ if and only if h(p) + g(1 −p) > 0 for p ∈[0, 1]. (7) (7) Furthermore (cf. [9]), we have Furthermore (cf. [9]), we have 0 ≤H(u,g,h)(X) ≤ess sup X for X ∈X+, (8) H(u,g,h)(c · 1lΩ) = c for c ∈R (8) and and H(u,g,h)(X + c · 1lΩ) = H(u,g,h)(X) + c for X ∈X+, c ∈[0, ∞). These properties are usually referred to as: non-excessive loading, no unjus- tiﬁed risk loading and translation invariance (or consistency), respectively. Furthermore, as the generalized Choquet integral is monotone (cf. [9, Lemma 1]), from (5) one can easily derive that the principle of equivalent utility under the Cumulative Prospect Theory is monotone, that is H(u,g,h)(X) ≤H(u,g,h)(Y ) for X, Y ∈X+, X ≤Y. (9) (9) It turns out that, under the Expected Utility model, every functional of equivalent utility can be uniquely extended from the family of all binary risks, i.e. risks taking exactly two non-negative values with positive probabilities, to X+ (cf. e.g. [5, Theorem 6]). In fact, in [5] only the case w = 0 has been considered but, making a straightforward substitution, from [5, Theorem 6] one can easily derive the analogous result for w > 0. In a recent paper [3], some aspects of the extension problem for functionals of equivalent utility under the Cumulative Prospect Theory have been investigated. In particular, the functionals whose restriction to the family of binary risks reduces either to the net principle or to the exponential principle, have been characterized. It follows from the results in [3] that under the Cumulative Prospect Theory a counterpart of [5, Theorem 6] does not hold. (2) Therefore, the following question naturally arises: does there exist a reasonable, in a sense, family of risks such that, under the Cumulative Prospect Theory, every functional of equivalent utility deﬁned on this family can be uniquely extended to X+. The aim of this paper is to show that the family of ternary risks, taking exactly three non-negative values, one of them being 0, with positive probabilities, possesses such a property. (2) where u : R →R is a continuous strictly increasing utility function. One can prove that, for every X ∈X+, there exists a unique real number Hu(X) such that (2) is valid. Therefore, Eq. (2) determines a functional on X+, called the principle of equivalent utility. For more details concerning the properties of the principle of equivalent utility under the Expected Utility Theory we refer e.g. to [1,2,8,14]. The principle of equivalent utility under the Rank-Dependent Utility model and under the Cumulative Prospect Theory, has been introduced and consid- ered by Heilpern [7] and Kaluszka and Krzeszowiec [9,10], respectively. Under the ﬁrst of these models, the premium H(u,g)(X) for a risk X ∈X+ is deﬁned as a solution of the equation Eg[u(w + H(u,g)(X) −X)] = u(w), (3) (3) where u : R →R is a continuous strictly increasing utility function and Eg is the Choquet integral with respect to a probability distortion function g. Let us recall that g : [0, 1] →[0, 1] is called a probability distortion function, provided it is non-decreasing and satisﬁes the boundary conditions g(0) = 0 and g(1) = 1. For every bounded random variable X, the Choquet integral with respect to the probability distortion function g is deﬁned as follows Eg[X] = 0 −∞ (g(P(X > t)) −1) dt + ∞ 0 g(P(X > t)) dt. (4) (4) It has been proved in [4] that, if g is a continuous probability distortion function and u : R →R is a continuous strictly increasing utility function, with u(0) = 0, then for every X ∈X+ the number H(u,g)(X) is uniquely determined by (3). ( ) Under the Cumulative Prospect Theory, the premium H(u,g,h)(X) for a risk X ∈X+ is deﬁned as a solution of the equation Egh[u(w + H(u,g,h)(X) −X)] = u(w) (5) ounded random variable X, Egh[u(w + H(u,g,h)(X) −X)] = u(w) (5) (5) where, for every bounded random variable X, where, for every bounded random variable X, Egh[X] = Eg[max{X, 0}] −Eh[max{−X, 0}] (6) Egh[X] = Eg[max{X, 0}] −Eh[max{−X, 0}] (6) (6) is the generalized Choquet integral related to the probability distortion func- tions g (for gains) and h (for losses). 2. Auxiliary results Assume that (Ω, Σ, P) is a non-atomic probability space and X+ is the family of all non-negative bounded random variables on (Ω, Σ, P). According to (5) the premium for a given risk depends only on a probability distribution of J. Chudziak 220 AEM the risk. Therefore, in what follows we identify the risks with their probability distributions. Let P3 := {p = (p1, p2, p3) : p1, p2, p3 ∈(0, 1) : p1 + p2 + p3 = 1}. Since (Ω, Σ, P) is non-atomic, for every x1, x2, x3 ∈R, with x1 < x2 < x3, and every p = (p1, p2, p3) ∈P3, there exists a random variable X on the space (Ω, Σ, P) such that P(X = xi) = pi for i ∈{1, 2, 3} (cf. e.g. [12, Lemma 2.7.1]). We denote such a random variable by ⟨x1, x2, x3; p⟩. Furthermore, X (3) denotes the family of all such random variables and Since (Ω, Σ, P) is non-atomic, for every x1, x2, x3 ∈R, with x1 < x2 < x3, and every p = (p1, p2, p3) ∈P3, there exists a random variable X on the space (Ω, Σ, P) such that P(X = xi) = pi for i ∈{1, 2, 3} (cf. e.g. [12, Lemma 2.7.1]). We denote such a random variable by ⟨x1, x2, x3; p⟩. Furthermore, X (3) denotes the family of all such random variables and X (3) 0 := {⟨0, x, y; p⟩: 0 < x < y, p ∈P3}. In the whole section we assume that w ∈[0, ∞), u : R →R is a strictly increasing continuous function with u(0) = 0 and g, h : [0, 1] →[0, 1] are continuous distortion functions such that g(p), h(p) ∈(0, 1) for p ∈(0, 1). Then (7) is satisﬁed and so, as we have already noted, the principle of equivalent utility H(u,g,h) is uniquely deﬁned by (5). 2. Auxiliary results 93 (2019) Extension problem for principles of equivalent utility 221 (i) ψp(x, y) > 0 (19) if and only if (g(p1 + p2) −g(p1))u(y −x) + g(p1)u(y) < u(w); (20) (ii) (i) ψp(x, y) > 0 (19) if and only if (i) ψp(x, y) > 0 (19) ψp(x, y) > 0 (19) if and only if if and only if (g(p1 + p2) −g(p1))u(y −x) + g(p1)u(y) < u(w); (20) ) ψp(x, y) ≤0 ≤φp(x, y) (21) if d l if y f (g(p1 + p2) −g(p1))u(y −x) + g(p1)u(y) < u(w); (20) (g(p1 + p2) −g(p1))u(y −x) + g(p1)u(y) < u(w); (20) (ii) ψp(x, y) ≤0 ≤φp(x, y) ψp(x, y) ≤0 ≤φp(x, y) (21) (21) if and only if if and only if h(p3)u(x −y) + g(p1)u(x) ≤u(w) ≤(g(p1 + p2) −g(p1))u(y −x) + g(p1)u(y); (22) (iii) (iii) (iii) φp(x, y) < 0 (23) φp(x, y) < 0 (23) if and only if u(w) < h(p3)u(x −y) + g(p1)u(x). (24) (24) Furthermore, the left (right) inequality in (21) is strict if and only if so is the right (left) inequality in (22). Proof. Let X := ⟨0, x, y; p⟩and let dX : R →R be given by dX(t) = Egh[u(w + t −X)] −u(w) for t ∈R. Since the generalized Choquet integral is monotone and u is strictly increas- ing, dX is non-decreasing. Furthermore, as g(p), h(p) ∈(0, 1) for p ∈(0, 1), applying [4, Lemma 3.3] we conclude that dX is injective. Hence, dX is strictly increasing. Moreover, we have 0 < u(y −x) < u(y), u(x −y) < 0 < u(x), u(y −X) = ⟨0, u(y −x), u(y); p3, p2, p1⟩ and u(x −X) = ⟨u(x −y), 0, u(x); p3, p2, p1⟩. Thus, considering (10) and (11), we obtain dX(y−w) = Egh[u(y−X)]−u(w) = (g(p1+p2)−g(p1))u(y−x)+g(p1)u(y)−u(w) and Thus, considering (10) and (11), we obtain and dX(x −w) = Egh[u(x −X)] −u(w) = h(p3)u(x −y) + g(p1)u(x) −u(w), respectively. On the other hand, considering (5), from (14) and (15) we derive that dX(x −w) = Egh[u(x −X)] −u(w) = h(p3)u(x −y) + g(p1)u(x) −u(w), respectively. On the other hand, considering (5), from (14) and (15) we derive that dX(x −w) = Egh[u(x −X)] −u(w) = h(p3)u(x −y) + g(p1)u(x) −u(w), respectively. On the other hand, considering (5), from (14) and (15) we derive that dX(φp(x, y) + x −w) = dX(ψp(x, y) + y −w) = dX(H(u,g,h)(X)) = 0. 2. Auxiliary results Applying (4) and (6), we get that, if X = ⟨x1, x2, x3; p1, p2, p3⟩∈X (3), then Egh[X] = (1−g(p2+p3))x1+(g(p2+p3)−g(x3))x2+g(p3)x3 if x1 ≥0, (10) Egh[X] = h(p1)x1 + (g(p2 + p3) −g(p3))x2 + g(p3)x3 if x1 < 0 ≤x2, (11) Egh[X] = h(p1)x1 + (h(p1 + p2) −h(p1))x2 + g(p3)x3 if x2 < 0 ≤x3 (12) and and and Egh[X] = h(p1)x1 + (h(p1 + p2) −h(p1))x2 + (1 −h(p1 + p2))x3 if x3 < 0 Egh[X] = h(p1)x1 + (h(p1 + p2) −h(p1))x2 + (1 −h(p1 + p2))x3 if x3 < 0. Let T := {(x, y) ∈(0, ∞)2 : x < y} (13) and, for every p ∈P3, let φp, ψp : T →R be deﬁned as follows (13) φp(x, y) = w + H(u,g,h)(⟨0, x, y; p⟩) −x for (x, y) ∈T, (14) ψp(x, y) = w + H(u,g,h)(⟨0, x, y; p⟩) −y for (x, y) ∈T. (15) Obviously, we have ψp(x, y) < φp(x, y) for (x, y) ∈T, p ∈P3, ψp(x, y) < φp(x, y) for (x, y) ∈T, p ∈P3, and and and ψp(x, y) −φp(x, y) = x −y for (x, y) ∈T, p ∈P3. (16) Moreover, in view of (8), we get w −x ≤φp(x, y) ≤w + y −x for (x, y) ∈T, p ∈P3 (17) and and ψp(x, y) −φp(x, y) = x −y for (x, y) ∈T, p ∈P3. (16) Moreover, in view of (8), we get w −x ≤φ (x y) ≤w + y −x for (x y) ∈T p ∈P3 (17) (16) Moreover, in view of (8), we get w −x ≤φp(x, y) ≤w + y −x for (x, y) ∈T, p ∈P3 (17) and Moreover, in view of (8), we get w −x ≤φp(x, y) ≤w + y −x for (x, y) ∈T, p ∈P3 (17) and w −y ≤ψp(x, y) ≤w for (x, y) ∈T, p ∈P3. (18) w −y ≤ψp(x, y) ≤w for (x, y) ∈T, p ∈P3. (18) Lemma 2.1. Let (x, y) ∈T and p = (p1, p2, p3) ∈P3. Then: w −y ≤ψp(x, y) ≤w for (x, y) ∈T, p ∈P3. (18) Lemma 2.1. Let (x, y) ∈T and p = (p1, p2, p3) ∈P3. Then: Vol. 2. Auxiliary results Therefore, as dX is strictly increasing, we obtain (i)–(iii) as well as the second part of the assertion. □ dX(φp(x, y) + x −w) = dX(ψp(x, y) + y −w) = dX(H(u,g,h)(X)) = 0. dX(φp(x, y) + x −w) = dX(ψp(x, y) + y −w) = dX(H(u,g,h)(X)) = 0. Therefore, as dX is strictly increasing, we obtain (i)–(iii) as well as the second part of the assertion. □ dX(φp(x, y) + x −w) = dX(ψp(x, y) + y −w) = dX(H(u,g,h)(X)) = 0. Therefore, as dX is strictly increasing, we obtain (i)–(iii) as well as the second part of the assertion. □ herefore, as dX is strictly increasing, we obtain (i)–(iii) as well as the second art of the assertion. □ Corollary 2.2. Assume that (x, y) ∈T and p = (p1, p2, p3) ∈P3. J. Chudziak AEM J. Chudziak 222 AEM (i) If (20) holds, then (1 −g(p1 + p2))u(ψp(x, y)) + (g(p1 + p2) −g(p1))u(φp(x, y)) + g(p1)u(φp(x, y) + x) = u(w). (25) (i) If (20) holds, then (1 −g(p1 + p2))u(ψp(x, y)) + (g(p1 + p2) −g(p1))u(φp(x, y)) + g(p1)u(φp(x, y) + x) = u(w). (25) ( ) (1 −g(p1 + p2))u(ψp(x, y)) + (g(p1 + p2) −g(p1))u(φp(x, y)) + g(p1)u(φp(x, y) + x) = u(w). (25 (25) (ii) If (22) is valid, then (ii) If (22) is valid, then h(p3)u(ψp(x, y)) + (g(p1 + p2) −g(p1))u(φp(x, y)) + g(p1)u(φp(x, y) + x) = u(w). (26) h(p3)u(ψp(x, y)) + (g(p1 + p2) −g(p1))u(φp(x, y)) + g(p1)u(φp(x, y) + x) = u(w). (26) h(p3)u(ψp(x, y)) + (g(p1 + p2) −g(p1))u(φp(x, y)) ( (26) + g(p1)u(φp(x, y) + x) = u(w). (26) (iii) If (24) holds, then (iii) If (24) holds, then h(p3)u(ψp(x, y)) + (h(p2 + p3) −h(p3))u(φp(x, y)) h(p3)u(ψp(x, y)) + (h(p2 + p3) −h(p3))u(φp(x, y)) (27) + g(p1)u(φp(x, y) + x) = u(w). + g(p1)u(φp(x, y) + x) = u(w). Proof. Let X := ⟨0, x, y; p⟩. Then, in view of (14)-(15), we get w + H(u,g,h)(X) −X = ⟨ψp(x, y), φp(x, y), φp(x, y) + x; p3, p2, p1⟩. Proof. Let X := ⟨0, x, y; p⟩. Then, in view of (14)-(15), we get Proof. Let X := ⟨0, x, y; p⟩. Then, in view of (14)-(15), we get w + H(u,g,h)(X) −X = ⟨ψp(x, y), φp(x, y), φp(x, y) + x; p3, p2, p1⟩. ⟨ ⟩ ( ) ( ) w + H(u,g,h)(X) −X = ⟨ψp(x, y), φp(x, y), φp(x, y) + x; p3, p2, p1⟩. w + H(u,g,h)(X) −X = ⟨ψp(x, y), φp(x, y), φp(x, y) + x; p3, p2, p1⟩. Moreover, as w ∈[0, ∞), from (8) and (14) we derive that φp(x, y) + x = w + H(u,g,h)(X) ≥0. Therefore, making use of (5) and (10)–(12), in view of Lemma 2.1, we obtain assertions (i)–(iii). □ Moreover, as w ∈[0, ∞), from (8) and (14) we derive that φp(x, y) + x = w + H(u,g,h)(X) ≥0. Therefore, making use of (5) and (10)–(12), in view of Lemma 2.1, we obtain assertions (i)–(iii). □ Lemma 2.3. Assume that w > 0 and put L := limx→∞u(x). Lemma 2.3. Assume that w > 0 and put L := limx→∞u(x). (a) If L = ∞, then for every p ∈P3 there exists (x, y) ∈T such that p x ∞( ) (a) If L = ∞, then for every p ∈P3 there exists (x, y) ∈T such that ψp(x, y) < 0 < φp(x, y). ψp(x, y) < 0 < φp(x, y). (28) (28) b) If L < ∞and p = (p1, p2, p3) ∈P3 is such that (b) If L < ∞and p = (p1, p2, p3) ∈P3 is such that (b) If L < ∞and p = (p1, p2, p3) ∈P3 is such that g(1 −p3) > u(w)/L, (29) g(1 −p3) > u(w)/L, (29) (29) then (28) is valid for some (x, y) ∈T. ( ) f ( y) (c) If L < ∞and (21) is satisﬁed for some p = (p1, p2, p3) ∈P3 and (x, y) ∈ T, then (29) holds. (28) holds for some (x, y) ∈T. (iii) If (24) holds, then (c) If L < ∞and (21) is satisﬁed for some p = (p1, p2, p3) ∈P3 and (x, y) ∈ T, then (29) holds. (c) If L < ∞and (21) is satisﬁed for some p = (p1, p2, p3) ∈P3 and (x, y) T, then (29) holds. Proof. (a) If L = ∞, then taking x ∈(0, w) and suﬃciently big x ∈(0, w), we conclude that (x, y) ∈T and (22) holds, with both inequalities being strict. Thus, according to Lemma 2.1(ii), (28) is valid for such (x, y). Proof. (a) If L = ∞, then taking x ∈(0, w) and suﬃciently big x ∈(0, w), we conclude that (x, y) ∈T and (22) holds, with both inequalities being strict. Thus, according to Lemma 2.1(ii), (28) is valid for such (x, y). (b) Assume that L < ∞and p ∈P3 is such that (29) holds. Then, for every x ∈(0, ∞), we have (b) Assume that L < ∞and p ∈P3 is such that (29) holds. Then, for every x ∈(0, ∞), we have lim y→∞((g(p1+p2)−g(p1))u(y−x)+g(p1)u(y)) = g(p1+p2)L = g(1−p3)L > u(w). im →∞((g(p1+p2)−g(p1))u(y−x)+g(p1)u(y)) = g(p1+p2)L = g(1−p3)L > u(w). Thus, for every x ∈(0, w) and suﬃciently big y ∈(w, ∞), (22) is valid, with both inequalities being strict. Hence, applying Lemma 2.1(ii), we conclude that (28) holds for some (x, y) ∈T. Vol. 93 (2019) Extension problem for principles of equivalent utility Vol. 93 (2019) Extension problem for principles of equivalent utility 223 (c) Assume that L < ∞and (21) holds for some p = (p1, p2, p3) ∈P3 and (x, y) ∈T. Then, according to Lemma 2.1(ii), (22) is satisﬁed and so u(w) ≤(g(p1 +p2)−g(p1))u(y −x)+g(p1)u(y) ≤g(p1 +p2)u(y) < g(1−p3)L. Thus, (29) is valid. □ (c) Assume that L < ∞and (21) holds for some p = (p1, p2, p3) ∈P3 and (x, y) ∈T. Then, according to Lemma 2.1(ii), (22) is satisﬁed and so u(w) ≤(g(p1 +p2)−g(p1))u(y −x)+g(p1)u(y) ≤g(p1 +p2)u(y) < g(1−p3)L ( , y) , g ( ), ( ) u(w) ≤(g(p1 +p2)−g(p1))u(y −x)+g(p1)u(y) ≤g(p1 +p2)u(y) < g(1−p3)L. Thus, (29) is valid. □ Lemma 2.4. For every p ∈P3, the functions φp and ψp are continuous. Proof. Let p ∈P3. In view of (16), it is enough to show that φp is continuous. Furthermore, it follows from (9) and (14) that φp is monotone in y for each x. (iii) If (24) holds, then Thus, in order to prove the continuity of φp, it suﬃces to show that it is continuous in x and y separately. Since in both cases similar arguments work, we prove only the continuity of φp in x. Suppose that, for some y ∈ (0, ∞), φp(·, y) is not continuous at the point x ∈(0, y). Then there exists a sequence (xn : n ∈N) of elements of (0, y) such that limn→∞xn = x but (φp(xn, y) : n ∈N) does not tend to φp(x, y). According to (17), the sequence (φp(xn, y) : n ∈N) is bounded, so there exists a subsequence (xnk : k ∈N) of the sequence (xn : n ∈N) such that limk→∞φp(xnk, y) =: d ̸= φp(x, y). Note that, in view of (16), we have lim k→∞ψp(xnk, y) = d + x −y. (30) (30) Moreover, the following three cases are possible: 1. ψp(x, y) ≥0, 1. ψp(x, y) ≥0, 2. ψp(x, y) < 0 < φp(x, y), 3. φp(x, y) ≤0. 3. φp(x, y) ≤0. Case 1 If ψp(x, y) > 0, then in view of Lemma 2.1(i), (20) is valid and so, for suﬃciently big k ∈N, we get Case 1 If ψp(x, y) > 0, then in view of Lemma 2.1(i), (20) is valid and so, for suﬃciently big k ∈N, we get (g(p1 + p2) −g(p1))u(y −xnk) + g(p1)u(y) < u(w). Furthermore, applying Corollary 2.2(i), we obtain (25) and Furthermore, applying Corollary 2.2(i), we obtain (25) and (1 −g(p1 + p2))u(ψp(xnk, y)) + (g(p1 + p2) −g(p1))u(φp(xnk, y)) (3 (1 −g(p1 + p2))u(ψp(xnk, y)) + (g(p1 + p2) −g(p1))u(φp(xnk, y)) (31 (31) (31) + g(p1)u(φp(xnk, y) + xnk) = u(w) (31) for suﬃciently big k ∈N. Letting k →∞in (31) and subtracting the equality obtained this way from (25), in view of (30), we get for suﬃciently big k ∈N. Letting k →∞in (31) and subtracting the equality obtained this way from (25), in view of (30), we get for suﬃciently big k ∈N. (iii) If (24) holds, then Letting k →∞in (31) and subtracting the equality obtained this way from (25), in view of (30), we get (1 −g(p1 + p2))(u(ψp(x, y)) −u(d + x −y)) (1 −g(p1 + p2))(u(ψp(x, y)) −u(d + x −y)) + (g(p1 + p2) −g(p1))(u(φp(x, y)) −u(d)) + (g(p1 + p2) −g(p1))(u(φp(x, y)) −u(d)) + (g(p1 + p2) −g(p1))(u(φp(x, y)) −u(d)) + g(p1)(u(φp(x, y) + x) −u(d + x)) = 0. On the other hand g is non-decreasing, with g(p) ∈(0, 1) for p ∈(0, 1), and u is strictly increasing. Thus, making use of (16), we conclude that the left hand 224 J. Chudziak AEM AEM side of the last equality is positive whenever d < φp(x, y), and it is negative whenever d > φp(x, y). This yields a contradiction. side of the last equality is positive whenever d < φp(x, y), and it is negativ whenever d > φp(x, y). This yields a contradiction. Suppose that ψp(x, y) = 0. Then, in view of (16), φp(x, y) = y −x > 0 and so, applying Lemma 2.1(i) and Corollary 2.2(i), we obtain h(p3)u(x −y) + g(p1)u(x) < u(w) (32) (32) and (g(p1 + p2) −g(p1))u(y −x) + g(p1)u(y) = u(w). (33) (33) If d > y −x, then making use of (30), we get limk→∞ψp(xnk, y) > 0. Hence, for suﬃciently big k ∈N, we have ψp(xnk, y) > 0 and so, according to Lemma 2.1(i) and Corollary 2.2(i), (31) holds. Thus, passing in (31) to the limit as k →∞and subtracting the obtained equality from (33), in view of (30), we get (g(p1 + p2) −g(p1))(u(y −x) −u(d)) + g(p1)(u(y) −u(d + x)) −(1 −g(p1 + p2))u(d + x −y) = 0. + g(p1)(u(y) −u(d + x)) −(1 −g(p1 + p2))u(d + x −y) = 0. However, as u is strictly increasing and g is non-decreasing, with g(p) ∈(0, 1) for p ∈(0, 1), the left hand side of this equality is negative, which gives a contradiction. If d < y −x, then in view of (30), we get limk→∞ψp(xnk, y) < 0. Thus ψp(xnk, y) < 0 for suﬃciently big k ∈N. On the other hand, making use of (32), for suﬃciently big k ∈N, we get p( k ) 32), for suﬃciently big k ∈N, we get h(p3)u(xnk −y) + g(p1)u(xnk) < u(w) whence, by Lemma 2.1(ii), φp(xnk, y) > 0. (iii) If (24) holds, then Therefore, applying Lemma 2.1(ii) and Corollary 2.2(ii), we get whence, by Lemma 2.1(ii), φp(xnk, y) > 0. Therefore, applying Lemma 2.1(ii) and Corollary 2.2(ii), we get h(p3)u(ψp(xnk, y)) + (g(p1 + p2) −g(p1))u(φp(xnk, y)) + g(p1)u(φp(xnk, y) + xnk) = u(w) (34) h(p3)u(ψp(xnk, y)) + (g(p1 + p2) −g(p1))u(φp(xnk, y)) ψp(xnk, y)) + (g(p1 + p2) −g(p1))u(φp(xnk, y)) + g(p1)u(φp(xnk, y) + xnk) = u(w) (34) (34) + g(p1)u(φp(xnk, y) + xnk) = u(w) (34) + g(p1)u(φp(xnk, y) + xnk) = u + g(p1)u(φp(xnk, y) + xnk) = u(w) for suﬃciently big k ∈N. Letting in the last equality k →∞and subtracting the obtained equality from (33), in view of (30), we obtain for suﬃciently big k ∈N. Letting in the last equality k →∞and subtracting the obtained equality from (33), in view of (30), we obtain (g(p1 + p2) −g(p1))(u(y −x) −u(d)) + g(p1)(u(y) −u(d + x)) −h(p3)u(d + x −y) = 0. Since d < y −x, arguing as previously, we conclude that the left hand side of this equality is positive, which yields a contradiction. Since d < y −x, arguing as previously, we conclude that the left hand side of this equality is positive, which yields a contradiction. Case 2 Applying Lemma 2.1(ii) and Corollary 2.2(ii), we obtain (22), with both inequalities being strict, and (26). Hence, we have Case 2 Applying Lemma 2.1(ii) and Corollary 2.2(ii), we obtain (22), with both inequalities being strict, and (26). Hence, we have h(p3)u(xnk−y)+g(p1)u(xnk) < u(w) < (g(p1+p2)−g(p1))u(y−xnk)+g(p1)u(y) (p3)u(xnk−y)+g(p1)u(xnk) < u(w) < (g(p1+p2)−g(p1))u(y−xnk)+g(p1)u(y) Vol. 93 (2019) Vol. 93 (2019) Extension problem for principles of equivalent utility 225 for suﬃciently big k ∈N. Therefore, according to Corollary 2.2(ii), for suﬃ- ciently big k ∈N, we get h(p3)u(ψp(xnk, y)) + (g(p1 + p2) −g(p1))u(φp(xnk, y)) + g(p1)u(φp(xnk, y) + xnk) = u(w). h(p3)u(ψp(xnk, y)) + (g(p1 + p2) −g(p1))u(φp(xnk, y)) + g(p1)u(φp(xnk, y) + xnk) = u(w). Thus, making use of (26) and arguing as in the previous case, we obtain h(p3)(u(ψp(x, y)) −u(d + x −y)) + (g(p1 + p2) −g(p1))(u(φp(x, y)) −u(d)) + g(p1)(u(φp(x, y) + x) −u(d + x)) = 0, which gives a contradiction. which gives a contradiction. Case 3 If φp(x, y) < 0, then according to Lemma 2.1(iii), we have (24). Thus u(w) < h(p3)u(xnk −y) + g(p1)u(xnk) for suﬃciently big k ∈N. (iii) If (24) holds, then Furthermore, applying Corollary 2.2(iii), we obtain (27) and h(p3)u(ψp(xnk, y)) + (h(p2 + p3) −h(p3))u(φp(xnk, y)) + g(p1)u(φp(xnk, y) + xnk) = u(w) for suﬃciently big k ∈N. Hence, repeating the arguments from the ﬁrst case, we get h(p3)(u(ψp(x, y)) −u(d + x −y)) + (h(p2 + p3) −h(p3))(u(φp(x, y)) −u(d)) + g(p1)(u(φp(x, y) + x) −u(d + x)) = 0, which yields a contradiction. which yields a contradiction. If φp(x, y) = 0, then in view of (16), ψp(x, y) = x−y < 0. Hence, according to Lemma 2.1(ii) and Corollary 2.2(ii), we have u(w) < (g(p1 + p2) −g(p1))u(y −x) + g(p1)u(y) (35) (35) and and h(p3)u(x −y) + g(p1)u(x) = u(w). (36) follows from (35) that, for suﬃciently big k ∈N, h(p3)u(x −y) + g(p1)u(x) = u(w). (36) 5) that, for suﬃciently big k ∈N, h(p3)u(x −y) + g(p1)u(x) = u(w). (36) (36) It follows from (35) that, for suﬃciently big k ∈N, It follows from (35) that, for suﬃciently big k ∈N, It follows from (35) that, for suﬃciently big k ∈N, u(w) < (g(p1 + p2) −g(p1))u(y −xnk) + g(p1)u(y) u(w) < (g(p1 + p2) −g(p1))u(y −xnk) + g(p1)u(y) whence, in view of Lemma 2.1(iii), ψp(xnk, y) < 0. Therefore, if d > 0, then for suﬃciently big k ∈N, we have ψp(xnk, y) < 0 < φp(xnk, y) and so, according to Lemma 2.1(ii) and Corollary 2.2(ii), (34) holds. Passing to the limit in (34) J. Chudziak AEM 226 AEM as k →∞and subtracting the equality obtained this way from (36), in view of (30), we get as k →∞and subtracting the equality obtained this way from (36), in view of (30), we get h(p3)(u(x −y) −u(d + x −y)) + g(p1)(u(x) −u(d + x)) −(g(p1 + p2) −g(p1))u(d) = 0. −(g(p1 + p2) −g(p1))u(d) = 0. Since d > 0, the left hand side of this equality is negative and so we get a contradiction. If d < 0, then φp(xnk, y) < 0 for suﬃciently big k ∈N. Thus, applying Lemma 2.1(iii) and Corollary 2.2(iii), we conclude that h(p3)u(ψp(xnk, y)) + (h(p2 + p3) −h(p3))u(φp(xnk, y)) + g(p1)u(φp(xnk, y) + xnk) = u(w) for suﬃciently big k ∈N. Hence, arguing as previously, we obtain h(p3)(u(x −y) −u(d + x −y)) + g(p1)(u(x) −u(d + x)) −(h(p2 + p3) −h(p3))u(d) = 0. for suﬃciently big k ∈N. (iii) If (24) holds, then Hence, arguing as previously, we obtain h(p3)(u(x −y) −u(d + x −y)) + g(p1)(u(x) −u(d + x)) for suﬃciently big k ∈N. Hence, arguing as previously, we obtain ciently big k ∈N. Hence, arguing as previously, we obtain ﬃciently big k ∈N. Hence, arguing as previously, we obtain h(p3)(u(x −y) −u(d + x −y)) + g(p1)(u(x) −u(d + x)) −(h(p2 + p3) −h(p3))u(d) = 0. owever, as d < 0, the left hand side of the last equality is positive. So, we ave a contradiction. However, as d < 0, the left hand side of the last equality is positive. So, we have a contradiction. This proves the continuity of φp(·, y) at x. □ □ Lemma 2.5. Let p ∈P3. emma 2.5. Let p ∈P3. Lemma 2.5. Let p ∈P3. Lemma 2.5. Let p ∈P3. (a) If w = 0, then for every y ∈(0, ∞), 0 is an interior point of φp((0, y) × {y}). (b) If w > 0, then for every y ∈(0, w), w is an interior point of φp((0, y) × { }) (a) If w = 0, then for every y ∈(0, ∞), 0 is an interior point of φp((0, y) × {y}). (a) If w = 0, then for every y ∈(0, ∞), 0 is an interior point of φp((0, y) × {y}). (b) If w > 0, then for every y ∈(0, w), w is an interior point of φp((0, y) × {y}). (b) If w > 0, then for every y ∈(0, w), w is an interior point of φp((0, y) × {y}). roof. Note that, for every y ∈(0, ∞), we have lim x→y−(h(p3)u(x −y) + g(p1)u(x)) = g(p1)u(y), (37) lim x→0+(h(p3)u(x −y) + g(p1)u(x)) = h(p3)u(−y) (38) (37) (38) and and lim x→0+((g(p1 + p2) −g(p1))u(y −x) + g(p1)u(y)) = g(p1 + p2)u(y). (39) lim x→0+((g(p1 + p2) −g(p1))u(y −x) + g(p1)u(y)) = g(p1 + p2)u(y). (39) (39) Therefore, if w = 0, then taking an arbitrary y ∈(0, ∞), from (37) we derive that, for x ∈(0, y) suﬃciently close to y, (24) is valid. Thus, according to Lemma 2.1(iii), (23) holds for some x ∈(0, y). Furthermore, as u is strictly in- creasing, making use of (38) and (39), we obtain that, for x ∈(0, y) suﬃciently close to 0, (22) is satisﬁed, with both inequalities being strict. So, applying Lemma 2.1(ii), we conclude that φp(x, y) > 0 for some x ∈(0, y). Hence, as φp is continuous, 0 belongs to the interior of φp((0, y) × {y}). 93 (2019) Extension problem for p Vol. 93 (2019) Extension problem for principles of equivalent utility 227 Vol. 93 (2019) 227 Extension problem for principles of equivalent utility Now assume that w > 0 and ﬁx y ∈(0, w). Then, for every x ∈(0, y), (20) is satisﬁed and so, according to Corollary 2.2(i), (25) holds. Suppose that φp(x, y) ≥w for x ∈(0, y). Then, in view of (16) and (17), we get lim φp(x, y) = lim ψp(x, y) = w. Now assume that w > 0 and ﬁx y ∈(0, w). Then, for every x ∈(0, y), is satisﬁed and so, according to Corollary 2.2(i), (25) holds. Lemma 2.5. Let p ∈P3. Chudziak 228 AEM If w > 0, then for suﬃciently small p1, p2 ∈(0, 1), (20) holds and so, applying Corollary 2.2(i), we get If w > 0, then for suﬃciently small p1, p2 ∈(0, 1), (20) holds and so, applying Corollary 2.2(i), we get (1 −g(p1 + p2))u(ψ(p1,p2,1−p1−p2)(x, y)) + (g(p1 + p2) −g(p1))u(φ(p1,p2,1−p1−p2)(x, y)) (1 −g(p1 + p2))u(ψ(p1,p2,1−p1−p2)(x, y)) + (g(p1 + p2) −g(p1))u(φ(p1,p2,1−p1−p2)(x, y)) + g(p1)u(φ(p1,p2,1−p1−p2)(x, y) + x) = u(w). + g(p1)u(φ(p1,p2,1−p1−p2)(x, y) + x) = u(w). Thus, repeating the previous arguments, we obtain (42). In this way we have proved that (42) holds for every (x, y) ∈T. Therefore, taking x0 ∈[w, ∞) and (x, y) ∈T, with y −x > x0 −w, in view of (16), we get Thus, repeating the previous arguments, we obtain (42). In this way we have proved that (42) holds for every (x, y) ∈T. Therefore, taking x0 ∈[w, ∞) and (x, y) ∈T, with y −x > x0 −w, in view of (16), we get lim p1,p2→0+ φ(1−p1−p2,p1,p2)(x, y) = lim p1,p2→0+(ψ(1−p1−p2,p1,p2)(x, y) + y −x) = w + y −x > x0. lim p1,p2→0+ φ(1−p1−p2,p1,p2)(x, y) = lim p1,p2→0+(ψ(1−p1−p2,p1,p2)(x, y) + y −x) = w + y −x > x0. Hence, there exists p ∈P3 such that φp(x, y) > x0. On the other hand, from Lemma 2.4 and Lemma 2.5 we deduce that φp(T) is connected and w is its interior point, respectively. Thus x0 ∈φp(T), which proves (40). Hence, there exists p ∈P3 such that φp(x, y) > x0. On the other hand, from Lemma 2.4 and Lemma 2.5 we deduce that φp(T) is connected and w is its interior point, respectively. Thus x0 ∈φp(T), which proves (40). p( ) ( ) Now, we show that (41) holds. To this end, ﬁx x0 ∈(−∞, w). Let (x, y) ∈T be such that x < w and y > max{w, w −x0}. Then, for suﬃciently small p2, p3 ∈(0, 1), we have h(p3)u(x −y) + g(1 −p2 −p3)u(x) < u(w) < (g(1 −p3) −g(1 −p2 −p3))u(y −x) + g(1 −p2 −p3)u(y) and so, applying Corollary 2.2(ii), we obtain h(p3)u(ψ(1−p2−p3,p2,p3)(x, y)) + (g(1 −p3) −g(1 −p2 −p3))u(φ(1−p2−p3,p2,p3)(x + g(1 −p2 −p3)u(φ(1−p2−p3,p2,p3)(x, y) + x) = u(w). and so, applying Corollary 2.2(ii), we obtain + g(1 −p2 −p3)u(φ(1−p2−p3,p2,p3)(x, y) + x) = u(w). + g(1 −p2 −p3)u(φ(1−p2−p3,p2,p3)(x, y) + x) = u(w). Lemma 2.5. Let p ∈P3. Suppose that , y) ≥w for x ∈(0, y). Then, in view of (16) and (17), we get lim x→y−φp(x, y) = lim x→y−ψp(x, y) = w. Hence, letting in (25) x →y−, we obtain g(p1)(u(w + y) −u(w)) = 0. Since u is strictly increasing and g(p1) > 0, this yields a contradiction. In this way we have proved that φp(x, y) < w for some x ∈(0, y). On the other hand, if φp(x, y) ≤w for x ∈(0, y), then applying (17), we get limx→0+ φp(x, y) = w. Thus, in view of (16), we have limx→0+ ψp(x, y) = w −y and so, letting in (25) x →0+, we obtain (1 −g(p1 + p2))(u(w −y) −u(w)) = 0. Since u is strictly increasing and g(p1 + p2) < 1, this gives a contradiction. Therefore φp(x, y) > w for some x ∈(0, y) and so, as φp is continuous, w is an interior point of φp((0, y) × {y}). □ Lemma 2.6. A family {φp(T) : p ∈P3} is a cover of [w, ∞) and a family {ψp(T) : p ∈P3} is a cover of (−∞, w), that is [w, ∞) ⊂ p∈P3 φp(T) (40) (40) and and (−∞, w) ⊂ p∈P3 ψp(T), (41) (41) respectively. Proof. In order to prove (40), ﬁx (x, y) ∈T. Since g and h are continuous, with g(0) = 0 and h(1) = 1, in the case where w = 0, for suﬃciently small p1, p2 ∈(0, 1), we have h(1−p1−p2)u(x−y)+g(p1)u(x)<u(w)<(g(p1+p2)−g(p1))u(y−x)+g(p1)u(y). (1−p1−p2)u(x−y)+g(p1)u(x)<u(w)<(g(p1+p2)−g(p1))u(y−x)+g(p1)u(y). Hence, applying Corollary 2.2(ii), we obtain Hence, applying Corollary 2.2(ii), we obtain h(1 −p1 −p2)u(ψ(p1,p2,1−p1−p2)(x, y)) + (g(p1 + p2) −g(p1))u(φ(p1,p2,1−p1−p2)(x, y)) + g(p1)u(φ(p1,p2,1−p1−p2)(x, y) + x) = u(w) + g(p1)u(φ(p1,p2,1−p1−p2)(x, y) + x) = u(w) for suﬃciently small p1, p2 ∈(0, 1). Thus, taking into account (17) and (18 we get lim p1,p2→0+ u(ψ(p1,p2,1−p1−p2)(x, y)) = u(w). Since u is a continuous, strictly increasing function, this implies that Since u is a continuous, strictly increasing function, this implies that (42) lim p1,p2→0+ ψ(p1,p2,1−p1−p2)(x, y) = w. (42) lim p1,p2→0+ ψ(p1,p2,1−p1−p2)(x, y) = w. J. Lemma 2.5. Let p ∈P3. Thus, arguing as previously, we get lim p2,p3→0+ u(φ(1−p2−p3,p2,p3)(x, y) + x) = u(w) lim p2,p3→0+ u(φ(1−p2−p3,p2,p3)(x, y) + x) = u(w) and so and so lim p2,p3→0+ φ(1−p2−p3,p2,p3)(x, y) = w −x. Hence, considering (16), we obtain Hence, considering (16), we obtain lim p2,p3→0+ ψ(1−p2−p3,p2,p3)(x, y) = w −y < x0. So, there exists p ∈P3 such that ψp(x, y) < x0. Furthermore, in view of (18), we get limy→0+ ψp(y/2, y) = w. Since, in view of Lemma 2.4, ψp(T) is connected, this means that x0 ∈φp(T) and so, (41) is proved. □ Vol. 93 (2019) Extension problem for principles of equivalent utility Vol. 93 (2019) Extension problem for principles of equivalent utility Vol. 93 (2019) Extension problem for principles of equivalent utility 229 Lemma 2.7. Let p = (p1, p2, p3) ∈P3 and Fp : T →R2 be deﬁned as follows Fp(x, y) = (φp(x, y), ψp(x, y)) for (x, y) ∈T. (43) Lemma 2.7. Let p = (p1, p2, p3) ∈P3 and Fp : T →R2 be deﬁned as follows Fp(x, y) = (φp(x, y), ψp(x, y)) for (x, y) ∈T. (43) (43) Then Fp(T) is open. Proof. It follows from Lemma 2.4 that Fp is continuous. We show that it is injective. Suppose that Fp(x1, y1) = Fp(x2, y2) for some (x1, y1), (x2, y2) ∈T. Then Proof. It follows from Lemma 2.4 that Fp is continuous. We show that it is injective. Suppose that Fp(x1, y1) = Fp(x2, y2) for some (x1, y1), (x2, y2) ∈T. Then φp(x1, y1) = φp(x2, y2) (44) (44) and ψp(x1, y1) = ψp(x2, y2). (45 ψp(x1, y1) = ψp(x2, y2). (45) Therefore, applying Lemma 2.1 and Corollary 2.2, we obtain Therefore, applying Lemma 2.1 and Corollary 2.2, we obtain Therefore, applying Lemma 2.1 and Corollary 2.2, we obtain g(p1)u(φp(x1, y1) + x1) = u(w) −γu(φp(x1, y1)) −δu(ψp(x1, y1)) = u(w) −γu(φp(x2, y2)) −δu(ψp(x2, y2)) = g(p1)u(φp(x2, y2) + x2), where γ := g(p1 + p2) −g(p1) whenever φp(x1, y1) ≥0, h(p2 + p3) −h(p3) otherwise and and δ := 1 −g(p1 + p2) whenever ψp(x1, y1) > 0, h(p3) otherwise. Thus, as u is strictly increasing and g(p1) > 0, we get Thus, as u is strictly increasing and g(p1) > 0, we get φp(x1, y1) + x1 = φp(x2, y2) + x2. Hence, in view of (44), we obtain x1 = x2. Lemma 2.5. Let p ∈P3. Furthermore, considering (16), (44) and (45), we conclude that Hence, in view of (44), we obtain x1 = x2. Furthermore, considering (16), (44) and (45), we conclude that y1 = φp(x1, y1) −ψp(x1, y1) + x1 = φp(x2, y2) −ψp(x2, y2) + x2 = y2, which completes the proof of the injectivity of Fp. which completes the proof of the injectivity of Fp. Now, as Fp is continuous and injective, applying the Invariant Domain Theorem, we obtain that Fp(T) is open. □ The following lemma, concerning the solutions of the general linear equation on a region, will play an important role in the proof of our main results. For more details concerning the general linear equation we refer to [11, Chapter 13.10]. Lemma 2.8. Assume that D is a nonempty, open and connected subset of R2, f : R →R is a nonconstant continuous function, a, b, A, B ∈R\{0} and c, C ∈R. Then f satisﬁes the equation f(ax + by + c) = Af(x) + Bf(y) + C for (x, y) ∈D (46) J. Chudziak AEM 230 J. Chudziak J. Chudziak 230 if and only if there exist γ ∈R\{0} and β1, β2 ∈R such that f(x) = γa A x + β1 for x ∈D1, (47) f(x) = γb B x + β2 for x ∈D2 (48) (47) (48) and and f(x) = γ(x −c) + Aβ1 + Bβ2 + C for x ∈{as + bt + c : (s, t) ∈D}, (49) where f(x) = γ(x −c) + Aβ1 + Bβ2 + C for x ∈{as + bt + c : (s, t) ∈D}, (49 D1 := {x ∈R : (x, y) ∈D for some y ∈R} and D2 := {y ∈R : (x, y) ∈D for some x ∈R}. Proof. Assume that f satisﬁes (46). Replacing in (46) x and y by x/a and y/b, respectively, we conclude that a triple of functions (f1, f2, f3), where f1(x) = Af(x/a) for x ∈R, f2(x) = Bf(x/b) for x ∈R and f3(x) = f(x + c) −C for x ∈R, satisﬁes the Pexider equation f3(x + y) = f1(x) + f2(y) for (x, y) ∈(a, b) · D, f3(x + y) = f1(x) + f2(y) for (x, y) ∈(a, b) · D, where (a, b) · D := {(as, bt) : (s, t) ∈D}. Lemma 2.5. Let p ∈P3. (52) (52) Hence, if αx and αx0 were diﬀerent, then we would have Hence, if αx and αx0 were diﬀerent, then we would have u1(z) = βx0 −βx αx −αx0 for z ∈Ux ∩Ux0, which is not possible, as Ux ∩Ux0 is a nonempty open set and u1 is strictly increasing. Thus αx0 = αx and so, in view of (52), βx0 = βx. Then, we have αx0u1(z) + βx0 = αx1u1(z) + βx1 for z ∈Ux0 ∩Ux1. Hence, arguing as previously, we obtain that αx0 = αx1 and βx0 = βx1. There- fore, αx = αx1 and βx = βx1. Repeating this procedure, we conclude ﬁnally that αx = αy and βx = βy. □ Lemma 2.5. Let p ∈P3. Moreover, fi for i ∈{1, 2, 3} are nonconstant continuous functions and, as D is nonempty, open and connected, so is (a, b) · D. Therefore, applying [11, Theorem 13.3.5] and [13, Theorem 1], we obtain that there exist γ ∈R\{0} and δ1, δ2 ∈R such that f1(x) = γx + δ1 for x ∈a · D1 := {as : s ∈D1}, f2(x) = γx + δ2 for x ∈b · D2 := {bt : t ∈D2} and f3(x) = γx + δ1 + δ2 for x ∈{as + bt : (s, t) ∈D}. f3(x) = γx + δ1 + δ2 for x ∈{as + bt : (s, t) ∈D}. Hence, considering the deﬁnitions of f1, f2 and f3, we get (47)–(49) wit β1 := δ1/A and β2 := δ2/B. Hence, considering the deﬁnitions of f1, f2 and f3, we get (47)–(49) with β1 := δ1/A and β2 := δ2/B. The converse is easy to check □ 1/ β2 2/ e converse is easy to check. □ □ / / The converse is easy to check. We complete this section with one more result, which will be useful in our further considerations. Lemma 2.9. Let I ⊆R be an open interval and let u1, u2 : R →R be strictly increasing functions. Assume that, for every x ∈I, there exist αx ∈R\{0}, βx ∈R and rx ∈(0, ∞) such that u2(z) = αxu1(z) + βx for z ∈Ux := (x −rx, x + rx). (50) hen there exist α ∈R\{0} and β ∈R such that u2(z) = αxu1(z) + βx for z ∈Ux := (x −rx, x + rx). (50) Then there exist α ∈R\{0} and β ∈R such that u2(z) = αu1(z) + β for z ∈I. (51) (50) u2(z) = αu1(z) + β for z ∈I. (51) (51) Vol. 93 (2019) Extension problem for principles of equivalent utility 231 Proof. It is enough to show that, for every x, y ∈I, we have αx = αy and βx = βy. To this end, ﬁx x, y ∈I. Let zx ∈Ux and zy ∈Uy. According to [6, Lemma 2.4], there exist n ∈N and x0, x1, ..., xn ∈I such that zx ∈Ux0, zy ∈Uxn and Uxi−1 ∩Uxi ̸= ∅for i ∈{1, ..., n}. Thus, in view of (50), we get αxu1(z) + βx = αx0u1(z) + βx0 for z ∈Ux ∩Ux0. 3. Main results The following three theorems, concerning the extension problem for functionals of equivalent utility under the Cumulative Prospect Theory, are the main results of the paper. Theorem 3.1. Let w ∈[0, ∞). Assume that, for i ∈{1, 2}, ui : R →R is a strictly increasing continuous function with ui(0) = 0 and gi, hi : [0, 1] →[0, 1] are strictly increasing continuous probability distortion functions. Furthermore, assume that H(u1,g1,h1) and H(u2,g2,h2) are the functionals of equivalent utility, deﬁned by (5), such that H(u1,g1,h1)(X) = H(u2,g2,h2)(X) for X ∈X (3) 0 . (53) 0 or limx→∞u1(x) = ∞, then g1 = g2, h1 = h2 and there exists ∞) such that (53) u2(x) = αu1(x) for x ∈R. (54) u2(x) = αu1(x) for x ∈R. u2(x) = αu1(x) for x ∈R. (54) (b) If w > 0 and L := limx→∞u1(x) < ∞, then g1 = g2 and there exist α, β ∈(0, ∞) such that h2(p) = β αh1(p) for p ∈[0, 1 −g−1 1 (u1(w)/L)] (55) (55) and and u2(x) = αu1(x) for x ∈(−∞, 0), βu1(x) for x ∈[0, ∞). (56) (56) J. Chudziak AEM AEM 232 Proof. Let T be given by (13) and let, for every p ∈P3 and i ∈{1, 2}, φ(i) p , ψ(i) p : T →R be deﬁned as follows Proof. Let T be given by (13) and let, for every p ∈P3 and i ∈{1, 2}, φ(i) p , ψ(i) p : T →R be deﬁned as follows φ(i) p (x, y) = w + H(ui,gi,hi)(⟨0, x, y; p⟩) −x for (x, y) ∈T, ψ(i) p (x, y) = w + H(ui,gi,hi)(⟨0, x, y; p⟩) −y for (x, y) ∈T, respectively. Then, in view of (53), for every p ∈P3, we have φp := φ(1) p = φ(2) p and ψp := ψ(1) p = ψ(2) p . Furthermore, for every p ∈P3, deﬁne Fp : T →R2 by (43) respectively. Then, in view of (53), for every p ∈P3, we have φp := φ(1) p = φ(2) p and ψp := ψ(1) p = ψ(2) p . Furthermore, for every p ∈P3, deﬁne Fp : T →R2 by (43). ( ) First we show that (56) holds for some α, β ∈(0, ∞). To this end, ﬁx x0 ∈R\{0}. If x0 ≥w, then in view of (40), there exists p = (p1, p2, p3) ∈P3 such that x0 ∈φp(T). 3. Main results Moreover, applying Lemma 2.7, we obtain that the set {y ∈R : (x0, y) ∈Fp(T)} is nonempty and open. Thus, (x0, y0) ∈Fp(T) for some y0 ∈R\{0}. In the case where y0 > 0, there is rx0 > 0 such that ( ) First we show that (56) holds for some α, β ∈(0, ∞). To this end, ﬁx x0 ∈R\{0}. If x0 ≥w, then in view of (40), there exists p = (p1, p2, p3) ∈P3 such that x0 ∈φp(T). Moreover, applying Lemma 2.7, we obtain that the set {y ∈R : (x0, y) ∈Fp(T)} is nonempty and open. Thus, (x0, y0) ∈Fp(T) for some y0 ∈R\{0}. In the case where y0 > 0, there is rx0 > 0 such that B((x0, y0), rx0) := (x0 −rx0, x0 + rx0) × (y0 −rx0, y0 + rx0) ⊂Fp(T) ∩(0, ∞)2. Therefore, taking (x, y) ∈B((x0, y0), rx0) and putting (s, t) := F −1 p (x, y), in view of (43), we get ψp(s, t) > 0. Hence, according to Lemma 2.1(i), for i ∈{1 2} we have B((x0, y0), rx0) := (x0 −rx0, x0 + rx0) × (y0 −rx0, y0 + rx0) ⊂Fp(T) ∩(0, ∞)2. Therefore, taking (x, y) ∈B((x0, y0), rx0) and putting (s, t) := F −1 p (x, y), in view of (43), we get ψp(s, t) > 0. Hence, according to Lemma 2.1(i), for i ∈{1, 2}, we have (gi(p1 + p2) −gi(p1))ui(t −s) + gi(p1)ui(t) < ui(w) and so, applying Corollary 2.2(i), we obtain and so, applying Corollary 2.2(i), we obtain ui(φp(s, t) + s) = 1 gi(p1) [ui(w) −(1 −gi(p1 + p2))ui(ψp(s, t)) −(gi(p1 + p2) −gi(p1))ui(φp(s, t))] = 1 gi(p1) [ui(w) −(1 −gi(p1 + p2))ui(y) −(gi(p1 + p2) −gi(p1))ui(x)]. = 1 gi(p1) [ui(w) −(1 −gi(p1 + p2))ui(ψp(s, t)) −(gi(p1 + p2) −gi(p1))ui(φp(s, t))] = 1 gi(p1) [ui(w) −(1 −gi(p1 + p2))ui(y) −(gi(p1 + p2) −gi(p1))ui(x)]. Thus, setting a := g1(p1)−g1(p1+p2) g1(p1) , A := g2(p1)−g2(p1+p2) g2(p1) , b := g1(p1+p2)−1 g1(p1) , B := g2(p1+p2)−1 g2(p1) , c := u1(w) g1(p1) and C := u2(w) g2(p1), for every (x, y) ∈B((x0, y0), rx0), we get u−1 1 (au1(x) + bu1(y) + c) = u−1 2 (Au2(x) + Bu2(y) + C). f i u−1 1 (au1(x) + bu1(y) + c) = u−1 2 (Au2(x) + Bu2(y) + C). Therefore, a function f := u2 ◦u−1 1 satisﬁes Eq. 3. Main results (46) with f := u2 ◦u−1 1 (57) f := u2 ◦u−1 1 (57) satisﬁes Eq. (46) with D := {(u1(x), u1(y)) : (x, y) ∈B((x0, y0), rx0)}. Moreover, as u1 is a continuous injection, D is open and connected. Thus, according to Lemma 2.8, there exist αx0 ∈R\{0} and βx0 ∈R such that f(x) = αx0x + βx0 for x ∈u1((x0 −rx0, x0 + rx0)). Moreover, as u1 is a continuous injection, D is open and connected. Thus, according to Lemma 2.8, there exist αx0 ∈R\{0} and βx0 ∈R such that Moreover, as u1 is a continuous injection, D is open and connected. Thus, according to Lemma 2.8, there exist αx0 ∈R\{0} and βx0 ∈R such that f(x) = αx0x + βx0 for x ∈u1((x0 −rx0, x0 + rx0)). f(x) = αx0x + βx0 for x ∈u1((x0 −rx0, x0 + rx0)). Vol. 93 (2019) Vol. 93 (2019) Extension problem for principles of equivalent utility 233 Hence, in view of (57), we get u2(x) = αx0u1(x) + βx0 for x ∈(x0 −rx0, x0 + rx0). (58) In the case where y0 < 0 there exists rx0 > 0 such that Hence, in view of (57), we get u2(x) = αx0u1(x) + βx0 for x ∈(x0 −rx0, x0 + rx0). (58) In the case where y0 < 0 there exists rx0 > 0 such that Hence, in view of (57), we get ( ), g u2(x) = αx0u1(x) + βx0 for x ∈(x0 −rx0, x0 + rx0). (58) (58) In the case where y0 < 0 there exists rx0 > 0 such that B((x0, y0), rx0) ⊂Fp(T) ∩[(0, ∞) × (−∞, 0)]. Furthermore, arguing as previously, we obtain that the function f, deﬁned by (57), satisﬁes Eq. (46) with a, A, c, C and D as above, b := −h1(p3) g1(p1) and B := −h2(p3) g2(p1) . Hence, (58) holds with some αx0 ∈R\{0} and βx0 ∈R. B := −h2(p3) g2(p1) . Hence, (58) holds with some αx0 ∈R\{0} and βx0 ∈R. If x0 < w, then in view of (41), there exists p ∈P3 such that x0 ∈ψp(T) . Thus, repeating the previous procedure, we conclude that (58) is satisﬁed with some αx0 ∈R\{0} and βx0 ∈R. . In this way we have proved that for every x ∈R\{0} there exist αx ∈R\{0}, βx ∈R and rx ∈(0, ∞) such that (50) is valid. 3. Main results Thus, as u1 and u2 are strictly increasing and continuous, with u1(0) = u2(0) = 0, applying Lemma 2.9 with I = (0, ∞) and then with I = (−∞, 0), we obtain that there exist α, β ∈(0, ∞) such that (56) holds. In the remaining part of the proof we consider two cases: 1. w = 0, 1. w = 0, 2. w > 0. 2. w > 0. 2. w > 0. Case 1 Let p = (p1, p2, p3) ∈P3 and y ∈(0, ∞). Then, according to Lemma 2.5, φp(x, y) = 0 for some x ∈(0, y). Thus, in view of (16), we have ψp(x, y) = x −y < 0. So, applying Lemma 2.1(ii) and Corollary 2.2(ii), we obtain hi(p3)ui(x −y) + gi(p1)ui(x) = 0 for i ∈{1, 2}. Hence, considering (56), we get hi(p3)ui(x −y) + gi(p1)ui(x) = 0 for i ∈{1, 2}. Hence, considering (56), we get Hence, considering (56), we get h2(p3) g2(p1) = − u2(x) u2(x −y) = −β α u1(x) u1(x −y) = β α h1(p3) g1(p1) . Since p = (p1, p2, p3) ∈P3 is ﬁxed arbitrarily, this means that h2(p3) h1(p3) = β α g2(p1) g1(p1) for p1, p3 ∈(0, 1), p1 + p3 < 1. (59) (59) Thus, taking q1, q2 ∈(0, 1) and p ∈(0, min{1 −qi : i ∈{1, 2}}), we obtain h2(q1) h1(q1) = β α g2(p) g1(p) = h2(q2) h1(q2), which means that the function h1/h2 is constant on (0, 1). Since, for i ∈{1, 2}, hi is continuous, with hi(1) = 1, this implies that h1 = h2. Furthermore, from (59) we derive that g2(p) g1(p) = α β for p ∈(0, 1). Therefore, using the fact that, for i ∈{1, 2}, gi is continuous and gi(1) = 1, we get β = α. Hence g1 = g2 and, considering (56), we obtain (54). This completes the proof in the case w = 0. 234 J Ch 23 J. Chudziak 234 AEM Case 2 Fix p = (p1, p2, p3) ∈P3 and y ∈(0, w). Then, in view of Lemma 2.5, there exists x ∈(0, y) such that φp(x, y) = w. Moreover, making use of (16), we get ψp(x, y) = w+x−y > 0. Thus, applying Lemma 2.1(i) and Corollary 2.2(i), for i ∈{1, 2}, we obtain (1−gi(p1 +p2))ui(w +x−y)+(gi(p1 +p2)−gi(p1))ui(w)+gi(p1)ui(w +x) = ui(w). 3. Main results Hence Hence 1 −gi(p1 + p2) gi(p1) = ui(w) −ui(w + x) ui(w + x −y) −ui(w) for i ∈{1, 2}. Therefore, since p = (p1, p2, p3) ∈P3 is ﬁxed arbitrarily, in view of (56), we get 1 −g1(p1 + p2) g1(p1) = 1 −g2(p1 + p2) g2(p1) for p1, p2 ∈(0, 1), p1 + p2 < 1. Thus, as g1 and g2 are continuous, taking p1 ∈(0, 1) and letting p2 →0+, we obtain that g1(p1) = g2(p1). Since gi(0) = 0 and gi(1) = 1 for i ∈{1, 2}, this implies that g1 = g2. Thus, as g1 and g2 are continuous, taking p1 ∈(0, 1) and letting p2 →0+, we obtain that g1(p1) = g2(p1). Since gi(0) = 0 and gi(1) = 1 for i ∈{1, 2}, this implies that g1 = g2. Suppose that p = (p1, p2, p3) ∈P3 is such that (28) holds for some (x, y) ∈ T. Then, in view of Lemma 2.1(ii) and Corollary 2.2(ii), for i ∈{1, 2}, we get hi(p3)ui(ψp(x, y))+(gi(p1+p2)−gi(p1))ui(φp(x, y))+gi(p1)ui(φp(x, y)+x) = ui(w). Since g1 = g2, making use of (56), from the last equality one can easily derive that β h2(p3) = β αh1(p3). (60) (60) Now, if limx→∞u1(x) = ∞, then applying Lemma 2.3(a), we conclude that (60) holds for every p3 ∈(0, 1). Since, for i ∈{1, 2}, hi is continuous with hi(1) = 1, letting in (60) p3 →1−we obtain that β = α. Thus, in view of (56) and (60), we get (54) and h1 = h2, respectively. If L := limx→∞u1(x) < ∞, then according to Lemma 2.3(b), (60) holds for every p3 ∈(0, 1) such that g1(1 −p3) > u1(w)/L. As h1 and h2 are continuous, this implies (55). □ Theorem 3.2. Let w ∈[0, ∞). Assume that, for i ∈{1, 2}, ui : R →R is a strictly increasing continuous function with ui(0) = 0 and gi, hi : [0, 1] →[0, 1] are strictly increasing continuous probability distortion functions. Furthermore, let H(ui,gi,hi) for i ∈{1, 2} be the functionals of equivalent utility, deﬁned by (5). 3. Main results If (i) g1 = g2, h1 = h2 and (54) holds with some α ∈(0, ∞) or (ii) w > 0, L := limx→∞u1(x) < ∞, g1 = g2 and (55), (56) hold with some α, β ∈(0, ∞), then (ii) w > 0, L := limx→∞u1(x) < ∞, g1 = g2 and (55), (56) hold with some α, β ∈(0, ∞), th H(u1,g1,h1)(X) = H(u2,g2,h2)(X) for X ∈X+. (61) (61) Vol. 93 (2019) Extension problem for principles of equivalent utility 235 235 Proof. Since the generalized Choquet integral is positively homogeneous (cf. [9, Lemma 1]), in view of (5), in the case of (i), we get roof. Since the generalized Choquet integral is positively homogeneous (cf. , Lemma 1]), in view of (5), in the case of (i), we get Eg2h2[u2(w + H(u1,g1,h1)(X) −X)] = αEg1h1[u1(w + H(u1,g1,h1)(X) −X)] Eg2h2[u2(w + H(u1,g1,h1)(X) −X)] = αEg1h1[u1(w + H(u1,g1,h1)(X) −X)] = αu1(w) = u2(w) for X ∈X+, = αu1(w) = u2(w) for X ∈X+, = αu1(w) = u2(w) for X ∈X+, which implies (61). p ( ) Assume that (ii) holds and ﬁx X ∈X+. We claim that P(X > w + H(u1,g1,h1)(X)) ≤1 −g−1 1 (u1(w)/L). (62) (62) Suppose that this is not true. Then, as the space (Ω, Σ, P) is non-atomic, there exists B ∈Σ such that Suppose that this is not true. Then, as the space (Ω, Σ, P) is non-atomic, there exists B ∈Σ such that B ⊂A := {X > w + H(u1,g1,h1)(X)} and and 1 −g−1 1 (u1(w)/L) < P(B) < P(A). (63) (63) Hence, setting X := w · 1lA\B + (w + H(u1,g1,h1)(X)) · 1lB, X := w · 1lA\B + (w + H(u1,g1,h1)(X)) · 1lB, we get X ≤X which, in view of (9), gives we get X ≤X which, in view of (9), gives we get X ≤X which, in view of (9), gives we get X ≤X which, in view of (9), gives H(u1,g1,h1)(X) ≤H(u1,g1,h1)(X). (64) H(u1,g1,h1)(X) ≤H(u1,g1,h1)(X). (64) (64) Furthermore, we have X = ⟨0, w, w + H(u1,g1,h1)(X); p⟩∈X (3) 0 , (65) (65) where p := (1 −P(A), P(A\B), P(B)). 3. Main results Let T be of the form (13) and let φ(1) p , ψ(1) p : T →R be given by φ(1) p (x, y) = w + H(u1,g1,h1)(⟨0, x, y; p⟩) −x for (x, y) ∈T, ψ(1) p (x, y) = w + H(u1,g1,h1)(⟨0, x, y; p⟩) −y for (x, y) ∈T, respectively. Then, considering (8), (64) and (65), we obtain ψ(1) p (w, w + H(u1,g1,h1)(X)) ψ(1) p (w, w + H(u1,g1,h1)(X)) = w + H(u1,g1,h1)(X) −(w + H(u1,g1,h1)(X)) = H(u1,g1,h1)(X) −H(u1,g1,h1)(X) ≤0 ≤H(u1,g1,h1)(X) = φ(1) p (w, w + H(u1,g1,h1)(X)). J. Chudziak AEM 236 Therefore, according to Lemma 2.3(c), we have g1(1 −P(B)) > u1(w)/L and so Therefore, according to Lemma 2.3(c), we have g1(1 −P(B)) > u1(w)/L and so so P(B) < 1 −g−1 1 (u1(w)/L), which contradicts (63). In this way (62) is proved. which contradicts (63). In this way (62) is proved. ( ) It follows from (62) that ( ) y ( ) It follows from (62) that P(−u2(w + H(u1,g1,h1)(X) −X) > t) ≤1 −g−1 1 (u1(w)/L) for t ∈[0, ∞). Thus, in view of (4), (55) and (56), we get P(−u2(w + H(u1,g1,h1)(X) −X) > t) ≤1 −g−1 1 (u1(w)/L) for t ∈[0, ∞). Thus, in view of (4), (55) and (56), we get P(−u2(w + H(u1,g1,h1)(X) −X) > t) ≤1 −g−1 1 (u1(w)/L) for t ∈[0, ∞). Thus, in view of (4), (55) and (56), we get Eh2[max{−u2(w + H(u1,g1,h1)(X) −X), 0}] = ∞ 0 h2(P(−u2(w + H(u1,g1,h1)(X) −X) > t)) dt = β α ∞ 0 h1(P(−αu1(w + H(u1,g1,h1)(X) −X) > t)) dt = β ∞ 0 h1(P(−u1(w + H(u1,g1,h1)(X) −X) > t)) dt = βEh1[max{−u1(w + H(u1,g1,h1)(X) −X), 0}]. Furthermore, since the Choquet integral is positively homogeneous, making use of (56), we obtain Furthermore, since the Choquet integral is positively homogeneous, making use of (56), we obtain Eg2[max{u2(w + H(u1,g1,h1)(X) −X), 0}] Eg2[max{u2(w + H(u1,g1,h1)(X) −X), 0}] Eg2[max{u2(w + H(u1,g1,h1)(X) −X), 0}] = βEg2[max{u1(w + H(u1,g1,h1)(X) −X), 0}]. Thus, as g1 = g2, considering (5) and (6), we conclude that Eg2h2[u2(w + H(u1,g1,h1)(X) −X)] = βEg2[max{u1(w + H(u1,g1,h1)(X) −X), 0}] Thus, as g1 = g2, considering (5) and (6), we conclude that Eg2h2[u2(w + H(u1,g1,h1)(X) −X)] = βEg1h1[u1(w + H(u1,g1,h1)(X) −X)] = βu1(w) = u2(w) and so H(u1,g1,h1)(X) = H(u2,g2,h2)(X). □ From Theorems 3.1 and 3.2 we derive the following result. Theorem 3.3. 3. Main results Let w ∈[0, ∞). Assume that, for i ∈{1, 2}, ui : R →R is a strictly increasing continuous function with ui(0) = 0 and gi, hi : [0, 1] →[0, 1] are strictly increasing continuous probability distortion functions. Moreover, let H(ui,gi,hi) for i ∈{1, 2} be the functionals of equivalent utility, deﬁned by (5). If (53) holds then H(u1,g1,h1)(X) = H(u2,g2,h2)(X) for X ∈X+. Remark 3.4. According to Theorem 3.3, every functional of equivalent utility, deﬁned by (5), can be uniquely extended from the family X (3) 0 to X+. It has been already mentioned, in the Introduction, that such a functional need not be uniquely extended from the family of all binary risks. We complete the paper with a suitable example. Vol. 93 (2019) Extension problem for principles of equivalent utility 237 Example 3.5. Let w ∈[0, ∞) and let u1, u2 : R →R be of the form u1(x) = x for x ∈R and and u2(x) = √w −√w −x for x ∈(−∞, w], √w + √x −w for x ∈(w, ∞), respectively. Furthermore, let gi, hi : [0, 1] →[0, 1] for i ∈{1, 2} be given by g1(p) = h1(p) = p for p ∈[0, 1] u2(x) = √w −√w −x for x ∈(−∞, w], √w + √x −w for x ∈(w, ∞), √ + √ ( , ), respectively. Furthermore, let gi, hi : [0, 1] →[0, 1] for i ∈{1, 2} be given by g1(p) = h1(p) = p for p ∈[0, 1] respectively. Furthermore, let gi, hi : [0, 1] →[0, 1] for i ∈{1, 2} be given by g1(p) = h1(p) = p for p ∈[0, 1] and g2(p) = h2(p) = √p √p + √1 −p for p ∈[0, 1]. Then, according to [3, Theorems 3.1-3.2], H(u1,g1,h1) and H(u2,g2,h2) coincide on the family of all binary risks. On the other hand, since limx→∞u1(x) = ∞, applying Theorem 3.1, we conclude that H(u1,g1,h1) and H(u2,g2,h2) do not coincide on X (3) 0 . Open Access. This article is distributed under the terms of the Creative Commons At- tribution 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. 3. Main results Publisher’s Note Springer Nature remains neutral with regard to jurisdic- tional claims in published maps and institutional aﬃliations. References 1] Bowers, N.L., Gerber, H.U., Hickman, J.C., Jones, D.A., Nesbitt, C.J.: Actuarial Math- ematics. The Society of Actuaries, Itasca (1986) [2] B¨uhlmann, H.: Mathematical Models in Risk Theory. Springer, New York (1970) [3] Chudziak, J.: On functional equations stemming from actuarial mathematics. Aequ. Math. 92, 471–486 (2018) [4] Chudziak, J.: On existence and uniqueness of the principle of equivalent utility under Cumulative Prospect Theory. Insur. Math. Econ. 79, 243–246 (2018) [5] Chudziak, J.: On applications of inequalities for quasideviation means in actuarial math ematics. Math. Inequal. Appl. 21, 601–610 (2018) [6] Chudziak, M., Sobek, B.: Generalized Pexider equation on an open domain. Results Math. 71, 1359–1372 (2017) [7] Heilpern, S.: A rank-dependent generalization of zero utility principle. Insur. Math. Econ. 33, 67–73 (2003) [8] Kaas, R., Goovaerts, M., Dhaene, J., Denuit, M.: Modern Actuarial Risk Theor Springer, Berlin (2008) [9] Kaluszka, M., Krzeszowiec, M.: Pricing insurance contracts under Cumulative Prospect Theory. Insur. Math. Econ. 50, 159–166 (2012) [10] Kaluszka, M., Krzeszowiec, M.: On iterative premium calculation principles under Cu- mulative Prospect Theory. Insur. Math. Econ. 52, 435–440 (2013) AEM AEM 238 J. Chudziak [11] Kuczma, M.: An Introduction to the Theory of Functional Equations and Inequalities. Cauchy’s Equation and Jensen’s Inequality, 2nd edn. Birkh¨auser, Berlin (2009) [11] Kuczma, M.: An Introduction to the Theory of Functional Equations and Inequalitie [ ] Cauchy’s Equation and Jensen’s Inequality, 2nd edn. Birkh¨auser, Berlin (2009) [12] Rachev, S.T., Klebanov, L.B., Stoyanov, S.V., Fabozzi, F.J.: The Methods of Distances in the Theory of Probability and Statistics. Springer, New York (2013) ( ) [13] Rad´o, F., Baker, J.A.: Pexider’s equation and aggregation of allocations. Aequ. Math. 32, 227–239 (1987) [13] Rad´o, F., Baker, J.A.: Pexider’s equation and aggregation of allocations. Aequ. Math. 32, 227–239 (1987) ( ) [14] Rolski, T., Schmidli, H., Schmidt, V., Teugels, J.: Stochastic Processes for Insurance and Finance. Wiley, New York (1999) ( ) [14] Rolski, T., Schmidli, H., Schmidt, V., Teugels, J.: Stochastic Processes for Insurance and Finance. Wiley, New York (1999) Jacek Chudziak Jacek Chudziak Faculty of Mathematics and Natural Sciences University of Rzesz´ow ul. Pigonia 1 35-310 Rzeszow Poland e-mail: chudziak@ur.edu.pl Received: March 30, 2018 Jacek Chudziak Faculty of Mathematics and Natural Sciences University of Rzesz´ow ul. Pigonia 1 35-310 Rzeszow Poland e-mail: chudziak@ur.edu.pl Received: March 30, 2018 Faculty of Mathematics and Natural Sciences
https://openalex.org/W3199168273	https://link.springer.com/content/pdf/10.1007/s40145-021-0486-x.pdf	English	null	Electronic structure and enhanced photoelectrocatalytic performance of RuxZn1−xO/Ti electrodes	Journal of advanced ceramics	2,021	cc-by	9,400	* Corresponding author. E-mail: yqshao1989@163.com Electronic structure and enhanced photoelectrocatalytic performance of RuxZn1–xO/Ti electrodes Received: January 4, 2021; Revised: April 16, 2021; Accepted: April 17, 2021 © The Author(s) 2021. Abstract: Modification is one of the most important and effective methods to improve the photoelectrocatalytic (PEC) performance of ZnO. In this paper, the RuxZn1–xO/Ti electrodes were prepared by thermal decomposition method and the effect of Ru content on those electrodes’ electronic structure was analyzed through the first-principles calculation. Various tests were also performed to observe the microstructures and PEC performance. The results showed that as the Ru4+ transferred into ZnO lattice and replaced a number of Zn2+, the conduction band of ZnO moved downward and the valence band went upward. The number of photogenerated electron–hole pairs increased as the impurity levels appeared in the band gap. In addition, ZnO nanorods exhibited a smaller grain size and a rougher surface under the effect of Ru. Meanwhile, the RuO2 nanoparticles on the surface of ZnO nanorods acted as the electron-transfer channel, helping electrons transfer to the counter electrode and delaying the recombination of the electron–hole pairs. Specifically, the RuxZn1–xO/Ti electrodes with 9.375 mol% Ru exhibited the best PEC performance with a rhodamine B (RhB) removal rate of 97%, much higher than the combination of electrocatalysis (EC, 12%) and photocatalysis (PC, 50%), confirming the synergy of photoelectrocatalysis. Keywords: RuxZn1–xO/Ti electrode; first-principles calculation; electric collector; photoelectric synergistic catalysis Keywords: RuxZn1–xO/Ti electrode; first-principles calculation; electric collector; photoelectric synergistic catalysis effectively degrade organic dyes has become a hot topic [5–9]. One of the most widely studied methods would be the semiconductor material-based photocatalysis (PC) technology. And ZnO, an atoxic direct band gap semiconductor with a wide band gap of 3.37 eV and an exciton binding energy of 60 mV, has been considered as one of the most ideal materials for wastewater treatment due to its excellent PC activity, great physical and chemical stability as well as high cost efficiency [10–13]. It has been reported that compared with TiO2, ZnO exhibited higher PC efficiency in terms Journal of Advanced Ceramics 2021, 10(5): 1025–1041 Journal of Advanced Ceramics 2021, 10(5): 1025–1041 ISSN 2226-4108 CN 10-1154/TQ https://doi.org/10.1007/s40145-021-0486-x Research Article 1 Introduction It was estimated that 15% of untreated organic dyes widely used in various fields have been discharged into water directly [1–4]. Those dyes could exist in the ecosystem stably, devastate its self-degradation ability, and make people suffer higher risk of intoxication, mutation, and cancerization. Therefore, how to cost- www.springer.com/journal/40145 www.springer.com/journal/40145 J Adv Ceram 2021, 10(5): 1025–1041 1026 disparity between Ru4+ and Zn2+ is smaller than 7% making it possible to prepared Ru-doped ZnO, and the minute difference in electronegativity between the two atoms helps form solid solutions with greater solubility [36,37]. We finally chose Ru to modify ZnO and analyzed its impact on the electronic structure and the photoelectrocatalysis (PEC) performance of composite- oxide electrodes. of the degradation of several organic contaminants in both acidic and basic medium [14–16]. However, the high recombination rate of photogenerated electron–hole pairs and oversized band gap have significantly limited its application. Hence, how to reduce the recombination rate and enlarge the light-absorption area have received much attention [17–20]. In recent years, an external bias potential placed between the anode and cathode has proven an effective method to promote the electron–hole separation rate. Under its effect, the fore-mentioned recombination rate was reduced, and more electrons and holes were allowed to participate in the reaction of oxidative cracking of organic dye as the photogenerated electrons were forced to move to the counter electrode through an external circuit, while the holes remained in the valence band thanks to the external electric field. In addition, Ma et al. [17] designed a dual-cocatalyst for spatial separation, of which Co–Pi as the outmost hole-transfer layer and Pt as the bottom electron collector and transport layer, and thereby photogenerated electron–hole pairs were separated effectively and twice higher current density was obtained. However, neither the external bias potential nor the electron collector could increase the yield of photogenerated electron–hole pairs, the light-absorption range was still relatively narrow. An efficient way to solve that problem was doping metal or non-metal elements into materials and changing their electronic structure and band gap [21–25]. Ashebir et al. [26] reported that both Mn- and Ag-doped ZnO nanoparticles showed a decreased band gap and higher PC performance relative to pure ZnO. According to Qiu et al. 1 Introduction [27] who studied the effect of N-doping on the PC performance of ZnO, the forbidden band width decreased significantly and the PC activity was enhanced after the N 2p impurity energy level appeared. Doping could also change the microstructure of the material [24,28,29]. Etacheri et al. [30] revealed that Mg-doped ZnO exhibited a significant c-axis compression and decreased grain size, and its specific surface area was twice as large as that of pure ZnO. 2.1.2 Preparation of RuxZn1–xO/Ti electrodes The precursors RuCl3·xH2O (containing 37% Ru by mass) and ZnCl2 were used to prepare the mixed solutions with Ru contents of 0, 3.125, 6.25, 9.375, and 12.5 mol%, which were stirred evenly by ultrasonic vibration. Then an appropriate amount of the mixed solution (3 μL·cm–2) was brushed on the Ti plates, which were cured under an infrared lamp at 100 ℃ for 10 min, and pre-oxidized in a muffle furnace at 500 ℃ for another 10 min, followed by air cooling. After repeating those steps for six times, the samples were annealed at 500 ℃ for 60 min, and then cooled in the air. The RuxZn1–xO/Ti electrodes with different Ru contents were prepared. 2 Experimental and calculation methods 2. 1 Preparation of RuxZn1–xO/Ti electrodes 2.1.1 Pretreatment of the Ti plates 2.1.1 Pretreatment of the Ti plates 2.1.1 Pretreatment of the Ti plates In order to strengthen the bonding force between the substrate and the coatings, enlarge the surface area, and extend the service life of the electrodes, the sandblasted 20 mm×20 mm×1.5 mm TA2 titanium plate was degreased, etched in 10 wt% H2SO4 for 2 h, and then cleaned with deionized water and preserved in absolute alcohol for further use. 2. 3 First-principles calculations According to the solid solution theory, both an ionic radius difference within 15% (Ru4+ 0.62 Å vs. Zn2+ 0.74 Å) and minute electronegativity disparity (Ru 2.20, Zn 1.65) are favorable factors, indicating the high possibility of forming RuxZn1–xO solid solution. Given that the ideal ZnO possesses a hexagonal wurtzite structure with a space group of P63/mc, a 2×2×2 supercell structure model with 64 atoms was constructed, in which a small number of Zn and Ru atoms exchanged places, as shown in Fig. 1. In this study, VASP software was adopted to calculate the electronic structure of the Ru-doped ZnO crystal based on the plane-wave density functional theory (DFT), where the k-point meshes of the Brillouin zone were obtained via 9×9×5 Monkhorst–Pack grid and the plane wave cut-off energy was 520 eV. 2. 2 Characterization techniques The microstructures of the RuxZn1–xO/Ti electrodes were observed by a scanning electron microscope (SEM, Supra 55, Carl Zeiss, Oberkochen, Germany) and the transmission electron microscope tests (TEM, TECNAI G2 F20, FEI, USA). The phase structures of the coatings were studied by X-ray diffractometer test (XRD, Ultima III, Rigaku, Tokyo, Japan) with Cu Kα radiation source and Ni filter, where the tube voltage and current were 36 kV and 30 mA respectively, and the angle range and scanning rate were 25°–60° and In general, the bias potential and electron collector could reduce the recombination rate of photogenerated electron–hole pairs effectively, and doping method could expand the light-absorption area and change the material’s microstructure [31]. Given the fact that noble metal oxide RuO2 is an excellent electrocatalyst with high conductivity [32–35], the atomic radius www.springer.com/journal/40145 www.springer.com/journal/40145 J Adv Ceram 2021, 10(5): 1025–1041 1027 10 mV and the frequency range was 0.05–105 Hz. The linear sweep voltammetric (LSV) tests were performed with a potential range of 0–2.5 V and a scanning rate of 10 mV·s–1. 2 (°)/min respectively. X-ray photoelectron spectroscopy (XPS) spectra were performed on the photoelectron spectroscopy system (Escalab 250, Thermo Scientific, USA), and the elemental peak fitting was performed via the Avantage software. The UV–Vis diffuse reflectance spectra (DRS) were tested through the UV2600 instrument with a wavelength range of 200–800 nm. Specific surface areas (SSA) were tested with the Micromeritics 3Flex specific surface analyzer, and the samples were degassed at 200 ℃ for 10 h. 2. 5 Photoelectrochemical measurement The PEC performance of the RuxZn1–xO/Ti electrodes was evaluated by the degradation rate of RhB, methyl orange (MO), and methylene blue (MB). The experiment was also done with the above three-electrode set. The electrolyte in a 500 mL cylindrical quartz beaker was the mixed solution of 0.1 M Na2SO4 and 20 mg·L–1 RhB, 0.1 M Na2SO4 and 20 mg·L–1 MO, or 0.1 M Na2SO4 and 20 mg·L–1 MB, respectively. A 100 W ultraviolet mercury lamp (386 nm) was placed directly opposite to the working electrode with a surface area of 4 cm2, and the light intensity (15 mW·cm–2) was monitored via a light intensity photometer (UV-M10- P/S, ORC, Japan). The solution was continuously stirred, and the potential was 2.5 V. The removal rate of RhB during each degradation period was evaluated via a Cary 50 UV–Vis spectrophotometer (Varian, USA) and the total organic carbon (TOC) tests were carried out through Shimadzu TOC-L ASI-L (Shimadzu, Japan). 3. 1 Microstructure morphology analysis Electrochemical tests were performed with a three- electrode system via the Metrohm AUTOLAB electrochemical workstation (AUT84638, PGSTAT302N). 0.1 M Na2SO4 solution was used as the electrolyte. The prepared RuxZn1–xO/Ti with different Ru contents was as working electrodes. The saturated calomel electrode (SCE) was as a reference electrode, and the large-area titanium plate was as a counter electrode. The amplitude for electrochemical impedance spectroscopy EIS (test) was As we seen from the SEM images of the RuxZn1–xO/Ti electrodes with different Ru contents shown in Fig. 2, the coating surface exhibited both large and small typical hexagonal prism ZnO grains [18,38]. According to Fig. 2(a), ZnO nanorods on the surface of pure ZnO coatings arranged orderly, possessing a relatively larger crystal grain diameter of 0.1–1 μm. However, as Ru content increased, ZnO nanorods tended to grow disorderly and the diameter gradually decreased with a Fig. 1 Ru-doped ZnO with hexagonal wurtzite crystal structure: (a) front view, (b) top view, and (c) left view. Fig. 1 Ru-doped ZnO with hexagonal wurtzite crystal structure: (a) front view, (b) top view, and (c) left view. www.springer.com/journal/40145 www.springer.com/journal/40145 1028 J Adv Ceram 2021, 10(5): 1025–1041 Fig. 2 SEM images of RuxZn1–xO/Ti electrodes: (a) 0 mol%, (b) 3.125 mol%, (c) 6.25 mol%, (d) 9.375 mol%, and (e) 12.5 mol%. SEM images of RuxZn1–xO/Ti electrodes: (a) 0 mol%, (b) 3.125 mol%, (c) 6.25 mol%, (d) 9.375 mol%, and (e) Fig. 2 SEM images of RuxZn1–xO/Ti electrodes: (a) 0 mol%, (b) 3.125 mol%, (c) 6.25 mol%, (d) 9.375 mol%, and (e) 12.5 mol%. few exceptions, i.e., adding Ru could reduce the grain size of ZnO, increase the irregularity degree of coatings, and thus enlarge the specific surface area. cos k D     (1) (1) where k is the Scherrer constant (0.89), λ is the X-ray wavelength of Cu Kα (0.154 nm), β is the full width at half maximum (FWHM) of the (101) crystal plane diffraction peak of ZnO, and θ is the diffraction angle. According to the results shown in Table 1, the grain size of ZnO decreased from 33 to 26 nm with the increase of Ru content, coinciding with the SEM analysis [30,40–42]. As shown in Fig. 3(b), the 2θ angles of 31.769°, 34.421°, and 36.252° corresponded to the (100), (002), and (101) crystal planes of hexagonal wurtzite phase ZnO, respectively. 3. 1 Microstructure morphology analysis Table 2 SSA of RuxZn1–xO/Ti coatings Table 1 Grain size of RuxZn1–xO/Ti coatings Content of Ru4+ (mol%) 0 3.125 6.25 9.375 12.5 Grain size (nm) 33 30 29 28 26 Table 2 SSA of RuxZn1–xO/Ti coatings Content of Ru4+ (mol%) 0 3.125 6.25 9.375 12.5 SSA (m2·g–1) 1.536 1.881 2.909 4.077 7.422 According to the results listed in Table 2, the specific surface area (SSA) of the of RuxZn1–xO/Ti electrodes increased gradually as the Ru content rose as adding Ru could reduce the grain size of ZnO nanorods and increase the coatings’ irregularity degree, which helped provide more active sites for PEC degradation. diffraction spots corresponded to the (101), (100), and (002) crystal planes of ZnO, respectively. According to Fig. 4(d), the RuO2 nanoparticles distributed on the ZnO nanorods’ surface uniformly, and this structure facilitated the transfer of electrons and the separation of photo-generated electrons and holes. The lattice spacing of 0.248 and 0.317 nm displayed in Fig. 4(e), corresponded to the (101) crystal plane of ZnO (JCPDS 36-1451) and the (110) crystal plane of RuO2 (JCPDS 18-1139), respectively. Figure 4(f) exhibits the polycrystalline electron diffraction pattern of ZnO and RuO2, where the two diffraction spots corresponded to the (101) crystal planes of ZnO and the (110) crystal plane of RuO2, respectively, consistent with the XRD analysis. diffraction spots corresponded to the (101), (100), and (002) crystal planes of ZnO, respectively. According to Fig. 4(d), the RuO2 nanoparticles distributed on the ZnO nanorods’ surface uniformly, and this structure facilitated the transfer of electrons and the separation of photo-generated electrons and holes. The lattice spacing of 0.248 and 0.317 nm displayed in Fig. 4(e), corresponded to the (101) crystal plane of ZnO (JCPDS 36-1451) and the (110) crystal plane of RuO2 (JCPDS 18-1139), respectively. Figure 4(f) exhibits the polycrystalline electron diffraction pattern of ZnO and RuO2, where the two diffraction spots corresponded to the (101) crystal planes of ZnO and the (110) crystal plane of RuO2, respectively, consistent with the XRD analysis. The microstructures of the RuxZn1–xO/Ti electrodes were analyzed by transmission electron microscopy (TEM). Figures 4(a) and 4(b) reveal that the wurtzite- type ZnO nanorods were piled up by the (001) planes, coinciding with its single-crystal characteristic [38]. The selected area electron diffraction (SAED) pattern shown in Fig. 4(c) further confirmed that the three Fig. 4 TEM and SAED patterns of RuxZn1–xO/Ti electrodes: (a–c) 0 mol% and (d–f) 9.375 mol%. www.springer.com/journal/40145 3. 1 Microstructure morphology analysis Compared with the pure ZnO electrodes, the 2θ of the three diffraction peaks of Ru-doped ones shifted to higher angles. The higher the Ru content, the larger the deviation degree, indicating that RuxZn1–xO solid solution could be formed after a number of Zn2+ in the ZnO lattice were replaced by Ru4+ with smaller radius. [41,43,44]. where k is the Scherrer constant (0.89), λ is the X-ray wavelength of Cu Kα (0.154 nm), β is the full width at half maximum (FWHM) of the (101) crystal plane diffraction peak of ZnO, and θ is the diffraction angle. According to the results shown in Table 1, the grain size of ZnO decreased from 33 to 26 nm with the increase of Ru content, coinciding with the SEM analysis [30,40–42]. As shown in Fig. 3(b), the 2θ angles of 31.769°, 34.421°, and 36.252° corresponded to the (100), (002), and (101) crystal planes of hexagonal wurtzite phase ZnO, respectively. Compared with the pure ZnO electrodes, the 2θ of the three diffraction peaks of Ru-doped ones shifted to higher angles. The higher the Ru content, the larger the deviation degree, indicating that RuxZn1–xO solid solution could be formed after a number of Zn2+ in the ZnO lattice were replaced by Ru4+ with smaller radius. [41,43,44]. Phase analysis of the RuxZn1–xO/Ti electrodes was carried out through the XRD tests. Pure ZnO electrodes only exhibited the diffraction peak of hexagonal wurtzite phase ZnO (space group: P63/mc, JCPDS 36-1451) as displayed in Fig. 3(a). Compared with pure ZnO, RuxZn1–xO/Ti electrode doped with Ru not only exhibited the diffraction peak of ZnO, but also the rutile phase RuO2 (space group: P42/mnm, JCPDS 18-1139) and tetragonal phase metal Ru (JCPDS 06-0663). In addition, the higher the Ru content, the more intensive the diffraction peaks of RuO2 and metallic Ru, while the intensity of ZnO diffraction peaks gradually decreased, suggesting that there might be more RuO2 particles covered on the surface of ZnO. The relatively sharper peak of ZnO indicated a higher degree of crystallization. The grain size of ZnO (D) was calculated by the following Scherrer equation (applied to the (101) crystal plane) [39]: www.springer.com/journal/40145 J Adv Ceram 2021, 10(5): 1025–1041 1029 Fig. 3 XRD patterns of RuxZn1–xO/Ti electrodes: (a) 2θ within 25°–60°; (b) the enlarged view of 2θ within 30°–38°. Fig. 3 XRD patterns of RuxZn1–xO/Ti electrodes: (a) 2θ within 25°–60°; (b) the enlarged view of 2θ within 30°–38°. 3. 1 Microstructure morphology analysis Fig. 4 TEM and SAED patterns of RuxZn1–xO/Ti electrodes: (a–c) 0 mol% and (d–f) 9.375 mol%. www.springer.com/journal/40145 www.springer.com/journal/40145 J Adv Ceram 2021, 10(5): 1025–1041 1030 12.5 mol% Ru had a red-shift by 0.12 eV, as the RuO2 content decreased and the RuOx/Ru content increased [50]. The high-resolution narrow scan spectra of Zn 2p in Fig. 5(c) exhibited two spin orbits of Zn 2p3/2 and Zn 2p1/2 with a spin separation energy of 23 eV. Pure ZnO electrodes displayed double peaks of Zn 2p3/2 and Zn 2p1/2 at 1021.41 and 1044.42 eV, respectively, corresponding to Zn2+ in ZnO [30,51]. Besides, those peaks had blue-shifted by 0.42 and 0.70 eV, when the Ru content was 9.375 mol% (1021.83 and 1044.85 eV) and 12.5 mol% (1022.11 and 1045.13 eV), respectively, due to the replacement between Zn2+ and Ru4+. After the formation of Zn–O–Ru bond, the electron density around Zn atoms reduced as the electronegativity of Ru (2.20) was greater than that of Zn (1.65). Accordingly, the binding energy between the nucleus and extranuclear electrons of Zn atoms was strengthened, while that of Ru atoms was weakened, which was also one reason for the red shift of Ru 3d characteristic peak [30,52,53]. The high-resolution narrow scan spectra of O 1s are shown in Fig. 5(d), where the O 1s characteristic peaks of pure ZnO electrode could be divided into two. One was at 531.70 eV resulted from the adsorbed O2 or OH– ions, while the other at 530.02 eV corresponded to O2– in ZnO. It is noted that besides the aforementioned peaks, the electrodes containing Ru exhibited another O 1s characteristic peak at 529.40 eV, which was coincided with O2– in RuO2. XPS analysis of RuxZn1–xO/Ti electrodes was carried out to identify the elements’ composition and valence, and the results were adjusted with C 1s of 284.8 eV. According to Fig. 5(a), the full spectra of RuxZn1–xO/Ti electrodes have exhibited all the characteristic peaks of Ru 3d, Zn 2p, O 1s, and C 1s. The high-resolution narrow scan spectra of Ru 3d shown in Fig. 5(b) displayed a pair of narrow characteristic peaks, corresponding to the two spin–orbital components of Ru 3d5/2 and Ru 3d3/2, respectively, which located at 280.48 and 284.98 eV with a spin separation energy of 4.5 eV when the Ru content was 9.375 mol% [45–47]. 3. 2 Band structure and density of state analysis The effect of Ru content on the electronic structure of RuxZn1–xO solid solution was analyzed by the first- principles calculation. The band and crystal structure characteristics were also explained in detail. According to the data listed in Table 4, the calculated results of this study were very close with other studies. The lattice parameters a, c, and unit cell volume (V0) of the solid solution, all decreased with the increase of Ru content, as the ionic radius of Ru (0.69 Å) was smaller than that of Zn (0.74 Å) [36,54]. The band structure and density of state (DOS) of RuxZn1–xO solid solution are shown in Fig. 6. Typically, the Fermi level is close to the central portion of the forbidden band [55]. As shown in Fig. 6(a), the minimum calculated energy gap (Eg) of pure ZnO samples was about 1.78 eV, though smaller than the experimental value (3.37 eV), the result was still competitive with previous studies by Hernández et al. [56] (1.87 eV) and Bendavid and Carter [54] (1.90 eV). A possible reason was that the DFT theory did not take strong Coulomb correlation and interaction between electrons into consideration, leading to an underestimated band gap value. As shown in Figs. 6(a)–6(e), after adding Ru, the conduction band of ZnO moved down, the valence band moved up, and the impurity level appeared and narrowed the band gap. The higher the Ru content, the narrower the band The UV-DRS tests were performed to observe the forbidden band width of the RuxZn1–xO/Ti coatings and the values were calculated through the following Tauc– plot method [59]: 1/ – ( ) n g h A h E    (2) (2) where α is the absorbance index; h is the Planck constant; is the frequency constant. A represents the slope of the Tauc edge; n equals to 2, determined by the semiconductor type; and Eg is the band gap, reflected by the intersection point of the linear part of the curve of 2 ( ) h  and the h axis. According to the results shown in Fig. 7, the band gap (3.12 eV) of pure ZnO was slightly smaller than the theoretical value (3.37 eV) because of the lack of oxygen [60]. 3. 1 Microstructure morphology analysis The Ru 3d characteristic peaks could be divided into five small peaks representing the form of different states of Ru, coinciding with the three existing forms of Ru (RA for Ru, RB for RuO2, and RC for RuOx/Ru, respectively, Fig. 5(b)) and C 1s. The peaks at 280.66 and 284.91 eV corresponded to Ru4+ in RuO2, and obtained by heat treatment after the coatings were exposed to the surrounding environment. The one located at 280.45 eV was consistent with the zero valence of Ru0, resulting from the disproportionation of RuCl3 or the insufficient oxygen supply during the heat treatment. The peak at 282.05 eV indicated the existence of RuOx/Ru anoxic ruthenium, and the peak of C 1s appeared at 284.8 eV [47–49]. As shown in Table 3, compared with the electrodes with 9.375 mol% Ru, the Ru 3d characteristic peak of those containing Fig. 5 XPS spectra of RuxZn1–xO/Ti coatings: (a) full spectra, (b) Ru 3d, (c) Zn 2p, and (d) O 1s. Fig. 5 XPS spectra of RuxZn1–xO/Ti coatings: (a) full spectra, (b) Ru 3d, (c) Zn 2p, and (d) O 1s. www.springer.com/journal/40145 J Adv Ceram 2021, 10(5): 1025–1041 1031 Table 3 Peak position and corresponding peak area ratio of XPS spectra of high-resolution Ru 3d and Zn 2p Ratio of the integral area of the peak (%) Composition (at%) Orbital/spin Peak position (eV) Ru 9.375 mol% Ru 12.5 mol% Ru 3d5/2 280.2±0.2 0.90 1.36 RuOx/Ru 3d5/2 281.9±0.2 1.88 5.64 RuO2 3d5/2 280.9±0.2 5.73 4.72 ZnO 2P3/2 1021.8±0.2 91.49 88.28 3. 2 Band structure and density of state analysis Table 3 Peak position and corresponding peak area ratio of XPS spectra of high-resolution Ru 3d and Zn 2p gap (1.44 eV for 3.125 mol%, 0.72 eV for 6.25 mol%, 0.39 eV for 9.375 mol%, and 0.24 eV for 12.5 mol%, respectively). In other words, the addition of Ru had a significant effect on the band structure of ZnO, reducing the electron-transfer energy barrier. According to the DOS of the RuxZn1–xO electrodes shown in Figs. 3. 1 Microstructure morphology analysis 6(f)–6(j), pure ZnO samples exhibited peaks in four areas: Peaks ≈ –19 eV were mainly caused by the O 2s orbit, those appeared within the range of –9.5 to –7.5 eV and –6.5 to –1.5 eV were resulted from the hybridization of O 2p and Zn 3d orbits, and peaks located at the conduction band of about 8 eV were mainly formed by Zn 4s and O 2p. A significant difference in the electronic structure between Ru-doped and pure ZnO samples was that an impurity level appeared in the forbidden bands and narrowed the band gap, due to the electronic states of Ru 4d. As the Ru content increased, more electron energy levels appeared near the Fermi level, the electrons became more active, and the conductivity was enhanced accordingly. However, with the band gap narrowing down, the recombination rate of photogenerated electron–hole pairs also increased. 3. 2 Band structure and density of state analysis www.springer.com/journal/40145 3. 2 Band structure and density of state analysis Besides, Table 4 Comparison of calculated and experimental values of parameters (a and c) and lattice volume (V0) of RuxZn1–xO/Ti solid solution Composition a (Å) c (Å) α, β, γ (°) V0 (Å) Ref. ZnO 3.250 5.207 90, 90, 120 47.600 JCPDS 36-1451 ZnO 3.250 5.232 90, 90, 120 — [57] ZnO 3.252 5.222 90, 90, 120 — [58] 0 mol% Ru 3.249 5.145 90, 90, 120 46.900 This work 3.125 mol% Ru 3.242 5.136 89.968, 90, 120.209 46.348 This work 6.25 mol% Ru 3.232 5.098 89.995, 90, 120.017 46.263 This work 9.375 mol% Ru 3.226 5.077 89.018, 90.888, 121.171 46.200 This work 12.5 mol% Ru 3.224 5.055 88.392, 91.499, 121.249 46.168 This work on of calculated and experimental values of parameters (a and c) and lattice volume (V0) of l ti Table 4 Comparison of calculated and experimental values of parameters (a an RuxZn1–xO/Ti solid solution www.springer.com/journal/40145 J Adv Ceram 2021, 10(5): 1025–1041 1032 www.springer.com/journal/40145 d structures and densities of state of the RuxZn1–xO solid solution: (a, f) 0 mol%, (b, g) 3.125 mol% 9.375 mol%, and (e, j) 12.5 mol%. Fig. 6 Band structures and densities of state of the RuxZn1–xO solid solution: (a, f) 0 mol%, (b, g) 3.125 mol%, (c, h) 6.25 mol%, (d, i) 9.375 mol%, and (e, j) 12.5 mol%. www.springer.com/journal/40145 www.springer.com/journal/40145 J Adv Ceram 2021, 10(5): 1025–1041 1033 Fig. 7 UV-DRS patterns of the RuxZn1–xO/Ti coatings: (a) 0 mol%, (b) 3.125 mol%, (c) 6.25 mol%, (d) 9.375 mol%, and (e) 12.5 mol%. Fig. 7 UV-DRS patterns of the RuxZn1–xO/Ti coatings: (a) 0 mol%, (b) 3.125 mol%, (c) 6.25 mol%, (d) 9.375 mol%, and (e) 12 5 mol% Fig. 7 UV-DRS patterns of the RuxZn1–xO/Ti coatings: (a) 0 mol%, (b) 3.125 mol%, (c) 6.25 mol%, (d) 9.375 mol%, and (e) 12.5 mol%. erns of the RuxZn1–xO/Ti coatings: (a) 0 mol%, (b) 3.125 mol%, (c) 6.25 mol%, (d) 9.375 mol%, and (e) the band gap decreased with the increase of Ru content, coinciding with the theoretical calculation results. excessive RuO2 particles would pile up on ZnO nanorods’ surface, narrowing its effective light-sensitive area. Besides, the narrowed band gap also resulted in an increasing electron–hole recombination rate. As we can see from Figs. 8(a) and 8(b), samples under UV irradiation exhibited stronger current densities due to the generation of photocurrent. www.springer.com/journal/40145 3. 3 Electrochemical measurements As shown in Fig. 8, the LSV test of the RuxZn1–xO/Ti electrodes without and with UV irradiation was performed at a scanning rate of 10 mV·s–1. It can be seen that when the potential exceeded 1.6 V, the current densities increased rapidly due to the electrolyzed water. With the rise of Ru content, the current densities increased first, then decreased, with or without UV irradiation, and reached the maximum when the content was 9.375 mol%. This was partly because pure ZnO coatings had a relatively larger band gap and poorer conductivity, leading to a lower current density when voltage remained the same. With the addition of Ru, coatings’ band gap decreased and the conductivity was improved, resulting in an increased number of active photo-generated electron-hole pairs which enhanced current density. However, with a Ru content of 12.5 mol%, Figure 9(a) shows the Nyquist plots of the RuxZn1–xO/ Ti electrodes, and the enlarged view of its high frequency region is displayed in Fig. 9(c), where the points and lines referred to the experimental and fitted data, respectively. Figure 9(b) exhibits the equivalent circuit, in which L represents the inductive reactance of the electrochemical system, Rs refers to the intersection of the high frequency region and the real axis, reflecting the sum of the solution resistance and the coatings’ internal resistance, Rf and Rct indicate the coating– substrate or coating–electrolyte resistance and the Faraday transfer resistance, respectively, and Zw is the Warburg impedance, representing the ionic diffusion Fig. 8 LSV curves of the RuxZn1–xO/Ti electrodes: (a) without and (b) with UV irradiation. Fig. 8 LSV curves of the RuxZn1–xO/Ti electrodes: (a) without and (b) with UV irradiation. www.springer.com/journal/40145 1034 J Adv Ceram 2021, 10(5): 1025–1041 Fig. 9 (a) Nyquist plots of the RuxZn1–xO/Ti electrodes; (b) electrical equivalent circuit for the fitting of the impedance spectra; (c) enlarged view of the high frequency region of the Nyquist plots; (d) Nyquist plots with and without UV irradiation. Fig. 9 (a) Nyquist plots of the RuxZn1–xO/Ti electrodes; (b) electrical equivalent circuit for the fitting of the impedance spectra; (c) enlarged view of the high frequency region of the Nyquist plots; (d) Nyquist plots with and without UV irradiation. confirmed that the addition of Ru could improve the TOC removal rate effectively, which exhibited an inverted U-shaped curve and reached to the maximum of 68% when the Ru content was 9.375 mol%. 3. 3 Electrochemical measurements This was partly because the shortage of wide forbidden band of pure ZnO coatings and related problems including relatively smaller light-sensitive area and lower yield of photogenerated carriers could be overcome by adding Ru. Meanwhile, the RuO2 particles growing on ZnO surface provided an efficient electron- transfer channel and reduced the recombination rate of photogenerated electron–hole pairs. And the coatings’ surface became less smooth with the addition of Ru, providing more active sites. However, with a Ru content of 12.5 mol%, excessive RuO2 particles wrapped around ZnO surface, hindering the irradiation of ultraviolet light. Though the forbidden band width of ZnO decreased after adding Ru, undersized band gap could accelerate the recombination rate of photogenerated electron–hole pairs in turn. That explained why the electrodes containing 9.375 mol% Ru exhibited the highest degradation rate. process taking place on the interface between the electrodes and electrolyte. Q reflects a constant phase angle element, and CdI refers to the double-layer capacitance [61–63]. According to the results from Fig. 9(c), Rs decreased all the way as the Ru content increased, indicating a reduced internal resistance. The diameter of the semicircle curve in the high-frequency region reflected the charge-transfer resistance, which decreased first, then increased, and reached to the minimum when the Ru content was 9.375 mol%, suggesting the electrodes’ highest separation rate and transfer efficiency. As shown in Fig. 9(d), compared with the impedance spectrum of electrodes without UV irradiation, those under the light exhibited a larger inclination degree at the low frequency region, indicating that the UV irradiation accelerated the ion-diffusion rate. 3. 4 Photoelectrocatalytic degradation of RhB As we can see from the UV–Vis absorption spectra and the removal rate of RhB during PEC degradation process exhibited in Figs. 10(a)–10(f), the strongest absorption peak of RhB located at 554 nm. The peaks’ intensity decreased as the degradation time increased, and the one corresponding to the electrode with 9.375 mol% Ru disappeared first. According to Fig. 10(f), as the Ru content rose, the removal rate increased first, from 71% of the pure ZnO electrode (120 min), and then decreased after reaching to the maximum of 97% (electrode containing 9.375 mol% Ru), indicating that doping appropriate amount of Ru could significantly improve electrodes’ degradation efficiency. The TOC test results shown in Fig. 10(g) In order to further illustrate the effect of Ru content on the degradation rate, the degradation kinetic analysis was carried out under the direction of the following first-order kinetic equation [64]: 0 ln t C C k         (3) (3) where C0 is the initial concentration of the RhB solution (20 mg·L–1), C is the concentration of RhB for a given degradation time, k is the reaction rate constant (min–1), and t is the reaction time. As the Ru content rose, k value increased first, then decreased, and reached www.springer.com/journal/40145 J Adv Ceram 2021, 10(5): 1025–1041 1035 Fig. 10 UV–Vis absorption spectra of RuxZn1–xO/Ti electrodes under PEC degradation with different intervals: (a) 0 mol%, (b) 3.125 mol%, (c) 6.25 mol%, (d) 9.375 mol%, and (e) 12.5 mol%; (f) removal ratio of RhB; (g) TOC retention rate; (h) degradation kinetics; (i) reaction rate constant. Fig. 10 UV–Vis absorption spectra of RuxZn1–xO/Ti electrodes under PEC degradation with different intervals: (a) 0 mol%, (b) 3.125 mol%, (c) 6.25 mol%, (d) 9.375 mol%, and (e) 12.5 mol%; (f) removal ratio of RhB; (g) TOC retention rate; (h) degradation kinetics; (i) reaction rate constant. Table 5 TON and TOF values calculated with the degradation amount of RhB (2 h) Content of Ru4+ (mol%) 0 3.125 6.25 9.375 12.5 TON 0.21 0.27 0.28 0.29 0.27 TOF (h–1) 0.11 0.13 0.14 0.15 0.13 Table 5 TON and TOF values calculated with the degradation amount of RhB (2 h) to the maximum of 0.03 when the content was 9.375 mol%, indicating the electrodes’ highest removal rate. Content of Ru4+ (mol%) 0 3.125 6.25 9.375 12.5 TON 0.21 0.27 0.28 0.29 0.27 TOF (h–1) 0.11 0.13 0.14 0.15 0.13 In order to further confirm the photoelectrocatalytic performance of RuxZn1–xO/Ti electrodes, the degradation amount of RhB (2 h) was adopted to calculate the TON and TOF values, and the calculation equations are shown below [65]: more quickly, which effectively improved the catalytic activity of the electrodes and enhanced the photo- electrocatalytic degradation efficiency. TON = CRhB/CCat (4) TOF = TON/t (5) To make further explanation, the PC and electrocatalysis (EC) degradation tests of RuxZn1–xO/Ti electrodes containing 9.375 mol% Ru were also performed, and the results are shown in Fig. 11. With a degradation time of 120 min, the removal rates of EC, PC, and PEC were 12%, 50%, and 97%, respectively. The much higher PEC efficiency confirmed the synergy effect of EC and PC degradation. According to the test results displayed in Fig. 11(g), after 120 min of degradation, the TOC removal rates for PEC, PC, and EC were 68%, 44%, and 10%, respectively. Compared with EC, the photogenerated electron–hole pairs generated during the PEC process, and the holes could decompose RhB where CRhB represents the concentration of the RhB involved in the reaction (mmol/L), CCat refers to the concentration of the catalyst in the solution (mmol/L), and t is the reaction time (h). The results are listed in Table 5. With the increase of Ru content, both the TON and TOF values increased first and then decreased, and reached the maximum when the Ru content was 9.375%. It is indicated that this electrode had the highest catalytic activity, which was consistent with the RhB catalytic degradation test. It is speculated that adding Ru could enlarge the number of photogenerated electrons produced by ZnO and help transfer electrons www.springer.com/journal/40145 J Adv Ceram 2021, 10(5): 1025–1041 1036 Fig. 11 UV–Vis absorption spectra of RuxZn1–xO/Ti electrodes with 9.375 mol% Ru at different catalytic methods: (a) EC, (b) PC, (c) PEC, (d) removal rate; (e) degradation kinetics; (f) reaction rate constant; (g) TOC retention rate; (h) UV–Vis absorption spectra after 120-min PEC degradation by Ti and RuxZn1–xO/Ti electrodes with 9.375 mol% Ru; (i) PEC removal rate. Fig. Table 5 TON and TOF values calculated with the degradation amount of RhB (2 h) 11 UV–Vis absorption spectra of RuxZn1–xO/Ti electrodes with 9.375 mol% Ru at different catalytic methods: (a) EC, (b) PC, (c) PEC, (d) removal rate; (e) degradation kinetics; (f) reaction rate constant; (g) TOC retention rate; (h) UV–Vis absorption spectra after 120-min PEC degradation by Ti and RuxZn1–xO/Ti electrodes with 9.375 mol% Ru; (i) PEC removal rate. are shown in Fig. 12. As we can see, the electrodes only took 60 and 40 min to remove 96% of MO and 98% of MB, respectively. It is sure that the RuxZn1–xO/ Ti electrodes have good PEC performance and can effectively remove a variety of organic substances. The degradation performance of the material prepared in this study was compared with those reported by other articles, which adopted the same preparation method or similar composition materials, shown in Table 6. We can see that the RuxZn1–xO/Ti electrodes could almost degrade the organics in a relatively shorter time, better than most of other materials containing ZnO. The main reason might be that doping Ru could increase the number of photogenerated electron–hole pairs and their separation rates at the same time. directly, and oxidize hydroxide ions on the electrodes’ surface into hydroxyl radicals, which would oxidize RhB in turn. Compared with PC, PEC was provided with an external power source, and the RuO2 particles on the ZnO surface acting as an electro-transfer channel, offered more active sites for electrocatalysis and helped separate the photogenerated electron–hole pairs. In addition, a bias potential of 2.5 V facilitated the oxygen evolution reaction, providing the oxygen needed to oxidize RhB. It can be seen from Figs. 11(e) and 11(f) that the regression curve basically accorded with first-order kinetics, and the rate constant of PEC (0.030 min–1) was much larger than that of EC (0.001 min–1) and PC (0.006 min–1), confirming the synergy effect of PEC degradation again. As shown in Figs. 11(h) and 11(i), the PEC degradation rate of Ti plate was only 15.3% after 120 min, and therefore, its effect on the RuxZn1–xO/Ti electrodes’ degradation rate could be ignored. 3. 5 Photoelectrocatalytic mechanism A preliminary schematic diagram of the PEC degradation mechanism of RuxZn1–xO/Ti electrodes based on a systematic analysis is presented in Fig. 13. With the addition of Ru, a number of Zn atoms were replaced and the substitutional solid solution was formed MO and MB were adopted to further identify the degradation ability of the RuxZn1–xO/Ti electrode containing 9.375 mol% Ru, and the experiment results www.springer.com/journal/40145 www.springer.com/journal/40145 J Adv Ceram 2021, 10(5): 1025–1041 1037 Fig. 12 UV–Vis absorption spectra of RuxZn1–xO/Ti electrodes containing 9.375 mol% Ru: (a) RhB, (b) MO, (c) MB, and (d) removal rate. Fig. 12 UV–Vis absorption spectra of RuxZn1–xO/Ti electrodes containing 9.375 mol% Ru: (a) RhB, (b) MO, (c) MB, and (d) removal rate Fig. 12 UV–Vis absorption spectra of RuxZn1–xO/Ti electrodes containing 9.375 mol% Ru: (a) RhB, (b) MO, (c) MB, and (d) removal rate. Table 6 Contrastive analysis of degradation performance between materials prepared in this work and other studies Table 6 Contrastive analysis of degradation performance between materials prepared in this work and other studies electron–hole pairs efficiently. Furthermore, those electrons could react with molecular oxygen and generate superoxide radicals, which then react with hydrogen ions to generate hydroxyl radicals in turn [71]. Meanwhile, holes could directly decompose organic dyes and oxidize hydroxide ions into hydroxyl radicals [72], which could degrade RhB into harmless compounds, such as H2O and CO2 (the specific reaction process was shown in Eqs. (6)–(13)) [73,74]. The RuxZn1–xO/Ti electrodes combined the advantages of ZnO (excellent photocatalytic ability) and RuO2 (good electron- transfer and electrocatalytic ability), which achieved a synergistic effect making the composite electrodes possess a degradation ability more powerful than the mere sum of photocatalysis and electrocatalysis. studies Reported material Organics Degradation time (min) Removal rate Ref. RuxZn1–xO/Ti RhB 120 97% This work RuxZn1–xO/Ti MO 60 96% This work RuxZn1–xO/Ti Mb 40 98% This work Ag/ZnO Phenol 180 100% [64] IrxZn1–x O/Ti RhB 80 99.4% [66] Ti/RuxTi1–x O MO 150 68% [61] Y/V/ZnO RhB 180 89% [39] CdO/ZnO 4-nitrophenol 60 60% [67] Cu/GZnO MO 80 89.1% [68] accordingly, which not only brought impurity levels to the forbidden band of ZnO, but also reduced the conduction band from E1 to E3, and increased the valence band from E2 to E4. That reduced the forbidden band width of ZnO and enlarged its light absorption range substantially, meaning that it could generate more photo-generated electron–hole pairs. 4 Conclusions [1] Weber EJ, Adams RL. Chemical- and sediment-mediated reduction of the azo dye disperse blue 79. Environ Sci Technol 1995, 29: 1163–1170. RuxZn1–xO/Ti electrodes with excellent PEC performance were prepared through simple thermal decomposition method. It has been found that Ru-doped ZnO exhibited a smaller forbidden band width as a number of Zn2+ and Ru4+ exchanged places in ZnO lattice, and the amount of photogenerated carriers increased accordingly. The rest of Ru existed on the ZnO nanorods’ surface in the form of RuO2 particles acting as an electron-transfer channel, together with the external bias, and it propelled photogenerated electrons to transfer to the counter electrode and reduced the recombination rate of electron–hole pairs. Meanwhile, with the addition of Ru, the grain size of ZnO nanorods decreased and the coatings’ surface became less smooth, leading to larger specific surface area as well as more active sites. The accessory oxygen evolution reaction could also accelerate the oxidation of the dye. However, with excessive Ru content, RuO2 particles could wrap ZnO nanorods around and hinder the irradiation of ultraviolet light, reducing the PEC efficiency. Indeed, the band gap decreased with the addition of Ru, but undersized band gap could accelerate the recombination rate of photogenerated electron–hole pairs. That explained why the electrodes with 9.375 mol% rather than those containing 12.5 mol% Ru exhibited the best PEC performance. [2] Zhang FL, Zhao JC, Shen T, et al. TiO2-assisted photodegradation of dye pollutants II. Adsorption and degradation kinetics of eosin in TiO2 dispersions under visible light irradiation. Appl Catal B: Environ 1998, 15: 147–156. [3] Maleki A. Fe3O4/SiO2 nanoparticles: An efficient and magnetically recoverable nanocatalyst for the one-pot multicomponent synthesis of diazepines. Tetrahedron 2012, 68: 7827–7833. [4] Maleki A. One-pot multicomponent synthesis of diazepine derivatives using terminal alkynes in the presence of silica- supported superparamagnetic iron oxide nanoparticles. Tetrahedron Lett 2013, 54: 2055–2059. [5] Afzaal M, Malik MA, O’Brien P. Preparation of zinc containing materials. New J Chem 2007, 31: 2029–2040. [6] Kamat PV. Meeting the clean energy demand: Nanostructure architectures for solar energy conversion. J Phys Chem C 2007, 111: 2834–2860. [7] Roshan S A, Joseph C, Ittyachen MA. Growth and characterization of a new metal-organic crystal: Potassium thiourea bromide. Mater Lett 2001, 49: 299–302. [8] Vayssieres L. Growth of arrayed nanorods and nanowires of ZnO from aqueous solutions. Adv Mater 2003, 15: 464–466. [9] Maleki A. 3. 5 Photoelectrocatalytic mechanism In addition, RuO2 nanoparticles attaching on the surface of ZnO nanorods could provide effective electron transport channels and electrocatalytically active sites [69,70]. Under the effect of an external electric field, photogenerated electrons could be quickly transferred to the counter electrode and separate photogenerated accordingly, which not only brought impurity levels to the forbidden band of ZnO, but also reduced the conduction band from E1 to E3, and increased the valence band from E2 to E4. That reduced the forbidden band width of ZnO and enlarged its light absorption range substantially, meaning that it could generate more photo-generated electron–hole pairs. In addition, RuO2 nanoparticles attaching on the surface of ZnO nanorods could provide effective electron transport channels and electrocatalytically active sites [69,70]. Under the effect of an external electric field, photogenerated electrons could be quickly transferred to the counter electrode and separate photogenerated + ZnO ZnO h e h      (6) 2 2 e O O     (7) 2 2 2 O 2H 2e H O       (8) 2 2 2 2 H O O OH OH O         (9) h OH OH     (10) + 2 2 h / OH RhB CO H O     (11) 2 2 2 RuO h H O RuO [ OH] H        (12) 2 2 2 2 RuO [ OH] RhB RuO CO H O      (13) + ZnO ZnO h e h      (6) 2 2 e O O     (7) 2 2 2 O 2H 2e H O       (8) 2 2 2 2 H O O OH OH O         (9) h OH OH     (10) + 2 2 h / OH RhB CO H O     (11) 2 2 2 RuO h H O RuO [ OH] H        (12) 2 2 2 2 RuO [ OH] RhB RuO CO H O      (13) (6) www.springer.com/journal/40145 J Adv Ceram 2021, 10(5): 1025–1041 1038 Fig. 13 Schematic microstructure model and PEC mechanism of RuxZn1–xO/Ti electrodes. Fig. 3. 5 Photoelectrocatalytic mechanism 13 Schematic microstructure model and PEC mechanism of RuxZn1–xO/Ti electrodes. 4 Conclusions One-pot three-component synthesis of pyrido[2′,1′: 2,3]imidazo[4,5-c]isoquinolines using Fe3O4@SiO2–OSO3H as an efficient heterogeneous nanocatalyst. RSC Adv 2014, 4: 64169–64173. Acknowledgements [10] Kumar SG, Rao KSRK. Zinc oxide based photocatalysis: Tailoring surface-bulk structure and related interfacial charge carrier dynamics for better environmental applications. RSC Adv 2015, 5: 3306–3351. The work was supported by the National Natural Science Foundation of China (83418083) and the Natural Science Foundation of Fujian Province (2019J01230). [11] Kim KJ, Kreider PB, Choi C, et al. Visible-light-sensitive [11] Kim KJ, Kreider PB, Choi C, et al. Visible-light-sensitive www.springer.com/journal/40145 J Adv Ceram 2021, 10(5): 1025–1041 1039 Na-doped p-type flower-like ZnO photocatalysts synthesized via a continuous flow microreactor. RSC Adv 2013, 3: 12702–12710. particles. Appl Phys A 2020, 126: 1–17. [25] Maleki A, Kari T. Novel leaking-free, green, double core/shell, palladium-loaded magnetic heterogeneous nanocatalyst for selective aerobic oxidation. Catal Lett 2018, 148: 2929–2934. [12] Maleki A. Green oxidation protocol: Selective conversions of alcohols and alkenes to aldehydes, ketones and epoxides by using a new multiwall carbon nanotube-based hybrid nanocatalyst via ultrasound irradiation. Ultrason Sonochem 2018, 40: 460–464. [26] Ashebir ME, Tesfamariam GM, Nigussie GY, et al. Structural, optical, and photocatalytic activities of Ag-doped and Mn-doped ZnO nanoparticles. J Nanomater 2018, 2018: 1–9. [13] Varzi Z, Maleki A. Design and preparation of ZnS– ZnFe2O4: A green and efficient hybrid nanocatalyst for the multicomponent synthesis of 2,4,5-triaryl-1H-imidazoles. Appl Organomet Chem 2019, 33: e5008. [27] Qiu YF, Fan HB, Tan GP, et al. Effect of nitrogen doping on the photo-catalytic properties of nitrogen doped ZnO tetrapods. Mater Lett 2014, 131: 64–66. [14] Lee JC, Park S, Park HJ, et al. Photocatalytic degradation of TOC from aqueous phenol solution using solution combusted ZnO nanopowders. J Electroceramics 2009, 22: 110–113. [28] Ditta MA, Farrukh MA, Ali S, et al. X-ray peak profiling, optical parameters and catalytic properties of pure and CdS doped ZnO–NiO nanocomposites. Russ J Appl Chem 2017, 90: 151–159. [15] Khassin AA, Yurieva TM, Kaichev VV, et al. Metal- support interactions in cobalt–aluminum co-precipitated catalysts: XPS and CO adsorption studies. J Mol Catal A: Chem 2001, 175: 189–204. [29] Maleki A, Kari T, Aghaei M. Fe3O4@SiO2@TiO2–OSO3H: An efficient hierarchical nanocatalyst for the organic quinazolines syntheses. J Porous Mater 2017, 24: 1481– 1496. [30] Etacheri V, Roshan R, Kumar V. Mg-doped ZnO nanoparticles for efficient sunlight-driven photocatalysis. ACS Appl Mater Interfaces 2012, 4: 2717–2725. [16] Maleki A, Varzi Z, Hassanzadeh-Afruzi F. Preparation and characterization of an eco-friendly ZnFe2O4@alginic acid nanocomposite catalyst and its application in the synthesis of 2-amino-3-cyano-4H-pyran derivatives. Polyhedron 2019, 171: 193–202. Acknowledgements [31] Soltaninejad V, Maleki A. A green, and eco-friendly bionanocomposite film (poly(vinyl alcohol)/TiO2/chitosan/ chlorophyll) by photocatalytic ability, and antibacterial activity under visible-light irradiation. J Photochem Photobiol A: Chem 2021, 404: 112906. [17] Ma CH, Liu ZF, Cai QJ, et al. ZnO photoelectrode simultaneously modified with Cu2O and Co–Pi based on broader light absorption and efficiently photogenerated carrier separation. Inorg Chem Front 2018, 5: 2571–2578. [32] Han JS, An HJ, Kim TW, et al. Effect of structure- controlled ruthenium oxide by nanocasting in electrocatalytic oxygen and chlorine evolution reactions in acidic conditions. Catalysts 2019, 9: 549. [18] Zhang SC, Liu ZF, Ruan MN, et al. Enhanced piezoelectric- effect-assisted photoelectrochemical performance in ZnO modified with dual cocatalysts. Appl Catal B: Environ 2020, 262: 118279. [33] Peng F, Zhou CM, Wang HJ, et al. The role of RuO2 in the electrocatalytic oxidation of methanol for direct methanol fuel cell. Catal Commun 2009, 10: 533–537. [19] Maleki A, Hajizadeh Z, Sharifi V, et al. A green, porous and eco-friendly magnetic geopolymer adsorbent for heavy metals removal from aqueous solutions. J Clean Prod 2019, 215: 1233–1245. [34] Yue H, Xue LZ, Chen F. Efficiently electrochemical removal of nitrite contamination with stable RuO2–TiO2/Ti electrodes. Appl Catal B: Environ 2017, 206: 683–691. [20] Maleki A, Mohammad M, Emdadi Z, et al. Adsorbent materials based on a geopolymer paste for dye removal from aqueous solutions. Arab J Chem 2020, 13: 3017– 3025. [35] Taheri-Ledari R, Valadi K, Maleki A. High-performance HTL-free perovskite solar cell: An efficient composition of ZnO NRs, RGO, and CuInS2 QDs, as electron-transporting layer matrix. Prog Photovolt: Res Appl 2020, 28: 956–970. [21] Wang XQ, Zhou YN, Li R, et al. Removal of Hg0 from a simulated flue gas by photocatalytic oxidation on Fe and Ce co-doped TiO2 under low temperature. Chem Eng J 2019, 360: 1530–1541. [36] Dalal B, Sarkar B, De SK. Itinerant to localized electronic behavior in phase segregated ruthenates. J Alloys Compd 2016, 667: 248–254. [22] Zhao SW, Zuo HF, Guo YR, et al. Carbon-doped ZnO aided by carboxymethyl cellulose: Fabrication, photoluminescence and photocatalytic applications. J Alloys Compd 2017, 695: 1029–1037. [37] Mariolacos K. The role of electronegativity in solid solution formation: An addendum. N Jb Miner Mh 2003, 2003: 215–221. [38] Liu ZF, E L, Ya J, et al. Growth of ZnO nanorods by aqueous solution method with electrodeposited ZnO seed layers. Appl Surf Sci 2009, 255: 6415–6420. Acknowledgements Design and preparation of a ternary composite of graphene oxide/carbon dots/ polypyrrole for supercapacitor application: Importance and unique role of carbon dots. Carbon 2017, 115: 134–146. [46] Kleiman-Shwarsctein A, Laursen AB, Cavalca F, et al. A general route for RuO2 deposition on metal oxides from RuO4. Chem Commun: Camb 2012, 48: 967–969. [63] Shao YQ, Yi ZY, He C, et al. Effects of annealing temperature on the structure and capacitive performance of nanoscale Ti/IrO2–ZrO2 electrodes. J Am Ceram Soc 2015, 98: 1485–1492. [47] Wang FZ, Xu Q, Tan ZA, et al. Efficient polymer solar cells with a solution-processed and thermal annealing-free RuO2 anode buffer layer. J Mater Chem A 2014, 2: 1318–1324. [64] Vaiano V, Matarangolo M, Murcia JJ, et al. Enhanced photocatalytic removal of phenol from aqueous solutions using ZnO modified with Ag. Appl Catal B: Environ 2018, 225: 197–206. [48] Ananth A, Dharaneedharan S, Gandhi MS, et al. Novel RuO2 nanosheets–facile synthesis, characterization and application. Chem Eng J 2013, 223: 729–736. [65] Costentin C, Drouet S, Robert M, et al. Turnover numbers, turnover frequencies, and overpotential in molecular catalysis of electrochemical reactions. Cyclic voltammetry and preparative-scale electrolysis. J Am Chem Soc 2012, 134: 11235–11242. [49] Cox PA, Goodenough JB, Tavener PJ, et al. The electronic structure of Bi2−xGdxRu2O7 and RuO2: A study by electron spectroscopy. J Solid State Chem 1986, 62: 360–370. [50] Kwak I, Kwon IS, Kim J, et al. IrO2–ZnO hybrid nanoparticles as highly efficient trifunctional electrocatalysts. J Phys Chem C 2017, 121: 14899–14906. [66] Feng KK, Lin YT, Guo J, et al. Study on the enhanced electron-hole separation capability of IrxZn1–xO/Ti electrodes with high photoelectrocatalysis efficiency. J Hazard Mater 2020, 393: 122488. [51] Jing LQ, Xu ZL, Shang J, et al. The preparation and characterization of ZnO ultrafine particles. Mater Sci Eng: A 2002, 332: 356–361. [52] Karamat S, Rawat RS, Tan TL, et al. Exciting dilute magnetic semiconductor: Copper-doped ZnO. J Supercond Nov Magn 2013, 26: 187–195. [67] Mostafa AM, Menazea AA. Laser-assisted for preparation ZnO/CdO thin film prepared by pulsed laser deposition for catalytic degradation. Radiat Phys Chem 2020, 176: 109020. [53] Kostova B, Konstantinov L. Factors determining optical absorption of variously doped single crystals of Bi12SiO20. J Phys: Conf Ser 2010, 253: 012029. [68] Beura R, Rajendran S, Gracia Pinilla MA, et al. Enhanced photo-induced catalytic activity of Cu ion doped ZnO–graphene ternary nanocomposite for degrading organic dyes. Acknowledgements [23] Zhao W, Zhong Q, Pan YX, et al. Systematic effects of S-doping on the activity of V2O5/TiO2 catalyst for low-temperature NH3–SCR. Chem Eng J 2013, 228: 815–823. [39] Alam U, Khan A, Raza W, et al. Highly efficient Y and V co-doped ZnO photocatalyst with enhanced dye sensitized visible light photocatalytic activity. Catal Today 2017, 284: 169–178. [24] Kayani ZN, Anjum M, Riaz S, et al. Role of Mn in biological, optical, and magnetic properties ZnO nano- www.springer.com/journal/40145 J Adv Ceram 2021, 10(5): 1025–1041 1040 study. J Magn Magn Mater 2009, 321: 2547–2549. study. J Magn Magn Mater 2009, 321: 2547–2549. [40] Gómez-Pozos H, González-Vidal JL, Torres GA, et al. Chromium and ruthenium-doped zinc oxide thin films for propane sensing applications. Sensors: Basel 2013, 13: 3432–3444. [57] Wu HC, Peng YC, Chen CC. Effects of Ga concentration on electronic and optical properties of Ga-doped ZnO from first principles calculations. Opt Mater 2013, 35: 509–515. [41] Khan MAM, Kumar S, Alhazaa AN, et al. Modifications in structural, morphological, optical and photocatalytic properties of ZnO: Mn nanoparticles by sol–gel protocol. Mater Sci Semicond Process 2018, 87: 134–141. [58] Lakel S, Elhamra F, Almi K, et al. First-principles investigation of electronic and optical properties and thermodynamic stability of Zn1−xBexO semiconductor alloy. Mater Sci Semicond Process 2015, 40: 803–810. [42] Son HS, Choi NJ, Kim KB, et al. Al-doped ZnO seed layer-dependent crystallographic control of ZnO nanorods by using electrochemical deposition. Mater Res Bull 2016, 82: 50–54. [59] Tauc J. Optical properties and electronic structure of amorphous germanium. Physica Status Solidi 1966, 3: 37– 46. [60] Liu HY, Zeng F, Lin YS, et al. Correlation of oxygen vacancy variations to band gap changes in epitaxial ZnO thin films. Appl Phys Lett 2013, 102: 181908. [43] Deraz NM. Effects of heat treatment on physicochemical properties of cerium based nickel system. J Anal Appl Pyrolysis 2012, 95: 56–60. [61] Chen ZJ, Zhu JQ, Zhang S, et al. Influence of the electronic structures on the heterogeneous photoelectrocatalytic performance of Ti/RuxSn1–xO2 electrodes. J Hazard Mater 2017, 333: 232–241. [44] Deraz NM. Effect of NiO content on structural, surface and catalytic characteristics of nano-crystalline NiO/CeO2 system. Ceram Int 2012, 38: 747–753. [45] Chan HYH, Takoudis CG, Weaver MJ. High-pressure oxidation of ruthenium as probed by surface-enhanced Raman and X-ray photoelectron spectroscopies. J Catal 1997, 172: 336–345. [62] Zhang X, Wang JM, Liu J, et al. Open Access This article is licensed under a Creative Commons www.springer.com/journal/40145 Acknowledgements J Water Process Eng 2019, 32: 100966. [54] Bendavid LI, Carter EA. First principles study of bonding, adhesion, and electronic structure at the Cu2O(111)/ ZnO(101¯0) interface. Surf Sci 2013, 618: 62–71. [69] Boudenne JL, Cerclier O, Galéa J, et al. Electrochemical oxidation of aqueous phenol at a carbon black slurry electrode. Appl Catal A: Gen 1996, 143: 185–202. [55] Shao YQ, Chen ZJ, Zhu JQ, et al. Relationship between electronic structures and capacitive performance of the electrode material IrO2–ZrO2. J Am Ceram Soc 2016, 99: 2504–2511. [70] Comninellis C. Electrocatalysis in the electrochemical conversion/combustion of organic pollutants for waste water treatment. Electrochimica Acta 1994, 39: 1857– 1862. [56] Hernández RG, Pérez WL, Rodríguez MJA. Electronic structure and magnetism in Ni0.0625Zn0.9375O: An ab initio [71] Pelegrini R, Peralta-Zamora P, de Andrade AR, et al. www.springer.com/journal/40145 J Adv Ceram 2021, 10(5): 1025–1041 1041 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. Electrochemically assisted photocatalytic degradation of reactive dyes. Appl Catal B: Environ 1999, 22: 83–90. Electrochemically assisted photocatalytic degradation of reactive dyes. Appl Catal B: Environ 1999, 22: 83–90. [72] Konstantinou IK, Albanis TA. TiO2-assisted photocatalytic degradation of azo dyes in aqueous solution: Kinetic and mechanistic investigations: A review. Appl Catal B: Environ 2004, 49: 1–14. 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. [73] Himebaugh RR, Smith MJ. Semi-micro tube method for chemical oxygen demand. Anal Chem 1979, 51: 1085–1087. [74] Wei L, Zhu H, Mao XH, et al. Electrochemical oxidation process combined with UV photolysis for the mineralization of nitrophenol in saline wastewater. Sep Purif Technol 2011, 77: 18–25. To view a copy of this licence, visit http://creativecommons. org/licenses/by/4.0/. Open Access This article is licensed under a Creative Commons www.springer.com/journal/40145
https://openalex.org/W2470088698	https://isprs-archives.copernicus.org/articles/XLI-B1/803/2016/isprs-archives-XLI-B1-803-2016.pdf	English	null	USING UAV TO DETECT SHORELINE CHANGES: CASE STUDY – POHRANOV POND, CZECH REPUBLIC	The international archives of the photogrammetry, remote sensing and spatial information sciences/International archives of the photogrammetry, remote sensing and spatial information sciences	2,016	cc-by	4,456	ABSTRACT: The paper describes utilization of an unmanned aerial vehicle (UAV) for detection of changes of shorelines. UAV is used as a cheep and on- demand available possibility how to collect remotely sensed data. Its utilization is limited by legal regulations and weather conditions. Paper deals with utilization of UAV for monitoring small water area and particularly study of changes of shorelines. Study contains other methods of classification. After classification will be data processed for next calculations. Indices regarding shoreline changes are in the study also. In conclusion, study contains obtainment findings and encouragement for the future. 1. INTRODUCTION first, a suitable way of utilization of an UAV for data collection is proposed. Next, collected data are processed to calculate shoreline changes. Only a part of the shoreline of a pound is used to demonstrate the proposed procedure within the case study. Studying changes of landscape is a very important issue in the modern society. It means that the society is interested in new findings which can help to understand how nature and its particular elements exist and behave. Understanding functions, relationships and rules can support landscape management and further sustainable development e.g. prevention of devastating impacts of floods. Monitoring of drought (which was the key problem of this summer season) is another example. Thus, many studies (e.g. Bortoleto et al., 2016; Qindong and Shengyan, 2016; Kotzee and Reyers, 2016; Dobešová et al. 2014) are focused on landscape changes. Some studies are particularly focused on water bodies (e.g. Gallop et al., 2015; Pechanec et al., 2015; Heine et al., 2015). The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLI-B1, 2016 XXIII ISPRS Congress, 12–19 July 2016, Prague, Czech Republic The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLI-B1, 2016 XXIII ISPRS Congress, 12–19 July 2016, Prague, Czech Republic Commission ICWG I/Vb KEY WORDS: Landscape, Pohranov pond, landscape indices, UAV, change the landscape 2. PREVIOUS RESEARCHES Detection of landscape changes is a very important part of landscape management. Remote sensing (including UAVs) is used to collect up-to-date data in various parts of electromagnetic spectrum. Data are processed by many different methods according to their content and expected results (e.g. Heine, 2015; Li et al., 2013; Nath and Deb, 2010). 2.1 Landscape changes Many researches focused on landscape changes have been published. Qindong and Shengyan (2016) analyzed landscape pattern changes in monitored period (1990 – 2013) and identified driving sources during that period. They used various landscape indices to calculate landscape changes. Remote sensing is very often used as a source of data for observation of landscape and terrain. There are many issues why this method is more reasonable than in situ observations, sampling and measurements and land surveying. It means the costs are lower (namely in case when bigger areas are monitored), accuracy and spatial resolution is adequate to aims of studies and finally data measured in various parts of electromagnetic spectrum are available. The last advantage is important for researches based on thermal imagery, various indices, etc. Satellites or aerial imagery is available for monitoring larger areas. Unmanned aerial vehicles (UAV) are increasingly used to monitor small areas, e.g. small water bodies (ponds). UAVs can provide results faster and usually with higher spatial resolution. Bortoleto et al. (2016) focused on index of restoration in landscapes. They proposed a mathematical index named SIR that describes suitability of individual habitat patches for restoration within a landscape. A model based on the SIR used a map of distance classes among fragments and a map of habitat quality established according to each land cover category. SIR was obtained as a result of calculation. USING UAV TO DETECT SHORELINE CHANGES: CASE STUDY – POHRANOV POND, CZECH REPUBLIC I. Čermáková a, J. Komárková a, P. Sedlák a, * a University of Pardubice, Faculty of Economics and Administration, 53210 Studentská 95 Pardubice, Czech Republic - st32689@student.upce.cz, (jitka.komarkova, pavel.sedlak)@upce.cz niversity of Pardubice, Faculty of Economics and Administration, 53210 Studentská 95 Pardubice, Czech Republic - st32689@student.upce.cz, (jitka.komarkova, pavel.sedlak)@upce.cz Commission ICWG I/Vb This contribution has been peer-reviewed. doi:10.5194/isprsarchives-XLI-B1-803-2016 2.2 Water bodies and shoreline changes Kotzee and Reyers (2016) dealt with floods in South Africa. Study was focused on analyses of three municipalities affected by a flood in the monitored area (South Africa). Principal component analysis was used to explore level of disaster planning, mitigation and next potential occurrences or increased number of occurrences of floods. Aim of the study is to extend research of the Faculty of Chemical Technology of University of Pardubice. This research is focused on using remote sensing for studying chemical processes and composition of various types of soils, e.g. of water sediments. Main part of this case study is a study of small water bodies which are monitored by an UAV. At This contribution has been peer-reviewed. doi:10.5194/isprsarchives-XLI-B1-803-2016 This contribution has been peer-reviewed. doi:10.5194/isprsarchives-XLI-B1-803-2016 803 The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLI-B1, 2016 XXIII ISPRS Congress, 12–19 July 2016, Prague, Czech Republic The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLI-B1, 2016 XXIII ISPRS Congress, 12–19 July 2016, Prague, Czech Republic The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLI-B1, 2016 XXIII ISPRS Congress, 12–19 July 2016, Prague, Czech Republic The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLI-B1, 2016 XXIII ISPRS Congress, 12–19 July 2016, Prague, Czech Republic Fig. 1: Shoreline changes between 1990- 2014, analysis provided in DSAS, source: Misra and Balaji (2015) Many articles were particularly focused on various water bodies and their shorelines. Gallop et al. (2015) studied long- term changes of shoreline in southwestern Australia. Shoreline changes were observed in the period of 34 years. Aerial photographs were used as a data source. It was realized that over 96 % beaches straightened during the time period. Figure 1 shows digitized vegetation lines and shore lines from 1974 to 2008. Empirical Orthogonal Function (EOF) helped to explain 45 % of variability between transects of beach. In this case, there were large changes in shoreline position that did not correspond to the indices of annual wave conditions. In future, there is big potential for improvement of this research by remote sensing and increased utilization of fuzzy-based computer algorithms. Fig. 1: Vegetation lines and shore lines from 1974 to 2008, source: Gallop et al. (2015) Fig. 2.3 Unmanned aerial vehicle data with a suitable resolution. UAV represents a more suitable way of data collection in this case. Fahlstrom and Gleason (2012) classified aircrafts that fly without pilots into three basic types. They are: UAV, remotely piloted vehicles (RPVs) and drones, respectively. All are unmanned so the name “unmanned aerial vehicle” or an UAV can be thought of as the most general term. Some people use terms RPV and UAV interchangeably. But, the remotely piloted vehicle is fully piloted or controlled from a remotely located position. On the other side, UAV may perform autonomous or pre-programmed missions. So, an RPV is always an UAV but an UAV need not always be an RPV. UAV provides data in necessary high distinction and costs are also lower. Tarot 690 is used for monitoring pond Pohranov. It can be characterised as follows: airscrew gear; 6 gears; 0.985 m average of impeller; 0.35 m height, 70 km/h maximum speed. This UAV has the following restrictions (conditions under which it cannot be used): temperature under -10 ºC; wind fasters than 10 m.s-1; fog with visibility under 100 m; creation frost on the airscrew; drizzle, rain and snowfall. Figure 4 shows the UAV used for monitoring Pohranov pond. Rathinam S. et al. (2007) used an UAV to detect and track a river by means of visible and near infra-red sensor Rathinam S. et al. (2007) used an UAV to detect and track a river by means of visible and near infra-red sensor Fig. 4: UAV used for monitoring pond Pohranov, source: authors Shorelines can be used to autonomously navigate UAVs as proposed by Baker (2010). A near-infrared sensor allows the vision system to distinguish water from land. The vision system then issues commands to the autopilot to follow the coastline or the riverbank. 2.2 Water bodies and shoreline changes doi:10.5194/isprsarchives-XLI-B1-803-2016 804 The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLI-B1, 2016 XXIII ISPRS Congress, 12–19 July 2016, Prague, Czech Republic The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLI-B1, 2016 XXIII ISPRS Congress, 12–19 July 2016, Prague, Czech Republic The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLI-B1, 2016 XXIII ISPRS Congress, 12–19 July 2016, Prague, Czech Republic The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLI-B1, 2016 XXIII ISPRS Congress, 12–19 July 2016, Prague, Czech Republic 3.3 Data processing and visualization Area of interest is located close to the city of Pardubice, in the Czech Republic. The case study studies part of a shoreline of the Pohranov pond, close to the municipality of Pohranov (see Figure 3.). Size of the pond is 0.4 km2 (Pardubický kraj, 2015). Video collected by the UAV camera is in the visible part of the spectrum only. Video provided from UAV must be at first processed to create one image from each observation. Primarily, images must be selected and created from the video. Freely available software Free Video to JPG Converter is used for this step. Merging all collected images into one picture is the next step. Image Composite Editor (freely available) is used for this step. A mosaic created from images is minimally distorted because only central parts are selected for merging. The central parts of images are insusceptible to distortion. The resulted images represent our monitored area and changes during monitoring time period. Georeferencing is the next step. Orthophoto data set coming from State Administration of Land Surveying and Cadastre (ČÚZK) is used as a reference data set. The data set is available in geoportal and it is distributed by web map service (WMS). ArcGIS for Desktop (version 10.2) is used for georeferencing because it supports both the required function and WMS services. Georeferencing with RMS = 0.012103 is considered as successful. Fig. 3: Placement of the city of Pardubice in the Czech Republic, source: authors Fig. 3: Placement of the city of Pardubice in the Czech Republic, source: authors 3. CASE STUDY The case study is focused on utilization of an UAV for monitoring of a small water area. The particular aim is to delineate a part of a shoreline of the chosen pond. Because of UAV utilization, there is no multi-spectral data available, so many indices used to detect landscape changes cannot be used. UAV is equipped with a common DJI video camera because no professional camera was available. A fast and cheap data collection with a commonly available equipment is the main objective of this research. NDWI cannot be used as well. This is the reason why landscape indices are used in the case study. Fig. 4: UAV used for monitoring pond Pohranov, source: authors Measurements must be done several times in different days to obtain a short time series. Time horizons were selected according to the above listed weather conditions and to cover a longer time period, namely: 7. 7. 2015, 18. 7. 2015, 23. 8. 2015 and 2. 11. 2015. Height of flight is 80 m (the height was set up in the software of UAV before flight) for all flights. Measurements must be done several times in different days to obtain a short time series. Time horizons were selected according to the above listed weather conditions and to cover a longer time period, namely: 7. 7. 2015, 18. 7. 2015, 23. 8. 2015 and 2. 11. 2015. Height of flight is 80 m (the height was set up in the software of UAV before flight) for all flights. 2.2 Water bodies and shoreline changes 1: Shoreline changes between 1990- 2014, analysis provided in DSAS, source: Misra and Balaji (2015) Water erosion of the river Trkmanka shoreline (a small stream in South Moravia, the Czech Republic) was studied by Pechanec et al. (2015). They focused on erosion susceptibility and shoreline changes because they assumed that water erosion destructively influences the soil surface. This method provides maximum volume of soil erosion caused by water. Tamborski et al. (2015) monitored Long Island in New York. The research was focused on submarine water discharge. Landsat data (TIR) could not be used because data was unable to resolve anomalies. Thus, thermal infrared images were used instead of TIR data. Data was provided by aerial vehicle and then it was analysed by water temperature. Study of shoreline was done manually, i.e. water was sampled near the beach and samples were analysed in a laboratory. Fig. 1: Vegetation lines and shore lines from 1974 to 2008, source: Gallop et al. (2015) Studies Misra and Balaji (2015); Adarsa et al. (2015) and Nandi et al. (2015) used Digital Shoreline Analysis System (DSAS) for detection of changes of shoreline in a monitored area. DSAS is a special extension which is focused on shoreline analysis. This software can calculate the rate of shoreline change. Example from this study is available in Figure 2. In the study of Pacheco et al. (2015), bathymetry for shallow water was primary proposed to monitor both beach and shoreface. LIDAR data (Light Detection and Ranging) were used. Depth-retrieval algorithm was proposed to process data including bathymetry based on LIDAR data. Several studies were directly focused on small water bodies, e.g. Heine et al. (2015), Zlinsky et al. (2011) and Works Jr and Gilmer (1976). Normalized Difference Water Index (NDWI) was proposed by McFeeters (1996) to delineate open water features and enhance their presence in remotely-sensed digital imagery. The NDWI uses reflected near-infrared radiation and visible green light to enhance the presence of water bodies and to eliminate the presence of soil and terrestrial vegetation features. This contribution has been peer-reviewed. doi:10.5194/isprsarchives-XLI-B1-803-2016 This contribution has been peer-reviewed. 3.2 Data collection Table 1: Shoreline changes in metres and percentage, source: authors Table 1: Shoreline changes in metres and percentage, source: authors Various indices can be used for calculation of shoreline changes. Because only data in the visible part of spectrum is available, only the most often used landscape indices are used in the case study. Fig. 6: Supervised classification in 7.7.2015, source: authors The simplest index regarding changes in the landscape is Coefficient of the Ecologic Stability (Kes) by Černohouz (2011). Kes calculates changes by data (areas) dividing on relatively stability and relatively instability: Kes = 𝑆 𝐿 where S = area of relatively stability (in metres), L = area of relatively instability (in metres). Resulting coefficient value is between zero and infinity. Nearly to zero value means that the area is ecological unstable, value higher more than three means ecological stability which do not need intervention of human beings. Fig. 6: Supervised classification in 7.7.2015, source: authors Fig. 7: Unsupervised classification in 7.7.2015, source: authors It is necessary to choose stability observation before calculation. First observation is chosen as a stabile one. After calculation it is found that both observations are stable. 16. 7. 2015 is Kes 7.38 and 23. 8. 2015 is Kes 13.63. It means that the area in the drought state is still stabile but there is a big fluctuation. So it is going to be monitored in next years. Černohouz (2011) also used index Kes modified by Agroproject. This index is calculated in percentage. Kes = 1.5∗𝐴+𝐵+0.5∗𝐶 0.2∗𝐷+0.8∗𝐸 3.2 Data collection Shorelines definition is the following step of all images processing. ArcGIS for Desktop is again used. The polylines The pond is surrounded by forest. It means that in situ observation is difficult. Satellite imaginary does not provide This contribution has been peer-reviewed. doi:10.5194/isprsarchives-XLI-B1-803-2016 This contribution has been peer-reviewed. doi:10.5194/isprsarchives-XLI-B1-803-2016 805 The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLI-B1, 2016 XXIII ISPRS Congress, 12–19 July 2016, Prague, Czech Republic The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLI-B1, 2016 XXIII ISPRS Congress, 12–19 July 2016, Prague, Czech Republic The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLI-B1, 2016 XXIII ISPRS Congress, 12–19 July 2016, Prague, Czech Republic are manually created in a form of new shapefiles (see Figure 5) are manually created in a form of new shapefiles (see Figure 5) Only three observations are used for indices calculation, because during the last observation the pond was drain (fish out) and it is a long time from the previous observation. Drain pond is available in Figure 8. Fig. 5: Define shorelines for every monitoring, source: authors Fig. 8: Launched pond Pohranov in 2.11.2015, source: authors Fig. 8: Launched pond Pohranov in 2.11.2015, source: authors Firstly, changes of size of shorelines in monitored time period are calculated. In the Table 1, there are visible changes in metres (Shape_change) and percentage (Shape_change_%) in monitored time period. The values regarding length are extracted from the software, values regarding change are calculated by authors. Fig. 5: Define shorelines for every monitoring, source: authors Extension of ArcGIS for Desktop – Image Analysis, is used for studying shoreline, and particularly their changes. This extension is used as a tool for supervised and unsupervised classification also. Supervised classification (Seed tool) and next creating polyline is available (see Figure 6). Unsupervised classification is available in the way shown by Figure 7. An approximate scale of figures 5,6 and 7 is 1 : 785 000 000. Next, resulting polygons are converted into polylines. Polylines must be repaired in both cases because shadows are visible in the images. Irregular texture is visible in both images. It decreases quality of classifications. In this case images must be repaired by observer. So, polylines created by observer are used for the following calculation of indices (Figure 5). Kes = 1.5∗𝐴+𝐵+0.5∗𝐶 0.2∗𝐷+0.8∗𝐸 Fig. 7: Unsupervised classification in 7.7.2015, source: authors This contribution has been peer-reviewed. doi:10.5194/isprsarchives-XLI-B1-803-2016 This contribution has been peer-reviewed. doi:10.5194/isprsarchives-XLI-B1-803-2016 806 The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLI-B1, 2016 XXIII ISPRS Congress, 12–19 July 2016, Prague, Czech Republic The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLI-B1, 2016 XXIII ISPRS Congress, 12–19 July 2016, Prague, Czech Republic The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLI-B1, 2016 XXIII ISPRS Congress, 12–19 July 2016, Prague, Czech Republic The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLI-B1, 2016 XXIII ISPRS Congress, 12–19 July 2016, Prague, Czech Republic A = percentage of area with the 5th level of quality (the best, stable system) methods how to observe changes of landscape. Importance of this method increases because there are many different possibilities of utilization of data collected by UAVs. The next important issue is availability of measurements – utilization of UAVs is limited by weather conditions only. B = percentage of area with the 4th level of quality C = percentage of area with the 3rd level of quality The research aims to extend research of the Faculty of Chemical Technology in the future (sampling and consequent chemical analyses of water samples to detect water quality and pollution). The main objective of this paper is to propose a suitable way of utilization of UAVs for detection of changes of small water bodies’ shorelines. Proposed procedure is applied to the Pohranov pond in Pardubice Region. The main output of the case study is the proposed procedure. The research can be extended to whole Pohranov pond and fully generalized in the future. Utilization of different sensors connected to an UAV can allow identification of water pollution and cooperation with the Faculty of Chemical Technology can allow verification of obtained results at first. As a final result, faster and cheaper way of pollution identification by UAVs will be proposed. D = percentage of area with the 2nd level of quality E = percentage of area with the 1st level of quality (the worst, unstable system) E = percentage of area with the 1st level of quality (the worst, unstable system) In the case that the coefficient is one or lower, the landscape is unstable, otherwise it is stable. For this calculation, the formula is simplified. n = count of classes n = count of classes Černohouz, O. Analýza změny využití krajiny v katastru Růžová. Ústí nad Labem, 2011. Master thesis. University of J. E. Purkyně. Calculation of the Index of Dominancy is the next step. This index represents relative substitution of single classes. Index of dominancy is counted by the following way: Dobešová Z. et al., 2014. International neural networks and GIS in modeling landscape changes. In: International Multidisciplinary Scientific GeoConference Surveying Geology and Mining Ecology Management 1(2014), pp. 651-658 D = Hmax – H For the second observation time, D is 0.54 and for the third observation time it is 0.59. These are higher numbers than it is obvious. Obvious are numbers near to zero which mean that in the landscape there are not dominant only several covers. In this case it means that landscape changes are stable in the monitored time period. Fahlstrom, P. G. and Gleason, T. J.. Introduction to UAV systems. 4th ed. Chichester: John Wiley & Sons, 2012, xxv, 280 s. Aerospace series. ISBN 978-1-119-97866-4. Gallop, S. L., et al., 2015. The impact of temperate reefs on 34 years of shoreline and vegetation line stability at Yanchep, southwestern Australia and implications for coastal setback. Marine Geology 369(2015), pp. 224-232. According to the calculated indices it can be concluded that shoreline changes in the monitored time period was not larger than it was expected. But there are some fluctuations which require more observations of the monitored area in the future. Geoportál ČÚZK: přístup k mapovým produktům a službám resortu Prague: ČÚZK, 2010 [access date: 2015-11-19]. available online: http://geoportal.cuzk.cz/ Guth J. and Kučera T., 1997. Monitoring of Land Cover Change with Using the Remote Sensing and GIS. Příroda 10(1997), pp. 107-124 Kes = 1.5∗𝐴+𝐵+0.5∗𝐶 0.2∗𝐷+0.8∗𝐸 Formula contains only stable area and unstable area, because the research is focused on shorelines and their changes only. It is found by the modified coefficient that in both time periods (the first observation is chosen as a stable one) is shoreline stable. Modified Kes is equal to 13.85 on 16. 7. 2015 and to 25.56 on 23. 8. 2015. According to Guth and Kučera (1997), Index of Diversity is used for detection of landscape changes too. The index informs about heterogeneity of landscape structures. Formula is as follows: Ai = relative area covers i-class of covers for n classes First observation is chosen as the stable one again. Then, data is divided into two classes. The first class is stable and the second class is unstable. In the case of this formula, it is necessary to recalculate data to percentage. Diversity in the second observation is 0.15 and in the third observation 0.11. This is a low value. It means that the land cover is more homogeneous (stable in this case). For the next index it is necessary to count Maximum Diversity: REFERENCES Adarsa J. et al., 2012. Morphological Change Study of Ghoramara Island, Eastern India Using Multi Temporal Satellite Data. Research Journal of Recent Sciences 1(10), pp. 72-81 Baker P. et al. 2010. Using shorelines for autonomous air vehicle guidance. Computer Vision and Image Understanding 114 (2010), pp. 723–729 Bortoleto L. A., 2016. Suitability index for restoration in landscapes: An alternative proposal for restoration projects. Ecological Indicators 1(2016), pp. 724-735 Hmax = log (n) n = count of classes ACKNOWLEDGMENTS H = - sum (Ai log Ai) H = - sum (Ai log Ai) The paper has been completed with the kind support of SGS project of Faculty of Economics and Administration, University of Pardubice. Ai = relative area covers i-class of covers for n classes 4 CONCLUSION Heine I. et al., 2015. Reconstruction of Lake Level Changes of Groundwater Fed Lakes in Northeastern Germany Using RapidEye Time Series. Water 7(2015), pp. 4175-4199 Utilization of UAVs for landscape and water monitoring is rapidly increasing. UAVs represent one of several available This contribution has been peer-reviewed. doi:10.5194/isprsarchives-XLI-B1-803-2016 This contribution has been peer-reviewed. doi:10.5194/isprsarchives-XLI-B1-803-2016 807 The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLI-B1, 2016 XXIII ISPRS Congress, 12–19 July 2016, Prague, Czech Republic Li W. et al., 2013. A Comparison of Land Surface Water Mapping Using the Normalized Difference Water Index from TM, ETM+ and ALI. Remote Sensing 5(2013), pp.5530-5549 Pechanec, V. et al., 2015. Decision support tool for the evaluation of landscapes. Ecological Informatics s. 4 available online: http://rj7gv6cf9w.search.serialssolutions.com//?sid=Elsevier:Scopus&genre=u nknown&issn=15749541&volume=&issue=&spage=&epage=&pages=&artn um=&date=2014&title=Ecological+Informatics&atitle=Decision+support+too l+for+the+evaluation+of+landscapes&aufirst=V.&auinit=V.&auinit1=V&aul ast=Pechanec&id=doi:10.1016%2fj.ecoinf.2015.06.006 Kotzee I. and Reyers B., 2016. Piloting a social-ecological index for measuring flood resilience: A composite index approach. Ecological Indicators 2(2016), pp. 45-53 McFeeters S. K., 1996. The use of the Normalized Difference Water Index (NDWI) in the delineation of open water features. International Journal of Remote Sensing 17(7), pp. 1425-1432. Qindong, F. and Shengyan, D., 2016. Landscape pattern changes at a county scale: A case study in Fengqiu, Henan Province, China from 1990 to 2013. Catena 2(2016), pp.152-160 Misra A. and Balaji R., 2015. A Study on the Shoreline Changes and LAND- use/ Land-cover along the South Gujarat Coastline. Procedia Engineering 6(116), pp. 381-389 Rathinam S. et al., 2007. Autonomous Searching and Tracking of a River using an UAV. In: Proceedings of the 2007 American Control Conference, New York City, USA, July 11-13, 2007, pp. 359-364. Nandi S. et al., 2015. Shoreline shifting and its prediction using remote sensing and GIS techniques: a case study of Sagar Island, West Bengal (India). Journal of Coastal Conservation 10(2015), pp. 1-20 Tamborski, J.J., et al., 2015. Identification and quantification of diffuse fresh submarine groundwater discharge via airborne thermal infrared remote sensing. Remote Sensing of Enviroment 46(171): pp. 202-217 Nath R. K., Deb S. K., 2010. Water-Body Area Extraction from High Resolution Satellite Images-An Introduction, Review, and Comparison. International Journal of Image Processing 6(3), pp. 353 -372 Work Jr E.A., Gilmer D.S., 1976. Utilization of Satellite Data for Invent Orying Prairie Ponds and Lakes. Photogrammetric Engineering and Remote Sensing 42(5), pp. 685-694 Pacheco A., et al., 2015. Retrieval of nearshore bathymetry from Landsat 8 images: A tool for coastal monitoring in shallow waters. This contribution has been peer-reviewed. doi:10.5194/isprsarchives-XLI-B1-803-2016 4 CONCLUSION Remote Sensing of Enviroment 46(159), pp. 102-116 Zlinszky A. et al., 2011. Initial Report of the Aimwetlab Project: Simultaneous Airborne Hyperspectral, LIDAR and Photogrammetric Survey of the Full Shoreline of Lake Balaton, Hungary. Geographia Technica 1(2011), pp. 101-117 Pardubický kraj: Region budoucnosti 2014/2015. Pardubice, 2015 [access date: 2015-11-24]. available online: www.pardubickykraj.cz This contribution has been peer-reviewed. doi:10.5194/isprsarchives-XLI-B1-803-2016 808
https://openalex.org/W2124259570	https://europepmc.org/articles/pmc3305337?pdf=render	English	null	Is Friedreich ataxia an epigenetic disorder?	Clinical epigenetics	2,012	cc-by	10,249	© 2012 Kumari and Usdin; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Friedreich ataxia (FRDA) is a debilitating and frequently fatal neurological disorder that is recessively inherited. It belongs to the group of genetic disorders known as the Repeat Expansion Diseases, in which pathology arises from the deleterious consequences of the inheritance of a tandem repeat array whose repeat number exceeds a critical threshold. In the case of FRDA, the repeat unit is the triplet GAA•TTC and the tandem array is located in the first intron of the frataxin (FXN) gene. Pathology arises because expanded alleles make lower than normal levels of mature FXN mRNA and thus reduced levels of frataxin, the FXN gene product. The repeats form a variety of unusual DNA structures that have the potential to affect gene expression in a number of ways. For example, triplex formation in vitro and in bacteria leads to the formation of persistent RNA:DNA hybrids that block transcription. In addition, these repeats have been shown to affect splicing in model systems. More recently, it has been shown that the region flanking the repeats in the FXN gene is enriched for epigenetic marks characteristic of transcriptionally repressed regions of the genome. However, exactly how repeats in an intron cause the FXN mRNA deficit in FRDA has been the subject of much debate. Identifying the mechanism or mechanisms responsible for the FXN mRNA deficit in FRDA is important for the development of treatments for this currently incurable disorder. This review discusses evidence for and against different models for the repeat-mediated mRNA deficit. Keywords: Friedreich ataxia, heterochromatin, histone modifications, transcription, splicing, tripl * Correspondence: ku@helix.nih.gov Section on Gene Structure and Disease, Laboratory of Cell and Molecular Biology, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0830, USA Open Access Open Access Is Friedreich ataxia an epigenetic disorder? Daman Kumari and Karen Usdin* Kumari and Usdin Clinical Epigenetics 2012, 4:2 http://www.clinicalepigeneticsjournal.com/content/4/1/2 Kumari and Usdin Clinical Epigenetics 2012, 4:2 http://www.clinicalepigeneticsjournal.com/content/4/1/2 Kumari and Usdin Clinical Epigenetics 2012, 4:2 http://www.clinicalepigeneticsjournal.com/content/4/1/2 Introduction Arrows indicate the location of the binding sites for serum response factor (SRF), activator protein 2 (AP2) [73], CCCTC-binding factor (CTCF) [25], an early growth response protein 3 (EGR3)-like factor [73] and an E-box binding protein [48] which have been shown to be positive regulators of FXN expression. Figure 1 The 5’ end of the frataxin (FXN) gene showing the minimal promoter, exon 1 and the promoter proximal end of intron 1. TSS1 and TSS2 refer to transcription start sites described in two different studies. TSS1 was identified based on a cDNA clone isolated from cardiac mRNA [4]. TSS2 is the major start site in lymphoblastoid cells identified by primer extension [52]. The positions of various interspersed repeated sequences are indicated by the rectangles outlined with black dashed lines. The dotted black arrow indicates the estimated extent of the FXN antisense transcript-1 (FAST-1) transcript based on nested PCR [25]. The solid black line superimposed on it is the region that could be amplified by a single round of PCR. The larger antisense transcript includes an open reading frame (ORF) with a non-canonical Kozak sequence whose significance is unknown. The ORF is intact in humans but truncated in closely related primates. Arrows indicate the location of the binding sites for serum response factor (SRF), activator protein 2 (AP2) [73], CCCTC-binding factor (CTCF) [25], an early growth response protein 3 (EGR3)-like factor [73] and an E-box binding protein [48] which have been shown to be positive regulators of FXN expression. Since most FRDA patients have at least one allele that contains a large repeat expansion, FRDA is considered to belong to a group of approximately 20 human genetic disorders known as the Repeat Expansion Diseases. In this group of diseases pathology arises from the conse- quences of inheritance of alleles with repeat numbers above a critical pathological threshold, which in the case of FRDA is approximately 90 repeats. The basis of the underlying expansion mutation responsible for these dis- orders is unknown, and problems with DNA replication, recombination and repair have all been suggested as possible mechanisms [6]. In principle, an FXN mRNA deficit could arise via an effect of the intronic repeats on the efficiency of tran- scription or some post-transcriptional event. However, no difference has been seen in the decay rate for the mature transcripts produced from normal and FRDA alleles [10]. Introduction onset with > 95% of patients becoming wheelchair bound by the age of 45. Early mortality due primarily to cardiac failure is not uncommon [2,3]. Friedreich ataxia (FRDA) (OMIM 229300; http://www. omim.org/entry/229300), first described in 1863 by Nikolaus Friedreich, is a relentlessly progressive disorder caused by mutations in the frataxin (FXN) gene. It is the most common heritable ataxia in Caucasians [1]. The major pathological changes include loss of myelinated axons in peripheral neurons, particularly in the dorsal root ganglia, the degeneration of posterior columns of the spinal cord and the loss of peripheral sensory nerve fibers. Myocardial muscle fibers also degenerate and are replaced by macrophages and fibroblasts. The net result of these and other changes include not only limb and gait abnormalities, but also hypertrophic cardiomyopa- thy, limb muscle weakness, absent lower limb reflexes and a positive extensor plantar response (Babinski sign). Decreased vibration sense, skeletal abnormalities, dysar- thria, and diabetes are common comorbid features. Many symptoms become apparent during adolescence. Loss of ambulation occurs roughly 15 years after disease The most common FRDA mutation is an expansion of the GAA•TTC repeat tract in intron 1 of the frataxin gene FRDA is inherited in an autosomal recessive fashion. The affected gene, frataxin (FXN) (OMIM 606829; http://omim.org/entry/606829), is located on chromo- some 9q13 in humans [4]. The first intron contains a GAA•TTC repeat tract embedded in the central poly(A) tract of an AluSq element from which it probably arose [5]. The GAA•TTC repeat tract, which is located approximately 1.3 kb downstream of the major FXN transcription start site (TSS), is polymorphic in the human population (Figure 1). While normal alleles have between 8 to 33 repeats, most individuals with FRDA have 2 FXN alleles each with > 90 repeats, the majority having 600 to 900 repeats [4]. A minority of patients (approximately 4%) are compound heterozygotes, having one allele with > 90 repeats and a second allele with a small deletion or point mutation in the FXN open read- ing frame. No cases of individuals with deletions or point mutations in both alleles are known [4]. Introduction Thus, the FXN mRNA deficit presumably results from events occurring at the level of transcrip- tion, and/or pre-mRNA stability or processing. The GAA•TTC repeats form an intrinsic block to transcription elongation in simple model systems In vitro transcription of templates containing as few as 11 GAA•TTC repeats produces less full-length RNA than templates with no repeats [11]. The repeats form a variety of unusual secondary structures under the same conditions (Figure 2). These structures include purine: purine:pyrimidine and pyrimidine:purine:pyrimidine tri- plexes [11-15] and a related structure known as sticky DNA [16]. It has been suggested that triplex formation could affect transcription by sequestering transcription factors or RNA polymerase (RNAP) [17,18]. It has also been suggested that a pre-existing triplex or sticky DNA blocks RNAP by making it more difficult for the tran- scription complex to unwind the template [17]. Introduction * Correspondence: ku@helix.nih.gov Section on Gene Structure and Disease, Laboratory of Cell and Molecular Biology, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0830, USA Kumari and Usdin Clinical Epigenetics 2012, 4:2 Page 2 of 10 http://www.clinicalepigeneticsjournal.com/content/4/1/2 Figure 1 The 5’ end of the frataxin (FXN) gene showing the minimal promoter, exon 1 and the promoter proximal end of intron 1. TSS1 and TSS2 refer to transcription start sites described in two different studies. TSS1 was identified based on a cDNA clone isolated from cardiac mRNA [4]. TSS2 is the major start site in lymphoblastoid cells identified by primer extension [52]. The positions of various interspersed repeated sequences are indicated by the rectangles outlined with black dashed lines. The dotted black arrow indicates the estimated extent of the FXN antisense transcript-1 (FAST-1) transcript based on nested PCR [25]. The solid black line superimposed on it is the region that could be amplified by a single round of PCR. The larger antisense transcript includes an open reading frame (ORF) with a non-canonical Kozak sequence whose significance is unknown. The ORF is intact in humans but truncated in closely related primates. Arrows indicate the location of the binding sites for serum response factor (SRF), activator protein 2 (AP2) [73], CCCTC-binding factor (CTCF) [25], an early growth response protein 3 (EGR3)-like factor [73] and an E-box binding protein [48] which have been shown to be positive regulators of FXN expression. Figure 1 The 5’ end of the frataxin (FXN) gene showing the minimal promoter, exon 1 and the promoter proximal end of intron 1. TSS1 and TSS2 refer to transcription start sites described in two different studies. TSS1 was identified based on a cDNA clone isolated from cardiac mRNA [4]. TSS2 is the major start site in lymphoblastoid cells identified by primer extension [52]. The positions of various interspersed repeated sequences are indicated by the rectangles outlined with black dashed lines. The dotted black arrow indicates the estimated extent of the FXN antisense transcript-1 (FAST-1) transcript based on nested PCR [25]. The solid black line superimposed on it is the region that could be amplified by a single round of PCR. The larger antisense transcript includes an open reading frame (ORF) with a non-canonical Kozak sequence whose significance is unknown. The ORF is intact in humans but truncated in closely related primates. FRDA results from a deficiency of FXN mRNA Expansion results in FXN mRNA levels that are 4% to 29% of normal [7]. There is an inverse relationship between repeat number and the amount of FXN mRNA produced. The FXN gene product, frataxin, is a small, highly conserved, acidic protein that is essential for life [8]. It is highly expressed in the dorsal root ganglia, the granular layer of the cerebellum as well as the heart, pancreas, thymus, brown fat, muscle and liver. Although the protein is nuclear encoded, it functions in the mito- chondria where it is thought to be involved in the bio- synthesis of iron-sulfur clusters (ISCs) [9], the complexes that serve as prosthetic groups for a variety of enzymes involved in energy and iron metabolism, purine synthesis and DNA repair. However, its precise role is currently unknown. However, whether the steady state levels of negative superhelicity present in mammalian chromosomes are high enough to allow the formation of such structures is unclear. b-Alanine-linked pyrrole-imidazole polyamides have been shown to bind GAA•TTC tracts with high Kumari and Usdin Clinical Epigenetics 2012, 4:2 Page 3 of 10 http://www.clinicalepigeneticsjournal.com/content/4/1/2 http://www.clinicalepigeneticsjournal.com/content/4/1/2 Figure 2 Examples of different structures formed by GAA•TTC repeats showing their constituent hydrogen bonding schemes. (A) A purine:purine:pyrimidine triplex [11,12]. This triplex involves Hoogsteen hydrogen bonding between a purine already in a Watson-Crick base pair with a pyrimidine, and a purine from a different region of the repeat. (B) A GAA hairpin [61]. Various hydrogen bonding schemes involving G•G, G•A and A•A base pairs have been proposed [74] including the one shown. However, the precise molecular details of the GAA hairpin are unknown. (C) A parallel duplex in which, unlike the antiparallel configuration of normal Watson-Crick duplex, the polarity of the two base-paired strands is the same [75]. In this configuration the base pairs involve a reverse Watson-Crick orientation and two, rather than three, hydrogen bonds. Figure 2 Examples of different structures formed by GAA•TTC repeats showing their constituent hydrogen bonding schemes. (A) A purine:purine:pyrimidine triplex [11,12]. This triplex involves Hoogsteen hydrogen bonding between a purine already in a Watson-Crick base pair with a pyrimidine, and a purine from a different region of the repeat. (B) A GAA hairpin [61]. Various hydrogen bonding schemes involving G•G, G•A and A•A base pairs have been proposed [74] including the one shown. However, the precise molecular details of the GAA hairpin are unknown. Kumari and Usdin Clinical Epigenetics 2012, 4:2 FRDA results from a deficiency of FXN mRNA Transcription through the repeat leaves the non- template purine-rich strand transiently unpaired. This strand can then fold back and interact with the duplex that has already reannealed behind the RNA polymerase (RNAP), thereby forming a triplex. Triplex formation, in turn, leaves the pyrimidine-rich strand in the second half of the repeat free to form a hybrid with the nascent RNA. This may be facilitated by the particular stability of an RNA:DNA hybrid containing a purine-rich RNA strand [76]. Evidence suggests that nucleation of this hybrid leads to unwinding of the triplex and the formation of a long persistent RNA:DNA hybrid that involves the whole repeat. The net result is the formation of a stable R loop in which the pyrimidine strand of the repeat is hybridized to the nascent transcript leaving the purine-rich strand unpaired. The RNAP becomes trapped on the template at the 3’ end of the repeat. Furthermore, studies of transcripts produced from the intact FXN gene did not detect any splicing abnormal- ities in FRDA cells [10,28]. However, since the existence of a very unstable splice isoform is difficult to defini- tively exclude, this issue is still unresolved. FRDA results from a deficiency of FXN mRNA Integrated Kumari and Usdin Clinical Epigenetics 2012, 4:2 http://www.clinicalepigeneticsjournal.com/content/4/1/2 Kumari and Usdin Clinical Epigenetics 2012, 4:2 http://www.clinicalepigeneticsjournal.com/content/4/1/2 Kumari and Usdin Clinical Epigenetics 2012, 4:2 http://www.clinicalepigeneticsjournal.com/content/4/1/2 Page 4 of 10 Figure 3 A triplex/RNA:DNA hybrid model for Friedreich ataxia (FRDA) [11,12,20]. Transcription through the repeat leaves the non- template purine-rich strand transiently unpaired. This strand can then fold back and interact with the duplex that has already reannealed behind the RNA polymerase (RNAP), thereby forming a triplex. Triplex formation, in turn, leaves the pyrimidine-rich strand in the second half showed a decreased splicing efficiency when the GAA- rich strand was transcribed but not the TTC-strand [30]. It was thus suggested that the deficit of mature FXN mRNA in FRDA results from aberrant mRNA spli- cing in which intron 1 is retained. The aberrant splicing seen with the minigene was attributed to the ability of the repeats to bind splicing factors such as the serine/ arginine (SR)-rich protein family and the proteins het- erogeneous nuclear ribonucleoprotein (hnRNP) A1 and hnRNP A2 as outlined in Figure 4, although how bind- ing of these factors would lead to intron retention is unclear. However, since total FXN mRNA abundance is reduced in FRDA cells, any mis-spliced transcript would have to be rapidly degraded for aberrant splicing to account for the mRNA deficit, which was not the case with the minigene tested [30]. In addition, the splicing abnormalities in the frataxin minigene were context and position dependent. This is important since in this con- struct both the repeat context and position differed from what is seen in the FXN locus. The FRDA GAA•TTC repeats have also been shown to reduce splicing in yeast [36]. This effect was attribu- ted to the increased length of the intron rather than any specific effects of the repeat per se. In yeast the largest known intron is < 1 Kb and in these organisms splicing efficiency is related to intron length [37]. However, many efficiently spliced human introns are much longer, with the human genome containing > 3000 genes with introns > 50 Kb. Since the FXN intron 1 of normal alleles is already 11 Kb and cases of FRDA are apparent with as few as 90 repeats, it seems unlikely that a change in intron length per se, is responsible for the reduced FXN expression in FRDA. Figure 3 A triplex/RNA:DNA hybrid model for Friedreich ataxia (FRDA) [11,12,20]. Expansion of the FRDA GAA•TTC repeat tract also causes epigenetic changes While it has been known for some time that a subset of Repeat Expansion Diseases are associated with hetero- chromatin formation, notably those disorders arising from CGG•CCG repeat expansion such as fragile X syn- drome (FXS) [38], the idea that the FRDA GAA•TTC repeats produce aberrant epigenetic modifications has only recently been appreciated. In part, the possibility that FRDA could be an epigenetic disorder was not initially entertained since unlike the affected gene in FXS, significant transcription still occurs from most FRDA alleles and early thinking in the field was that DNA methylation was required for epigenetic silencing constructs do show a consistent mRNA decrease in cells and mouse models [33-35], but whether this effect is mediated at the level of transcription elongation is unclear. FRDA results from a deficiency of FXN mRNA (C) A parallel duplex in which, unlike the antiparallel configuration of normal Watson-Crick duplex, the polarity of the two base-paired strands is the same [75]. In this configuration the base pairs involve a reverse Watson-Crick orientation and two, rather than three, hydrogen bonds. could also arise via bidirectional transcription through the repeat [24]. An antisense transcript, FXN antisense transcript-1 (FAST-1), has been identified in the FXN gene that could potentially contribute to such hybrids (see Figure 1). However, its 5’ end has not been mapped, its concentration is low and it is unclear at this time whether it includes the repeat [25]. affinity, to block sticky DNA formation and to increase FXN expression in cells from individuals with FRDA [19]. This would be consistent with a role for sticky DNA in FRDA. However, the specificity of these polyamides is uncertain and thus, the molecular basis of their effect is unclear. In addition to preformed triplexes, there is also evi- dence to suggest that triplexes formed during transcrip- tion in vitro lead to the formation of an RNA:DNA hybrid as illustrated in Figure 3[11,12,20]. This results in a block to transcription and trapping of RNAP on the template at the end of the repeat. Other sequences or conditions that favor the formation of R-loops also impede transcription [21,22]. Thus it is reasonable to think that an R-loop on a FRDA allele, however it is formed, could cause a block to transcription elongation. Single-stranded nicks in the template, perhaps arising from attempts to repair one of the structures formed by the repeat, can also increase the likelihood that R-loops will form during subsequent rounds of transcription [23]. Furthermore, work in vitro suggests that R-loops While direct proof of the formation of an R-loop by the FRDA GAA•TTC repeats in mammalian cells is lacking, other purine-rich repeats are known to do so [26]. In addi- tion, the promoter distal end of the repeat in human induced pluripotent cells generated from patient cells is known to bind the mismatch repair proteins MSH2 and MSH3 [27], which would be consistent with the formation of an unusual DNA conformation of some sort at this locus. While a consistent inhibition of transcription elonga- tion is seen with different RNAPs on naked DNA tem- plates in vitro [11,12,16,28,29], conflicting results have been seen with mammalian nuclear extracts and episomes in yeast and mammalian cells [16,28,30-32]. The GAA•TTC repeats affect splicing in model systems However, once the repeat number exceeds 90, splicing factors that are normally involved in the proper spl b i l li d h h l li i i d Thi ld b h l f bi di f li i f Figure 4 Altered splicing model for Friedreich ataxia (FRDA) [30]. On unaffected frataxin (FXN) alleles the repeat is too short to significantly impact splicing. However, once the repeat number exceeds 90, splicing factors that are normally involved in the proper splicing of the FXN gene become mislocalized such that normal splicing is prevented. This could be the result of binding of splicing factors to the repeat, preventing their normal assembly at the splice junctions. Alternatively, the unrestrained spread of these or other repeat-binding proteins, such as heterogeneous nuclear ribonucleoprotein (hnRNP) A1 [77], could block access of factors needed for proper splicing analogous to what has been proposed for HIV [78]. It may also be that the repeat sequesters serine/arginine (SR) proteins such as alternative splicing factor/splicing factor 2 (ASF/SF2). Since these proteins are is required for 5’ splice site selection and cleavage [79], this could lead to a local deficiency at the splice site and thus the failure to efficiently remove intron 1. [39-43]. Since the FRDA repeat contains no CpG resi- dues, the only dinucleotide subject to significant methy- lation in mammals, non-epigenetic mechanisms, like those described earlier, initially received more attention. that, despite the absence of methylatable residues, the FRDA repeats might trigger the formation of hetero- chromatin that could spread to adjacent sequences. j While the repeat itself cannot be methylated, DNA methylation could potentially occur secondarily to other chromatin changes in the region flanking the repeat. Consistent with that idea, we have shown that while DNA methylation is seen in the region flanking the repeat on normal alleles, perhaps due to spreading from adjacent Alu elements, more extensive DNA methyla- tion is seen in this region in patient cells [33,48]. A direct relationship between repeat length and the extent of DNA methylation has also been found in patient cells [49]. Since disease severity is related to repeat length, a direct relationship between disease severity and DNA methylation thus also exists. The GAA•TTC repeats affect splicing in model systems A so-called frataxin minigene construct, containing a CMV promoter, the FXN exon 1, part of intron 1 and all of exon 2, that was transfected into mammalian cells, Kumari and Usdin Clinical Epigenetics 2012, 4:2 Page 5 of 10 Kumari and Usdin Clinical Epigenetics 2012, 4:2 http://www.clinicalepigeneticsjournal.com/content/4/1/2 Figure 4 Altered splicing model for Friedreich ataxia (FRDA) [30]. On unaffected frataxin (FXN) alleles the repeat is too short to significantly impact splicing. However, once the repeat number exceeds 90, splicing factors that are normally involved in the proper splicing of the FXN gene become mislocalized such that normal splicing is prevented. This could be the result of binding of splicing factors to the repeat, preventing their normal assembly at the splice junctions. Alternatively, the unrestrained spread of these or other repeat-binding proteins, such as heterogeneous nuclear ribonucleoprotein (hnRNP) A1 [77], could block access of factors needed for proper splicing analogous to what has been proposed for HIV [78]. It may also be that the repeat sequesters serine/arginine (SR) proteins such as alternative splicing factor/splicing factor 2 (ASF/SF2). Since these proteins are is required for 5’ splice site selection and cleavage [79], this could lead to a local deficiency at the splice site and thus the failure to efficiently remove intron 1. Figure 4 Altered splicing model for Friedreich ataxia (FRDA) [30]. On unaffected frataxin (FXN) alleles the repeat is too short to significantly impact splicing. However, once the repeat number exceeds 90, splicing factors that are normally involved in the proper splicing of the FXN gene become mislocalized such that normal splicing is prevented. This could be the result of binding of splicing factors to the repeat, preventing their normal assembly at the splice junctions. Alternatively, the unrestrained spread of these or other repeat-binding proteins, such as heterogeneous nuclear ribonucleoprotein (hnRNP) A1 [77], could block access of factors needed for proper splicing analogous to what has been proposed for HIV [78]. It may also be that the repeat sequesters serine/arginine (SR) proteins such as alternative splicing factor/splicing factor 2 (ASF/SF2). Since these proteins are is required for 5’ splice site selection and cleavage [79], this could lead to a local deficiency at the splice site and thus the failure to efficiently remove intron 1. Figure 4 Altered splicing model for Friedreich ataxia (FRDA) [30]. On unaffected frataxin (FXN) alleles the repeat is too mpact splicing. The GAA•TTC repeats affect splicing in model systems However, it is now appreciated that even in those repeat expansion diseases where the repeat has a high density of CpG residues, such as FXS, DNA methylation is probably not the first step in heterochromatinization [44,45]. Furthermore, the expanded CTG•CAG repeats in myotonic dystrophy type 1 (DM1) are associated with heterochromatin despite their lack of CpG residues [46]. In addition, work with transgenic mice containing GAA•TTC repeats or CAG•CTG repeats showed that the repeats conferred variegation in the expression of a linked transgene, analogous to position effect variegation (PEV) in Drosophila [47]. These observations suggested Kumari and Usdin Clinical Epigenetics 2012, 4:2 http://www.clinicalepigeneticsjournal.com/content/4/1/2 Page 6 of 10 Not only is DNA methylation more extensive on FRDA alleles, but the methylation protection of 3 CpG residues that is seen upstream of the repeat on unaf- fected alleles is also lost [48]. One of these residues is within an E-box site that is important for maximal pro- moter activity in reporter assays in mouse myoblast cells. However, plasmids that are specifically methylated at this site do not show reduced transcription [48]. This suggests that loss of factor binding does not occur sec- ondarily to DNA methylation, but rather that protein binding normally protects those CpG residues from methylation. Thus, the loss of the normal methylation ‘footprint’ in FRDA cells likely reflects chromatin changes that restrict access of these factors to their nor- mal binding sites. Consistent with this view, FRDA patient alleles have been shown to be enriched for a variety of histone modifications characteristic of silenced genes including hypoacetylated H3 and H4 and dimethylation and trimethylation of histone H3 lysine 9 (H3K9) [48,50]. These histone modifications are highest in the regions flanking the repeat [50-52]. region of the FXN gene that includes the repeat [50-52], it may be that the trigger for these epigenetic modifica- tions is specifically related to some intrinsic property of the repeat itself as has been suggested for FXS [53]. This effect may be at the DNA level perhaps via the ability of the repeat to bind proteins that then recruit silencing fac- tors [54]. It could also be a consequence of the repair of DNA damage occurring in the repeat [55-57]. An unu- sual structure formed by the FRDA repeat may contri- bute to this process if it were trigger the DNA damage response. The GAA•TTC repeats affect splicing in model systems The binding of MSH2/MSH3 complexes to the region containing the repeat in patient cells lends weight to the idea that some sort of structure formed by the repeat is recognized by the cell as a site of DNA damage [27]. It is also possible that reduced transcription, result- ing perhaps from a triplex/RNA:DNA hybrid formation, leads to heterochromatic changes, as it does in some plant genes by favoring the recruitment of H3K27 tri- methylation (H3K27Me3) [58]. It could also be that heterochromatinization is RNA dependent perhaps involving a long non-coding RNA (lncRNA), as has been described for HOX genes and the lncRNA HOTAIR [59]. The non-coding RNA could be generated in cis or trans. Recent work has shown that the formation of a DNA:RNA triplex between a chromoso- mally located gene and ectopic RNA leads to enrichment of the DNA with H4K20Me3 and subsequent gene silen- cing [60]. Formation of such a triplex by the GAA•TTC repeat and either the repeat region in the sense or anti- sense transcript could thus lead to heterochromatin initiation within the repeat. Alternatively, if RNA con- taining a large number of GAA repeats can form hairpin- like GAA repeats in DNA [61], they may be source of double-stranded (ds)RNA for the RNA interference (RNAi) pathway. Transcripts containing the repeats may thus enter the RNAi pathway as has been demonstrated for the repeats responsible for FXS and DM1 [62,63]. Aberrant DNA methylation does not extend as far as the promoter in any of the patient cell lines that have been tested thus far. However, whether histone modifi- cations extend into the promoter is still controversial. The wide variation in the level of histone modifications seen in normal cells, the use of FRDA cell lines with very different repeat numbers and mRNA levels and dif- ferences in the experimental design and data analysis have added to the difficulty in reaching a consensus. However, to date there have been a number of reports of a histone profile typical of transcriptionally repressed genes on the affected FXN promoter in lymphoblastoid cells [25,52], the brains of affected individuals [33] and in a cell culture model [35]. Enrichment of repressive chromatin marks on the FXN promoter has also been reported in the brain and heart in transgenic mice mod- els of the disorder [33]. The GAA•TTC repeats affect splicing in model systems In addition, enrichment of the a and g isoforms of heterochromatin protein 1 (HP1), a non-chromosomal protein associated with heterochro- matin, on the promoter and the loss of CCCTC-binding factor (CTCF) binding to the promoter region in patient cells lends support to the idea that epigenetic changes originating in the repeat can spread to the 5’ end of the FXN gene [25]. How could these chromatin changes affect FXN transcription? Since the repeat-associated chromatin changes are located in both the transcriptional unit and in the promoter of at least some patient cells, they have, in principle, the poten- tial to affect expression of the FXN gene in a number of different ways. This effect could be exerted close to the start of transcription mediated by chromatin changes on the promoter. In addition, CTCF binding has been shown to play an important role in FXN expression [25]. So, sim- ply the loss of this factor from patient alleles could lead to reduced rates of transcription. Furthermore, even in the absence of altered promoter chromatin, histone and DNA methylation changes in the intron that lead to loss of bind- ing of important regulatory factors may affect transcrip- tion initiation or early steps in elongation. Since DNA What is the basis of the epigenetic changes? It has been suggested that the loss of CTCF binding is responsible for the observed histone changes on FRDA alleles [25]. However, this raises the question of what leads to the loss of CTCF binding. Since heterochroma- tin can be generated by the repeats embedded in a com- pletely different sequence context [35,47] and levels of the repressive histone modifications are highest in the Kumari and Usdin Clinical Epigenetics 2012, 4:2 http://www.clinicalepigeneticsjournal.com/content/4/1/2 Kumari and Usdin Clinical Epigenetics 2012, 4:2 http://www.clinicalepigeneticsjournal.com/content/4/1/2 Page 7 of 10 More recently it has been suggested that the reduced levels of Pol II are not due to reduced initiation but to reduced promoter proximal pausing [51]. This conclu- sion was based on the fact that no difference was seen in H3K4Me3 levels on unaffected and affected alleles at the 5’ end of the gene. However, in this study the region examined was upstream of what we now know to be the major TSS, in a part of the promoter that also did not show differences between affected and unaffected alleles in earlier reports [10,52]. Since H3K4Me3 is highest on nucleosomes immediately downstream of the TSS, the lower levels of H3K4Me3 that were seen on patient alleles just upstream of the repeat in the study of Kim et al. [51], in fact lend support to the idea that early events in transcription occurring prior to or during H3K4 tri- methylation are abnormal in FRDA. However, further work is needed to establish precisely what step or steps are affected. methylation in the body of a gene can affect the efficiency of transcription elongation [64], an effect on transcription through the intron is also possible. Do epigenetic changes account for the FXN mRNA deficit? The role of chromatin changes in causing the FXN mRNA deficit in FRDA is currently the subject of much debate. Histone deacetylase inhibitors have been shown to increase FXN expression in FRDA primary lympho- cytes and the brain and heart of a knock-in mouse model of the disorder [34,50]. The histone deacetylase, HDAC3, has been identified as an important target of these drugs [65]. The increase in FXN expression is accompanied by an increase in histone acetylation on FRDA alleles. However, it has been reported that while the histone methyltransferase inhibitor BIX-01294 reduced the levels of H3K9 dimethylation and trimethy- lation on FRDA alleles, no accompanying increase in FXN transcription was seen. What is the basis of the epigenetic changes? This has led to the sugges- tion that epigenetic changes are not responsible for the FXN deficit and that repeat expansion causes FRDA by forming a structural block to transcription elongation [10]. Whatever the cause of the reduced levels of Pol II on FRDA alleles, the lower levels of H3K36 trimethylation, a histone mark associated with transcription elongation, in the promoter proximal region [10,51,52], supports the idea that there is an effect of the repeat on transcription very close to the TSS more than 1 kb upstream of the repeat. Furthermore, the reduced levels of H3K79Me2, another mark of transcription elongation, found upstream of the repeat in patient cells [51], further strengthens the idea that there is reduced transcription in the region preceding the repeat. This idea would appear to be supported by the obser- vation that phosphorylation of serine 5, a mark charac- teristic of the initiating form of RNA polymerase II (Pol II Ser5-P), is present at similar levels at TSS1 [10], a transcription start site identified in early studies [4]. However, recent work has shown that the major TSS (TSS2) used in lymphoblastoid cells, the cell type used for these studies, is closer to the start of the FXN open reading frame than previously thought [52]. This is rele- vant since the initiating form of Pol II is typically found to have a narrow distribution at or downstream of the TSS [66]. When a region immediately downstream of TSS2 was examined, reduced levels of the initiating form of Pol II [52] as well as total Pol II [51] were seen in FRDA patient cells. A reduced level of H3K4 tri- methylation (H3K4Me3) was also seen the region in the region immediately downstream of TSS2 in patient cells [52]. Deposition of this histone mark occurs early in the transcription cycle primarily on the first nucleosome [67,68]. Trimethylation of H3K4 is thought to be required for both recruitment of the basal transcription machinery and for transcription initiation on genes that, like FXN, lack a TATA box [69]. In other genes, deposi- tion of this histone mark is thought to occur immedi- ately downstream of the promoter in a manner dependent on the levels of the initiating form of Pol II [69,70]. What is the basis of the epigenetic changes? In either event, the reduced level of H3K4Me3 seen on patient alleles suggests that a problem with transcription from FRDA templates is apparent very early in the transcription cycle, perhaps at the level of polymerase recruitment or transcription initiation. This is not to say that there is not a problem with transcription closer to the repeat as well. An additional effect of repeat expansion on Pol II elongation is sug- gested by the reduced accumulation of H3K36Me3 downstream of the repeat on FRDA alleles [10,51,52]. Whether this represents an effect of the histone changes and DNA hypermethylation in the vicinity of the repeat in patient cells or a chromatin-independent process remains to be seen. The relationship between GAA repeat number and the extent of intron DNA methylation raises the possibility that the epigenetic changes on smaller alleles may be smaller than on larger alleles and less likely to extend into the promoter. Thus the relative contribution of promoter-proximal and promoter-distal events may vary with repeat number. Conclusions Unaffected alleles are aberrantly methylated in the region flanking the repeat. Nonetheless, the 5’ end of the gene is associated with histones that are enriched for marks of active chromatin. In particular, acetylation of histone H3 and H4 is high. The net result is that the chromatin is open and permissive for transcription. Transcription factors including serum response factor (SRF), activator protein 2 (AP2) [73] and CCCTC-binding factor (CTCF) [25] associate with the 5’ end of the gene. An early growth response protein 3 (EGR3)-like factor binds to the 5’ end of intron 1 [73] and an E-box binding protein [48] bind to the region immediately upstream of the repeat. Under these conditions transcription initiation and elongation takes place normally. In contrast, FRDA alleles become associated with histones that are hypoacetylated and show more extensive DNA methylation in the region flanking the repeat. The net effect of these and other histone changes is the formation of a compact chromatin configuration. This reduces binding of transcription factors and both frataxin (FXN) transcription initiation and elongation are reduced. Loss of CTCF binding is correlated with an increase in the amount of FXN antisense transcript-1 (FAST-1) RNA that is transcribed antisense to FXN, but how this relates to silencing is unclear. TSS: transcription start site. Ac Acc Ac Ac A Ac Ac Ac c Ac Ac Ac A Ac Ac Ac A Ac Ac Ac A Me Me M Me Me Me Me Me e Me Me Figure 5 Diagrammatic representation of an epigenetic model for Friedreich ataxia (FRDA) Not shown to scale Unaffected alleles are Ac Acc Ac Ac A Ac Ac Ac c Ac Ac Ac A Ac Ac Ac A Ac Ac Ac A Me Me M Me Me Me Me Me e Me Me Figure 5 Diagrammatic representation of an epigenetic model for Friedreich ataxia (FRDA). Not shown to scale. Unaffected alleles are aberrantly methylated in the region flanking the repeat. Nonetheless, the 5’ end of the gene is associated with histones that are enriched for marks of active chromatin. In particular, acetylation of histone H3 and H4 is high. The net result is that the chromatin is open and permissive for transcription. Conclusions Transcription factors including serum response factor (SRF), activator protein 2 (AP2) [73] and CCCTC-binding factor (CTCF) [25] Ac Acc Ac Ac A Ac Ac Ac c Ac Ac Ac A Ac Ac Ac A Ac Ac Ac A Me Me Me M Me Me Me Me e Me Me Figure 5 Diagrammatic representation of an epigenetic model for Friedreich ataxia (FRDA). Not shown to s b l h l d h fl k h h l h ’ d f h d h h Figure 5 Diagrammatic representation of an epigenetic model for Friedreich ataxia (FRDA). Not shown to scale. Unaffected alleles are aberrantly methylated in the region flanking the repeat. Nonetheless, the 5’ end of the gene is associated with histones that are enriched for marks of active chromatin. In particular, acetylation of histone H3 and H4 is high. The net result is that the chromatin is open and permissive for transcription. Transcription factors including serum response factor (SRF), activator protein 2 (AP2) [73] and CCCTC-binding factor (CTCF) [25] associate with the 5’ end of the gene. An early growth response protein 3 (EGR3)-like factor binds to the 5’ end of intron 1 [73] and an E-box binding protein [48] bind to the region immediately upstream of the repeat. Under these conditions transcription initiation and elongation takes place normally. In contrast, FRDA alleles become associated with histones that are hypoacetylated and show more extensive DNA methylation in the region flanking the repeat. The net effect of these and other histone changes is the formation of a compact chromatin configuration. This reduces binding of transcription factors and both frataxin (FXN) transcription initiation and elongation are reduced. Loss of CTCF binding is correlated with an increase in the amount of FXN antisense transcript-1 (FAST-1) RNA that is transcribed antisense to FXN, but how this relates to silencing is unclear. TSS: transcription start site. Figure 5 Diagrammatic representation of an epigenetic model for Friedreich ataxia (FRDA). Not shown to scale. Unaffected alleles are aberrantly methylated in the region flanking the repeat. Nonetheless, the 5’ end of the gene is associated with histones that are enriched for marks of active chromatin. In particular, acetylation of histone H3 and H4 is high. The net result is that the chromatin is open and permissive for transcription. Conclusions An effect of the GAA•TTC repeat on events occurring > 1 kb away at the FXN promoter is difficult to reconcile with an effect of aberrant splicing. It is also difficult to reconcile with a direct effect of the formation of a tri- plex/R-loop unless problems occurring in the repeat lead to the buildup of stalled polymerases that stretches back to the promoter. Therefore, perhaps the most likely Kumari and Usdin Clinical Epigenetics 2012, 4:2 http://www.clinicalepigeneticsjournal.com/content/4/1/2 Kumari and Usdin Clinical Epigenetics 2012, 4:2 http://www.clinicalepigeneticsjournal.com/content/4/1/2 Page 8 of 10 of the transcription deficit. Such a mechanism would not necessarily preclude a role for triplexes/R-loops in events occurring at the promoter if, as discussed earlier, such structures contribute in some way to the formation of heterochromatin. explanation for the promoter proximal effects is that the repeat-mediated epigenetic changes generate a chroma- tin configuration that is less permissive for early steps in transcription as illustrated in Figure 5. That is that FRDA is, at least in part, a disorder of epigenetic dysre- gulation. The lack of an effect of BIX-01294 on FXN mRNA yield can be reconciled with this idea, if histone marks other than H3K9 methylation need to be removed before a chromatin conformation permissive for transcription is reestablished, as has been suggested for a number of other repressed genes [71,72]. If this is the case, it would suggest that histone deacetylase inhi- bitors, which are currently in clinical trials for treating FRDA, are probably acting on one of the direct causes Whether problems with Pol II elongation in the vici- nity of the repeat are epigenetically mediated or arise from a physical block to elongation like that formed by triplex/R-loops also remains an open question, with some data supporting a role for chromatin-mediated events and some data favoring a chromatin-independent mechanism. It may be that both mechanisms contribute to the FXN mRNA deficit in some way and further work will be necessary to understand the relative , y g FRDA, are probably acting on one of the direct causes y work will be necessary to understand the relativ Ac Acc Ac Ac A Ac Ac Ac c Ac Ac Ac A Ac Ac Ac A Ac Ac Ac A Me Me M Me Me Me Me Me e Me Me Figure 5 Diagrammatic representation of an epigenetic model for Friedreich ataxia (FRDA). Not shown to scale. References Ku S, Soragni E, Campau E, Thomas EA, Altun G, Laurent LC, Loring JF, Napierala M, Gottesfeld JM: Friedreich’s ataxia induced pluripotent stem cells model intergenerational GAATTC triplet repeat instability. Cell Stem Cell 2010, 7:631-637. 5. Clark RM, Dalgliesh GL, Endres D, Gomez M, Taylor J, Bidichandani SI: Expansion of GAA triplet repeats in the human genome: unique origin of the FRDA mutation at the center of an Alu. Genomics 2004, 83:373-383 28. Bidichandani SI, Ashizawa T, Patel PI: The GAA triplet-repeat expansion in Friedreich ataxia interferes with transcription and may be associated with an unusual DNA structure. Am J Hum Genet 1998, 62:111-121. 6. Pearson CE, Nichol Edamura K, Cleary JD: Repeat instability: mechanisms of dynamic mutations. Nat Rev Genet 2005, 6:729-742. 7. Campuzano V, Montermini L, Lutz Y, Cova L, Hindelang C, Jiralerspong S, Trottier Y, Kish SJ, Faucheux B, Trouillas P, Authier FJ, Dürr A, Mandel JL, Vescovi A, Pandolfo M, Koenig M: Frataxin is reduced in Friedreich ataxia patients and is associated with mitochondrial membranes. Hum Mol Genet 1997, 6:1771-1780. 29. Krasilnikova MM, Kireeva ML, Petrovic V, Knijnikova N, Kashlev M, Mirkin SM: Effects of Friedreich’s ataxia (GAA)n(TTC)n repeats on RNA synthesis and stability. Nucleic Acids Res 2007, 35:1075-1084. 30. Baralle M, Pastor T, Bussani E, Pagani F: Influence of Friedreich ataxia GAA noncoding repeat expansions on pre-mRNA processing. Am J Hum Genet 2008, 83:77-88. 8. Cossee M, Puccio H, Gansmuller A, Koutnikova H, Dierich A, LeMeur M, Fischbeck K, Dolle P, Koenig M: Inactivation of the Friedreich ataxia mouse gene leads to early embryonic lethality without iron accumulation. Hum Mol Genet 2000, 9:1219-1226. 31. Krasilnikova MM, Mirkin SM: Replication stalling at Friedreich’s ataxia (GAA)n repeats in vivo. Mol Cell Biol 2004, 24:2286-2295. 32. Ohshima K, Montermini L, Wells RD, Pandolfo M: Inhibitory effects of expanded GAA.TTC triplet repeats from intron I of the Friedreich ataxia gene on transcription and replication in vivo. J Biol Chem 1998, 273:14588-14595. 9. Rotig A, de Lonlay P, Chretien D, Foury F, Koenig M, Sidi D, Munnich A, Rustin P: Aconitase and mitochondrial iron-sulphur protein deficiency in Friedreich ataxia. Nat Genet 1997, 17:215-217. 10. Punga T, Buhler M: Long intronic GAA repeats causing Friedreich ataxia impede transcription elongation. EMBO Mol Med 2010, 2:120-129. 33. References 1. Cossee M, Schmitt M, Campuzano V, Reutenauer L, Moutou C, Mandel JL, Koenig M: Evolution of the Friedreich’s ataxia trinucleotide repeat expansion: founder effect and premutations. Proc Natl Acad Sci USA 1997, 94:7452-7457. 23. Roy D, Zhang Z, Lu Z, Hsieh CL, Lieber MR: Competition between the RNA transcript and the nontemplate DNA strand during R-loop formation in vitro: a nick can serve as a strong R-loop initiation site. Mol Cell Biol 2010, 30:146-159. 2. Harding AE: Early onset cerebellar ataxia with retained tendon reflexes: a clinical and genetic study of a disorder distinct from Friedreich’s ataxia. J Neurol Neurosurg Psychiatry 1981, 44:503-508. 2. Harding AE: Early onset cerebellar ataxia with retained tendon reflexes: a clinical and genetic study of a disorder distinct from Friedreich’s ataxia. J Neurol Neurosurg Psychiatry 1981, 44:503-508. 24. Reddy K, Tam M, Bowater RP, Barber M, Tomlinson M, Nichol Edamura K, Wang YH, Pearson CE: Determinants of R-loop formation at convergent bidirectionally transcribed trinucleotide repeats. Nucleic Acids Res 2011, 39:1749-1762. 3. Harding AE, Zilkha KJ: ’Pseudo-dominant’ inheritance in Friedreich’s ataxia. J Med Genet 1981, 18:285-287. 25. De Biase I, Chutake YK, Rindler PM, Bidichandani SI: Epigenetic silencing in Friedreich ataxia is associated with depletion of CTCF (CCCTC-binding factor) and antisense transcription. PLoS One 2009, 4:e7914. 4. Campuzano V, Montermini L, Moltò MD, Pianese L, Cossée M, Cavalcanti F, Monros E, Rodius F, Duclos F, Monticelli A, Zara F, Cañizares J, Koutnikova H, Bidichandani SI, Gellera C, Brice A, Trouillas P, De Michele G, Filla A, De Frutos R, Palau F, Patel PI, Di Donato S, Mandel JL, Cocozza S, Koenig M, Pandolfo M: Friedreich’s ataxia: autosomal recessive disease caused by an intronic GAA triplet repeat expansion. Science 1996, 271:1423-1427. 4. Campuzano V, Montermini L, Moltò MD, Pianese L, Cossée M, Cavalcanti F, Monros E, Rodius F, Duclos F, Monticelli A, Zara F, Cañizares J, Koutnikova H, Bidichandani SI, Gellera C, Brice A, Trouillas P, De Michele G, Filla A, De Frutos R, Palau F, Patel PI, Di Donato S, Mandel JL, Cocozza S, Koenig M, Pandolfo M: Friedreich’s ataxia: autosomal recessive disease caused by an intronic GAA triplet repeat expansion. Science 1996, 271:1423-1427. 26. Yu K, Chedin F, Hsieh CL, Wilson TE, Lieber MR: R-loops at immunoglobulin class switch regions in the chromosomes of stimulated B cells. Nat Immunol 2003, 4:442-451. 27. Conclusions Page 9 of 10 Kumari and Usdin Clinical Epigenetics 2012, 4:2 http://www.clinicalepigeneticsjournal.com/content/4/1/2 in R.R.Y triplex structures from Friedreich’s ataxia. Mol Cell 1999, 3:465-475. Authors’ contributions Both DK and KU contributed equally to the writing of this manuscript. Both authors read and approved the final manuscript. 19. Burnett R, Melander C, Puckett JW, Son LS, Wells RD, Dervan PB, Gottesfeld JM: DNA sequence-specific polyamides alleviate transcription inhibition associated with long GAA.TTC repeats in Friedreich’s ataxia. Proc Natl Acad Sci USA 2006, 103:11497-11502. contribution of these mechanisms to the FXN mRNA deficit responsible for FRDA. contribution of these mechanisms to the FXN mRNA deficit responsible for FRDA. in R.R.Y triplex structures from Friedreich’s ataxia. Mol Cell 1999, 3:465-475. 17. Kohwi Y, Kohwi-Shigematsu T: Altered gene expression correlates with DNA structure. Genes Dev 1991, 5:2547-2554. 18. Sakamoto N, Ohshima K, Montermini L, Pandol 18. Sakamoto N, Ohshima K, Montermini L, Pandolfo M, Wells RD: Sticky DNA, a self-associated complex formed at long GAATTC repeats in intron 1 of the frataxin gene, inhibits transcription. J Biol Chem 2001, 276:27171-27177. Received: 18 August 2011 Accepted: 30 January 2012 Published: 30 January 2012 y 21. Bentin T, Cherny D, Larsen HJ, Nielsen PE: Transcription arrest caused by long nascent RNA chains. Biochim Biophys Acta 2005, 1727:97-105. 22. Tous C, Aguilera A: Impairment of transcription elongation by R-loops in vitro. Biochem Biophys Res Commun 2007, 360:428-432. Competing interests This work was made possible by a grant to KU from the Intramural Program of NIDDK (DK057810). The Authors declare that there is no conflict of interest. 20. Grabczyk E, Mancuso M, Sammarco MC: A persistent RNA.DNA hybrid formed by transcription of the Friedreich ataxia triplet repeat in live bacteria, and by T7 RNAP in vitro. Nucleic Acids Res 2007, 35:5351-5359. Received: 18 August 2011 Accepted: 30 January 2012 Published: 30 January 2012 Conclusions Transcription factors including serum response factor (SRF), activator protein 2 (AP2) [73] and CCCTC-binding factor (CTCF) [25] associate with the 5’ end of the gene. An early growth response protein 3 (EGR3)-like factor binds to the 5’ end of intron 1 [73] and an E-box binding protein [48] bind to the region immediately upstream of the repeat. Under these conditions transcription initiation and elongation takes place normally. In contrast, FRDA alleles become associated with histones that are hypoacetylated and show more extensive DNA methylation in the region flanking the repeat. The net effect of these and other histone changes is the formation of a compact chromatin configuration. This reduces binding of transcription factors and both frataxin (FXN) transcription initiation and elongation are reduced. Loss of CTCF binding is correlated with an increase in the amount of FXN antisense transcript-1 (FAST-1) RNA that is transcribed antisense to FXN, but how this relates to silencing is unclear. TSS: transcription start site. Figure 5 Diagrammatic representation of an epigenetic model for Friedreich ataxia (FRDA). Not shown to scale. Unaffected alleles are aberrantly methylated in the region flanking the repeat. Nonetheless, the 5’ end of the gene is associated with histones that are enriched for marks of active chromatin. In particular, acetylation of histone H3 and H4 is high. The net result is that the chromatin is open and permissive for transcription. Transcription factors including serum response factor (SRF), activator protein 2 (AP2) [73] and CCCTC-binding factor (CTCF) [25] associate with the 5’ end of the gene. An early growth response protein 3 (EGR3)-like factor binds to the 5’ end of intron 1 [73] and an E-box binding protein [48] bind to the region immediately upstream of the repeat. Under these conditions transcription initiation and elongation takes place normally. In contrast, FRDA alleles become associated with histones that are hypoacetylated and show more extensive DNA methylation in the region flanking the repeat. The net effect of these and other histone changes is the formation of a compact chromatin configuration. This reduces binding of transcription factors and both frataxin (FXN) transcription initiation and elongation are reduced. Loss of CTCF binding is correlated with an increase in the amount of FXN antisense transcript-1 (FAST-1) RNA that is transcribed antisense to FXN, but how this relates to silencing is unclear. TSS: transcription start site. References Oberle I, Rousseau F, Heitz D, Kretz C, Devys D, Hanauer A, Boue J, Bertheas M, Mandel J: Instability of a 550-base pair DNA segment and abnormal methylation in fragile X syndrome. Science 1991, 252:1097-1102. 61. Heidenfelder BL, Makhov AM, Topal MD: Hairpin formation in Friedreich’s ataxia triplet repeat expansion. J Biol Chem 2003, 278:2425-2431. 62. Handa V, Saha T, Usdin K: The fragile X syndrome repeats form RNA hairpins that do not activate the interferon-inducible protein kinase, PKR, but are cut by Dicer. Nucleic Acids Res 2003, 31:6243-6248. 39. Bestor TH: Gene silencing. Methylation meets acetylation. Nature 1998, 393:311-312. 63. Krol J, Fiszer A, Mykowska A, Sobczak K, de Mezer M, Krzyzosiak WJ: Ribonuclease dicer cleaves triplet repeat hairpins into shorter repeats that silence specific targets. Mol Cell 2007, 25:575-586. 40. Chen X, Mariappan SV, Catasti P, Ratliff R, Moyzis RK, Laayoun A, Smith SS, Bradbury EM, Gupta G: Hairpins are formed by the single DNA strands of the fragile X triplet repeats: structure and biological implications. Proc Natl Acad Sci USA 1995, 92:5199-5203. 64. Lorincz MC, Dickerson DR, Schmitt M, Groudine M: Intragenic DNA methylation alters chromatin structure and elongation efficiency in mammalian cells. Nat Struct Mol Biol 2004, 11:1068-1075. 41. Jones PL, Wolffe AP: Relationships between chromatin organization and DNA methylation in determining gene expression. Semin Cancer Biol 1999, 9:339-347. 65. Xu C, Soragni E, Chou CJ, Herman D, Plasterer HL, Rusche JR, Gottesfeld JM: Chemical probes identify a role for histone deacetylase 3 in Friedreich’s ataxia gene silencing. Chem Biol 2009, 16:980-989. 42. Kass SU, Pruss D, Wolffe AP: How does DNA methylation repress transcription? Trends Genet 1997, 13:444-449. 66. Komarnitsky P, Cho EJ, Buratowski S: Different phosphorylated forms of RNA polymerase II and associated mRNA processing factors during transcription. Genes Dev 2000, 14:2452-2460. 43. Razin A: CpG methylation, chromatin structure and gene silencing-a three-way connection. EMBO J 1998, 17:4905-4908. 44. Eiges R, Urbach A, Malcov M, Frumkin T, Schwartz T, Amit A, Yaron Y, Eden A, Yanuka O, Benvenisty N, Ben-Yosef D: Developmental study of fragile X syndrome using human embryonic stem cells derived from preimplantation genetically diagnosed embryos. Cell Stem Cell 2007, 1:568-577. 67. Guenther MG, Levine SS, Boyer LA, Jaenisch R, Young RA: A chromatin landmark and transcription initiation at most promoters in human cells. Cell 2007, 130:77-88. 68. References Ng HH, Robert F, Young RA, Struhl K: Targeted recruitment of Set1 histone methylase by elongating Pol II provides a localized mark and memory of recent transcriptional activity. Mol Cell 2003, 11:709-719. 45. Tabolacci E, Moscato U, Zalfa F, Bagni C, Chiurazzi P, Neri G: Epigenetic analysis reveals a euchromatic configuration in the FMR1 unmethylated full mutations. Eur J Hum Genet 2008, 16:1487-1498. 69. Vermeulen M, Mulder KW, Denissov S, Pijnappel WW, van Schaik FM, Varier RA, Baltissen MP, Stunnenberg HG, Mann M, Timmers HT: Selective anchoring of TFIID to nucleosomes by trimethylation of histone H3 lysine 4. Cell 2007, 131:58-69. 46. Cho DH, Thienes CP, Mahoney SE, Analau E, Filippova GN, Tapscott SJ: Antisense transcription and heterochromatin at the DM1 CTG repeats are constrained by CTCF. Mol Cell 2005, 20:483-489. 70. Wang P, Lin C, Smith ER, Guo H, Sanderson BW, Wu M, Gogol M, Alexander T, Seidel C, Wiedemann LM, Ge K, Krumlauf R, Shilatifard A: Global analysis of H3K4 methylation defines MLL family member targets and points to a role for MLL1-mediated H3K4 methylation in the regulation of transcriptional initiation by RNA polymerase II. Mol Cell Biol 2009, 29:6074-6085. 47. Saveliev A, Everett C, Sharpe T, Webster Z, Festenstein R: DNA triplet repeats mediate heterochromatin-protein-1-sensitive variegated gene silencing. Nature 2003, 422:909-913. g 48. Greene E, Mahishi L, Entezam A, Kumari D, Usdin K: Repeat-induced epigenetic changes in intron 1 of the frataxin gene and its consequences in Friedreich ataxia. Nucleic Acids Res 2007, 35:3383-3390. 71. Kubicek S, O’Sullivan RJ, August EM, Hickey ER, Zhang Q, Teodoro ML, Rea S, Mechtler K, Kowalski JA, Homon CA, Kelly TA, Jenuwein T: Reversal of H3K9me2 by a small-molecule inhibitor for the G9a histone methyltransferase. Mol Cell 2007, 25:473-481. 49. Castaldo I, Pinelli M, Monticelli A, Acquaviva F, Giacchetti M, Filla A, Sacchetti S, Keller S, Avvedimento VE, Chiariotti L, Cocozza S: DNA methylation in intron 1 of the frataxin gene is related to GAA repeat length and age of onset in Friedreich ataxia patients. J Med Genet 2008, 45:808-812. 72. Link PA, Gangisetty O, James SR, Woloszynska-Read A, Tachibana M, Shinkai Y, Karpf AR: Distinct roles for histone methyltransferases G9a and GLP in cancer germ-line antigen gene regulation in human cancer cells and murine embryonic stem cells. Mol Cancer Res 2009, 7:851-862. 50. References Herman D, Jenssen K, Burnett R, Soragni E, Perlman SL, Gottesfeld JM: Histone deacetylase inhibitors reverse gene silencing in Friedreich’s ataxia. Nat Chem Biol 2006, 2:551-558. 51. Kim E, Napierala M, Dent SY: Hyperexpansion of GAA repeats affects post- initiation steps of FXN transcription in Friedreich’s ataxia. Nucleic Acids Res 2011, 39:8366-8377. 73. Li K, Singh A, Crooks DR, Dai X, Cong Z, Pan L, Ha D, Rouault TA: Expression of human frataxin is regulated by transcription factors SRF and TFAP2. PLoS One 2010, 5:e12286. 73. Li K, Singh A, Crooks DR, Dai X, Cong Z, Pan L, Ha D, Rouault TA: Expression of human frataxin is regulated by transcription factors SRF and TFAP2. PLoS One 2010, 5:e12286. 74. Chou SH, Chin KH, Wang AH: Unusual DNA duplex and hairpin motifs. Nucleic Acids Res 2003, 31:2461-2474. 52. Kumari D, Biacsi RE, Usdin K: Repeat expansion affects both transcription initiation and elongation in Friedreich ataxia cells. J Biol Chem 2011, 286:4209-4215. 74. Chou SH, Chin KH, Wang AH: Unusual DNA duplex and hairpin motifs. Nucleic Acids Res 2003, 31:2461-2474. 75. LeProust EM, Pearson CE, Sinden RR, Gao X: Unexpected formation of parallel duplex in GAA and TTC trinucleotide repeats of Friedreich’s ataxia. J Mol Biol 2000, 302:1063-1080. 53. Kumari D, Usdin K: The distribution of repressive histone modifications on silenced FMR1 alleles provides clues to the mechanism of gene silencing in fragile X syndrome. Hum Mol Genet 2010, 19:4634-4642. 76. Roberts RW, Crothers DM: Stability and properties of double and triple helices: dramatic effects of RNA or DNA backbone composition. Science 1992, 258:1463-1466. 54. Grewal SI, Moazed D: Heterochromatin and epigenetic control of gene expression. Science 2003, 301:798-802. p 55. O’Hagan HM, Mohammad HP, Baylin SB: Double strand breaks can initiate gene silencing and SIRT1-dependent onset of DNA methylation in an exogenous promoter CpG island. PLoS Genet 2008, 4:e1000155. 77. Zhu J, Mayeda A, Krainer AR: Exon identity established through differential antagonism between exonic splicing silencer-bound hnRNP A1 and enhancer-bound SR proteins. Mol Cell 2001, 8:1351-1361. 56. Matzke M, Aufsatz W, Kanno T, Daxinger L, Papp I, Mette MF, Matzke AJ: Genetic analysis of RNA-mediated transcriptional gene silencing. Biochim Biophys Acta 2004, 1677:129-141. 78. Damgaard CK, Tange TO, Kjems J: hnRNP A1 controls HIV-1 mRNA splicing through cooperative binding to intron and exon splicing silencers in the context of a conserved secondary structure. RNA 2002, 8:1401-1415. p y 57. References Al-Mahdawi S, Pinto RM, Ismail O, Varshney D, Lymperi S, Sandi C, Trabzuni D, Pook M: The Friedreich ataxia GAA repeat expansion mutation induces comparable epigenetic changes in human and transgenic mouse brain and heart tissues. Hum Mol Genet 2008, 17:735-746. 11. Grabczyk E, Usdin K: The GAATTC triplet repeat expanded in Friedreich’s ataxia impedes transcription elongation by T7 RNA polymerase in a length and supercoil dependent manner. Nucleic Acids Res 2000, 28:2815-2822. 34. Rai M, Soragni E, Jenssen K, Burnett R, Herman D, Coppola G, Geschwind DH, Gottesfeld JM, Pandolfo M: HDAC inhibitors correct frataxin deficiency in a Friedreich ataxia mouse model. PLoS One 2008, 3 e1958. 12. Grabczyk E, Usdin K: Alleviating transcript insufficiency caused by Friedreich’s ataxia triplet repeats. Nucleic Acids Res 2000, 28:4930-4937. 13. Jain A, Rajeswari MR, Ahmed F: Formation and thermodynamic stability of intermolecular (RRY) DNA triplex in GAA/TTC repeats associated with Freidreich’s ataxia. J Biomol Struct Dyn 2002, 19:691-699. 35. Soragni E, Herman D, Dent SY, Gottesfeld JM, Wells RD, Napierala M: Long intronic GAATTC repeats induce epigenetic changes and reporter gene silencing in a molecular model of Friedreich ataxia. Nucleic Acids Res 2008, 36:6056-6065. 14. Mariappan SV, Catasti P, Silks LA, Bradbury EM, Gupta G: The high- resolution structure of the triplex formed by the GAA/TTC triplet repeat associated with Friedreich’s ataxia. J Mol Biol 1999, 285:2035-2052. 36. Shishkin AA, Voineagu I, Matera R, Cherng N, Chernet BT, Krasilnikova MM, Narayanan V, Lobachev KS, Mirkin SM: Large-scale expansions of Friedreich’s ataxia GAA repeats in yeast. Mol Cell 2009, 35:82-92. 15. Potaman VN, Oussatcheva EA, Lyubchenko YL, Shlyakhtenko LS, Bidichandani SI, Ashizawa T, Sinden RR: Length-dependent structure formation in Friedreich ataxia (GAA)n*(TTC)n repeats at neutral pH. Nucleic Acids Res 2004, 32:1224-1231. 37. Klinz FJ, Gallwitz D: Size and position of intervening sequences are critical for the splicing efficiency of pre-mRNA in the yeast Saccharomyces cerevisiae. Nucleic Acids Res 1985, 13:3791-3804. 16. Sakamoto N, Chastain PD, Parniewski P, Ohshima K, Pandolfo M, Griffith JD, Wells RD: Sticky DNA: self-association properties of long GAA.TTC repeats 16. Sakamoto N, Chastain PD, Parniewski P, Ohshima K, Pandolfo M, Griffith JD, Wells RD: Sticky DNA: self-association properties of long GAA.TTC repeats Page 10 of 10 Kumari and Usdin Clinical Epigenetics 2012, 4:2 http://www.clinicalepigeneticsjournal.com/content/4/1/2 38. References Shanbhag NM, Rafalska-Metcalf IU, Balane-Bolivar C, Janicki SM, Greenberg RA: ATM-dependent chromatin changes silence transcription in cis to DNA double-strand breaks. Cell 2010, 141:970-981. 79. Zuo P, Manley JL: The human splicing factor ASF/SF2 can specifically recognize pre-mRNA 5’ splice sites. Proc Natl Acad Sci USA 1994, 91:3363-3367. 58. Buzas DM, Robertson M, Finnegan EJ, Helliwell CA: Transcription- dependence of histone H3 lysine 27 trimethylation at the Arabidopsis polycomb target gene FLC. Plant J 2011, 65:872-881. doi:10.1186/1868-7083-4-2 Cite this article as: Kumari and Usdin: Is Friedreich ataxia an epigenetic disorder? Clinical Epigenetics 2012 4:2. 59. Rinn JL, Kertesz M, Wang JK, Squazzo SL, Xu X, Brugmann SA, Goodnough LH, Helms JA, Farnham PJ, Segal E, Chang HY: Functional demarcation of active and silent chromatin domains in human HOX loci by noncoding RNAs. Cell 2007, 129:1311-1323. 60. Schmitz KM, Mayer C, Postepska A, Grummt I: Interaction of noncoding RNA with the rDNA promoter mediates recruitment of DNMT3b and silencing of rRNA genes. Genes Dev 2010, 24:2264-2269.
https://openalex.org/W3179969625	https://www.researchsquare.com/article/rs-599622/latest.pdf	English	null	Structural, Linear and Nonlinear Optical Properties of Some Al-free and Al-doped ZnO Nanopowder Samples	Research Square (Research Square)	2,021	cc-by	4,461	Abstract: This research work aims to prepare and characterization some Al-doped ZnO nanoparticles. The co-precipitation method was used for preparing the desired samples, where ZnO replaced by AlCl3. Then the resulted materials were characterized and their structural phases identified using the XRD technique, where eight crystalline phases were identified and then assigned to some of the interatomic planes, (100), (002), (101), (102), (110), (103), (112), and (201). Both the average crystalline size and the micro-strain were calculated for each sample, where it was found that both of them increase when Ai-content increases. UV-vis spectra showed an absorption peak centered at 3.34 eV which represents the optical bandgap of ZnO. Al-Impurities acted to improve and increase the value of the optical transmittance, especially in the visible light region. The increase of Al-content influenced, where their values increase when AL-content increases. the value of the calculated nonlinear refractive index may be considered promising for different optoelectronic applications. Keywords: ZnO, nanoparticles, optical properties, linear and nonlinear optics Corresponding author: Hosam M. Gomaa (Ph.D.), Email: H_goumaa@yahoo.com WhatsApp. +201001381061 Research Article Research Article Keywords: ZnO, nanoparticles, optical properties, linear and nonlinear optics Posted Date: July 6th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-599622/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Structural, Linear and nonlinear optical properties of some Al-free and Al-doped ZnO nanopowder samples N.M. Moussa1, F.M. Ebrahim1, Hosam Mohamed Gomaa2, K. Adly3, and M.Y Hassaan1 1Physics Department, faculty of science, Al azhar University, Cairo, Egypt 2Independent researcher, from Sakkara, Giza, Egypt 3 Physics Department, faculty of science. Suez Canal University, Ismailia, Egypt Structural, Linear and nonlinear optical properties of some Al-free and Al-doped ZnO nanopowder samples N.M. Moussa1, F.M. Ebrahim1, Hosam Mohamed Gomaa2, K. Adly3, and M.Y Hassaan1 1Physics Department, faculty of science, Al azhar University, Cairo, Egypt 2Independent researcher, from Sakkara, Giza, Egypt 3 Physics Department, faculty of science. Suez Canal University, Ismailia, Egypt Structural, Linear and nonlinear optical properties of some Al-free and Al-doped ZnO nanopowder samples Structural, Linear and nonlinear optical properties of some Al-free and Al-doped ZnO nanopowder samples 1. Introduction: Nanomaterials and Nanocomposites are of high importance in different fields because of their potential applications, as storage energy devices, optoelectronic instruments, and solar cells. for example, Nanoparticles based on ZnO have used as a drug-delivery active medium in UV-region semiconductor lasers [1-6], where zinc oxide is a semiconductor oxide that has a wide direct bandgap of about 3.37 eV, and a large exciton binding energy of about 60 m eV [2, 3], at room temperature. Large numbers of publications stated that Nanoparticles based on Zinc metal oxide 1 with or without another metal oxide or more is considered as one of the most unique ways that provide what called long-lasting superior protection [7-9]. Also, it was found that when ZnO nanopowder mixes with other different ions acts to improve the optical, electrical, and catalytic properties of these ions [9], especially, when ZnO nanopowder doped with Al, where the resulted compound is conductive and transparent in the visible region, which suggests it be used in the transparent conductive pastes [10–11]. Therefore, in recent years the most researchers in the area of material science focused to develop new technics and experimental methods to obtain and fabricate ZnO nanostructures, like the co-precipitation, sol-gel, hydrothermal, and spray pyrolysis [12-18], Among all developed techniques the co-precipitation technique has attracted more attention because of its simplicity as well as its low cost, and effectiveness. Through inspecting the related previous articles, it was found that ZnO nanopowder doped with Al were reported only by a small number of researchers especially their optical properties in the UV-vis-near Ir region. So, this study is performed to prepare Al-free and Al-doped ZnO nanopowder using the co- precipitation method, to estimate the effect of Al-doping on structural and optical properties of ZnO nanopowder. 2. Experimental Work: Pure and Al-doped ZnO nanoparticles were prepared by the chemical co-precipitation method. The materials used in this study without any purification include: Zinc acetate de hydrate {Zn (CH3COO)2 .2H2O}, sodium hydroxide (NaOH), Aluminum chloride Anhydrous (AlCl3). The experimental procedure for the preparation of pure ZnO and Al-doped ZnO samples are as follows: for the preparation of pure ZnO nanoparticles, 21.950 g of Zinc Acetate was dissolved in 100 ml distilled water and the solution mixed to be homogenous, then 16 g of sodium hydroxide was dis- solved in distilled water. Next NaOH solution was added drop wise to obtain homogenous mixed solution, yielding a white precipitate. The white precipitate was stirred at room temperature for 2 hours. After that the solution was washed several times with distilled water and ethanol. The white powder was dried at 70 ºC for 3 hours followed by further heating at 400 ºC for 4 hrs, finely the white powder was grind using agate mortar. For the synthesis, Al doped ZnO nanoparticles, (2%) of Aluminum chloride was dissolved in (20 ml) distilled water, and (21.51 g) of zinc acetate dissolved in (80 ml) distilled water solution after stirring both solutions for half an hour the AlCl3 was added dropwise to zinc acetate solution with continuous stirring then (16g) of sodium 2 hydroxide which dissolved in (100 ml) distilled water was added dropwise to this homogenous mixture to form a white precipitate. The solution with the white precipitate was processed as above to obtain Al-doped ZnO samples, table (1). The prepared samples were then characterized using different experimental methods, like the Powder X-ray diffractometer (XRD), at room temperature using a PW 1830 diffractometer with Cu Kα radiation (40 KV X 25 mA) and a graphite monochromatic, with 2θ values from 10 to 80 degrees. The optical measurements were obtained using Genway 6405-UV–visible Spectrophotometer which was used to get the optical UV–vis. Spectra, in the range 190 to 110 nm, at room temperature. While the FTIR spectra were recorded, at room temperature, using Fourier transform Infrared (FTIR) spectrometer in the range from 4000 to 400 cm−1. Table (1) Samples Composition Sample Code x wt (%) ZnO wt % AlCl3 wt% AZ1 0 100 0 AZ2 4 96 4 AZ3 6 94 6 AZ4 8 92 8 Table (1) Samples Composition 3.1 Structural phase identification: Figure (1) illustrates the normalized XRD patterns for the Aluminum-free sample and Aluminum- doped samples, where the pattern of each sample consists of eight- sharp peaks of different amplitudes and different positions. The existence of such a number of peaks means a variety of crystalline planes (hkl) that can be characterized according to their positions as in table (2). Based on the standard data on JCPDS card No. 36 –1451) all these data remark the structure matrix of Zinc oxide, ZnO, in other words, each sample has a pure hexagonal structure with well-developed crystallinity, where Al3+ ions were substituted into the sites of Zn2+ ions and/or incorporated into interstitial sites in the lattice without altering the hexagonal structure of ZnO [22]. it was observed when the content of Al3+ increased above 6% the positions of the crystalline phases/peaks exhibit a slight shift towards the higher diffraction's angle 2θ°, which may be attributed to the lattice shrinkage caused by the replacing of the Zn2+ (radius 0.74 ̊A) by Al3+ (radius 0.53 ̊A) [23-26]. 3 Williamson-Hall (W-H) model, equation (1), was used to determine the average crystallite size (D) and the micro-strain (ε) for all samples; Williamson-Hall (W-H) model, equation (1), was used to determine the average crystallite size (D) and the micro-strain (ε) for all samples; βcosθ = Kλ + 4ε sinθ D (1) βcosθ = Kλ + 4ε sinθ D (1) βcosθ = Kλ + 4ε sinθ D βcosθ = Kλ + 4ε sinθ D (1) 𝛽ℎ𝑘𝑙= 𝐾𝜆 𝑑ℎ𝑘𝑙 𝐶𝑜𝑠 𝜃ℎ𝑘𝑙 + 4 𝜀tan 𝜃ℎ𝑘𝑙 𝛽ℎ𝑘𝑙𝐶𝑜𝑠 𝜃ℎ𝑘𝑙= 𝐾𝜆 𝑑ℎ𝑘𝑙 + 4 𝜀Sin 𝜃ℎ𝑘𝑙 𝛽ℎ𝑘𝑙= 𝐾𝜆 𝑑ℎ𝑘𝑙 𝐶𝑜𝑠 𝜃ℎ𝑘𝑙 + 4 𝜀tan 𝜃ℎ𝑘𝑙 𝛽ℎ𝑘𝑙𝐶𝑜𝑠 𝜃ℎ𝑘𝑙= 𝐾𝜆 𝑑ℎ𝑘𝑙 + 4 𝜀Sin 𝜃ℎ𝑘𝑙 𝛽ℎ𝑘𝑙= 𝐾𝜆 𝑑ℎ𝑘𝑙 𝐶𝑜𝑠 𝜃ℎ𝑘𝑙 + 4 𝜀tan 𝜃ℎ𝑘𝑙 (2) 𝛽ℎ𝑘𝑙𝐶𝑜𝑠 𝜃ℎ𝑘𝑙= 𝐾𝜆 𝑑ℎ𝑘𝑙 + 4 𝜀Sin 𝜃ℎ𝑘𝑙 (3) (3) Where β is the full width at half maximum (FWHM) of the peak, θ is the Bragg's diffraction angle, K is the shape factor (K = 0.9), λ is the wavelength for CuKα radiation (λ = 1.54056 Å). Williamson [25-27] proposed the dependency of the diffraction line broadening on the crystallite size and strain contribution as shown in relation (2), which may further simplified to be as relation (3), Williamson Hall (W–H) equation. 3.1 Structural phase identification: By plotting thee right hand side of relation (3), 𝛽ℎ𝑘𝑙𝐶𝑜𝑠 𝜃ℎ𝑘𝑙, versus 4 Sin 𝜃ℎ𝑘𝑙, as shown in figure (2), both micro-strain ε and the crystal size can be obtained. Where the slope of the resulted line gives the micro-strain ε, while the y-intercept gives the crystallite size. Figure (3) depicts that the obtained average crystallite size increase approximately linearly when ZnO is replaced by AlCl3, where the Al-doped samples showed crystalline sizes larger than the Al-free one, which suggests the enhancement of crystalline quality. While figure (4) showed that the obtained micro-strain increases slightly when AlCl3 content increases, which may be due to lattice mismatch with increasing the Al concentration. Figure (5) exhibits Fourier-transform infrared spectroscopy, FTIR, for the Al-free sample and Al- doped samples in the wavelength range 400-4000 cm-1. The absorption peak at 477 cm-1 is corresponding to the stretching mode of ZnO vibration. The absorption peak around 570 cm-1 attributed to presence of Al in ZnO lattice. The stretching mode of vibration bands due to C=O is observed between 1600-1400 cm-1. A broad band of absorption were observed at 1097 cm-1 , 1384 cm-1 and 840 cm-1 effect the presence of H2O (O-H) and CO2 (C-O) that absorbed from the air, so can be ignored [28-29]. Those results confirmed the reducing the bonding force between acetate anion and zinc cations when the structural phase transformed from zinc acetate to ZnO, where the OH-groups gradually replaced with acetate groups, which removed completely to forming Zn(OH)2, and hence/finally ZnO could be formed with the release more of acetate anion [30]. 4 Table (2): detectable crystalline Planes 5 Table (2): detectable crystalline Planes Phase No. Position (2θ°) Crystalline Plane (hkl) Refs. 1 31.69 (100) [19-20-21] 2 34.36 (002) 3 36.18 (101) 4 47.46 (102) 5 56.52 (110) 6 62.80 (103) 7 67.88 (112) 8 69.01 (201) Figure (1): XRD patterns for all samples 10 20 30 40 50 60 70 AZ4 AZ3 AZ1 Intensity (a.u) 2qo AZ2 (100) (002) (101) (102) (110) (103) (112) (201) Phase No. Position (2θ°) Crystalline Plane (hkl) Refs. 3.2 UV-vis spectral analysis & Optoelectronic parameters: The study of non-centrosymmetric substances like ZnO nanopowder is of most importance in estimate the electronic structure according to the optical properties, which are of high importance in updating both the electronics and optoelectronic devices for the different applications. In the current study some optical parameters like the transmittance, and absorbance were measured, while some others like absorption coefficient, linear refractive, and nonlinear refractive index were calculated [31-32-33]. For the studied samples both the optical absorbance and optical transmittance were measured then normalized, by dividing by the highest value, to avoid the instrumental errors. Figure (6) shows the variation of the normalized optical absorbance with the change in the wavelength of the incident light, where all samples exhibit approximately the same cut-off wavelength around 293 nm, in addition to an absorption peak at 373 nm which represent the optical bandgap of ZnO in the UV region. Figure (6) also clarify the effect of All-content on the optical absorption, where the increase of Al-content act to increase the optical absorption, such increase may be due to the observed increase in the average crystal size, as shown in figure (3). Figure (7) illustrates that all the studied samples have a wide transmission window extended over all the visible range, which may suggest them for multi applications in this region. According to relation (4), [34], both the optical absorption of each sample and its thickness (t) were used to calculate its absorption coefficient α, which is an important factor for determining the energy bandgaps for both direct and indirect allowed transitions, by using Tauc’s relation (5), [35-36]; 𝛼= 2.303 ∗ 𝐴 𝑡 (4) 𝛼𝐸= 𝛼𝑜 (𝐸−𝐸𝑔)ϳ (5) 𝛼= 2.303 ∗ 𝐴 𝑡 𝛼𝐸= 𝛼𝑜 (𝐸−𝐸𝑔)ϳ (4) (5) 𝛼𝐸= 𝛼𝑜 (𝐸−𝐸𝑔)ϳ Where αo is a constant called band tailing parameter, Eg is the optical energy gap, and j is the power factor of the transition mode. The values of j for both direct and indirect transitions are ½ and 2, respectively. To determine the value of the energy bandgap for indirect allowed transition the quantity (αE)0.5 was plotted versus the photon energy E, as shown in figure (8), where the direct bandgap energy is equal to the intercept of the straight portion curve with the x-axis. 3.1 Structural phase identification: 1 31.69 (100) [19-20-21] 2 34.36 (002) 3 36.18 (101) 4 47.46 (102) 5 56.52 (110) 6 62.80 (103) 7 67.88 (112) 8 69.01 (201) 10 20 30 40 50 60 70 AZ4 AZ3 AZ1 Intensity (a.u) 2qo AZ2 (100) (002) (101) (102) (110) (103) (112) (201) 10 20 30 40 50 60 70 AZ4 AZ3 AZ1 Intensity (a.u) 2qo AZ2 (100) (002) (101) (102) (110) (103) (112) (201) Figure (1): XRD patterns for all samples 5 5 Figure (2): βCosθ versus 4Sinθ Figure (3): crystal size versesAlCl3 concentration 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 0.005 0.006 0.007 0.008 0.009 0.010 b Cosq 4 Sinq AZ1 AZ2 AZ3 AZ4 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 0.005 0.006 0.007 0.008 0.009 0.010 b Cosq 4 Sinq AZ1 AZ2 AZ3 AZ4 Figure (2): βCosθ versus 4Sinθ Figure (3): crystal size versesAlCl3 concentration 6 Figure (4): strain as a function of Al content. Figure (5) FTIR spectra for all the prepared samples. 0.00 0.02 0.04 0.06 0.08 2.3 2.4 2.5 2.6 2.7 e x 10-3 AlCl3 content AZ1 AZ2 AZ3 AZ4 0.00 0.02 0.04 0.06 0.08 2.3 2.4 2.5 2.6 2.7 e x 10-3 AlCl3 content AZ1 AZ2 AZ3 AZ4 Figure (4): strain as a function of Al content. Figure (5) FTIR spectra for all the prepared samples. Figure (4): strain as a function of Al content. Figure (4): strain as a function of Al content. Figure (5) FTIR spectra for all the prepared samples. 7 7 3.2 UV-vis spectral analysis & Optoelectronic parameters: And by the same method, it can determine the value of the bandgap energy for the direct allowed transition by plot (αE)2 versus E, as shown in figure (9). The calculations showed that the replacement of ZnO 8 by AlCl3 act to increase the optical bandgaps from 3.53 to 3.75 for the indirect allowed electronic transitions, and from 3.75 to 4.1 eV for the direct allowed electronic transitions. Figure (6): The normalized Absorbance, for all the prepared samples. 300 350 400 450 500 550 600 650 700 750 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 Optical absorbance l, nm AZ1 AZ2 AZ3 AZ4 3.34 eV Figure (6): The normalized Absorbance, for all the prepared samples. 3.2 UV-vis spectral analysis & Optoelectronic parameters: 𝐾(𝐸) = 𝛼 ℎ𝑐 4𝜋𝐸 (6) 𝑅= 100−𝐴−𝑇% 100 (7) (6) (7) 100 11 𝑛= 1+𝑅 1−𝑅+ √ 4𝑅 (1−𝑅)2 + 𝐾2 (8) 𝜀′ = 𝑛2 − 𝐾2 (9) 𝜀′′ = 2 𝑛𝐾 (10) 𝜀∗= 𝜀′ + 𝑗 𝜀′′ (11) 𝑛= 1+𝑅 1−𝑅+ √ 4𝑅 (1−𝑅)2 + 𝐾2 𝜀′ = 𝑛2 − 𝐾2 𝜀′′ = 2 𝑛𝐾 𝜀∗= 𝜀′ + 𝑗 𝜀′′ (8) (9) (10) (11) Figures 10(a-d) represents the vibration of 𝜺′ and 𝜺′′ with the energy of the incident photons for x = 0, 0.04, 0.06 and 0.08, respectively. Where the real component 𝜺′ increases while the imaginary component (ε'') decreased, in such a way where both of them show a peak in the same position (3.3 eV). Such a result can be used to obtain the average plasma frequency𝑓𝑜= 8 𝑥 1014 𝐻𝑧 which is higher value expresses the existence of a high concentration of free carriers [37]. 3.2 UV-vis spectral analysis & Optoelectronic parameters: Figure (7): The optical transmittance, for all the prepared samples 300 350 400 450 500 550 600 650 700 750 94 95 96 97 98 99 Optical transmittance T% l, nm AZ1 AZ2 AZ3 AZ4 3.34 eV 300 350 400 450 500 550 600 650 700 750 94 95 96 97 98 99 Optical transmittance T% l, nm AZ1 AZ2 AZ3 AZ4 3.34 eV Figure (7): The optical transmittance, for all the prepared samples 9 Figure (7): The optical transmittance, for all the prepared samples 9 Figure (8): (αE) 0.5 versus E=hν for all the prepared samples 1.5 2.0 2.5 3.0 3.5 4.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 3 4 5 6 7 (ahn)0.5 E, (eV) AZ1 AZ2 AZ3 AZ4 AZ4 E, (eV) 1.5 2.0 2.5 3.0 3.5 4.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 3 4 5 6 7 (ahn)0.5 E, (eV) AZ1 AZ2 AZ3 AZ4 AZ4 Figure (8): (αE) 0.5 versus E=hν for all the prepared samples Figure (8): (αE) 0.5 versus E=hν for all the prepared samples 10 Figure (9): (αE) 2 versus (E=hν), for all the prepared samples 1.5 2.0 2.5 3.0 3.5 4.0 1.5 2.0 2.5 3.0 3.5 4.0 0 500 1000 1500 2000 2500 3000 3500 E, (eV) (aE)2 E, (eV) AZ1 AZ2 AZ3 AZ4 AZ4 1.5 2.0 2.5 3.0 3.5 4.0 1.5 2.0 2.5 3.0 3.5 4.0 0 500 1000 1500 2000 2500 3000 3500 E, (eV) (aE)2 E, (eV) AZ1 AZ2 AZ3 AZ4 AZ4 Figure (9): (αE) 2 versus (E=hν), for all the prepared samples 10 Figure (9): (αE) 2 versus (E=hν), for all the prepared samples 10 Figure (10): (𝜺′) and (𝜺′′) as a functions of the photon energy Figure (10): (𝜺′) and (𝜺′′) as a functions of the photon energy Based on the measured optical parameters the optical dielectric relaxation can be organized in view of the real (𝜺′) and imaginary (𝜺′′) components of the optical dielectric constant ε* [37-38]. Where for unfree damper, the real component (𝜺′) characterize the damping of the light propagation thorough the material/medium. Also this component, which related to the energy stored within the medium, can be considered as accounts for electromagnetic dispersions. In other side, the imaginary component is considered as a damping factor describes the amount of energy loss and/or absorbed within the medium [37-38]. Conclusion: Four Nano composite powder samples have been prepared base on the chemical reaction formula, Zn1-x. Alx, where 0 ≤ x ≤ 0.1 in wt% using the co-precipitation method. The substitution of ZnO by Al setup seven crystalline phases/planes (100), (002), (101), (102), (110), (103), and (201). For characterizing the obtained samples the average crystalline size and the micro-strain were calculated and were found to be increase with increasing Al content. All the studied samples exhibited high optical transparency in the visible light region. Direct and indirect energy gaps, the absorption coefficient, and nonlinear refractive index were calculated and showed increasing values with increasing Al content. The obtained results are considered promising for different optoelectronic applications. 3.4 Nonlinear optical parameters 𝜒(1) = 𝑛2−1 4𝜋 (12) 𝜒(3) = 1.7 𝑥 10−10 (𝜒(1))4 (13) 𝑛2 = 12𝜋 𝜒(3) 𝑛 (14) 𝜒(1) = 𝑛2−1 4𝜋 (12) 𝜒(3) = 1.7 𝑥 10−10 (𝜒(1))4 (13) 𝑛2 = 12𝜋 𝜒(3) 𝑛 (14) 𝜒(1) = 𝑛2−1 4𝜋 𝜒(3) = 1.7 𝑥 10−10 (𝜒(1))4 𝑛2 = 12𝜋 𝜒(3) 𝑛 (12) (13) (14) Frequency conversion materials especially nanopowder semiconductors are important for nonlinear optical applications, so it is useful to identify the nonlinear properties of the studied samples to recognize if they are suitable for nonlinear devices and applications or not. For this promotion, it's favorable to use the Z-scan technique but it's not available for now at least so the set of the previous relations should be used to obtain the nonlinear parameters of the studied samples based on UV-vis measurements. Figure (11) shows the calculated nonlinear refractive index n2, where the obtained values for the Al-free sample and Al-doped samples is better than those reported previously [38-39], which may suggest these samples for nonlinear applications. Frequency conversion materials especially nanopowder semiconductors are important for nonlinear optical applications, so it is useful to identify the nonlinear properties of the studied samples to recognize if they are suitable for nonlinear devices and applications or not. For this promotion, it's favorable to use the Z-scan technique but it's not available for now at least so the set of the previous relations should be used to obtain the nonlinear parameters of the studied samples based on UV-vis measurements. Figure (11) shows the calculated nonlinear refractive index n2, where the obtained values for the Al-free sample and Al-doped samples is better than those reported previously [38-39], which may suggest these samples for nonlinear applications. 12 Figure (11): Nonlinear refractive index n2 300 350 400 450 500 550 600 650 700 750 0.0 2.0x10-13 4.0x10-13 6.0x10-13 8.0x10-13 1.0x10-12 AZ1 AZ2 AZ3 AZ4 n2 l, nm 300 350 400 450 500 550 600 650 700 750 0.0 2.0x10-13 4.0x10-13 6.0x10-13 8.0x10-13 1.0x10-12 AZ1 AZ2 AZ3 AZ4 n2 l, nm Figure (11): Nonlinear refractive index n2 Conflict of interest: All the authors declare that there is no conflict of interest. 13 4. References 1. Coleman, VA., Bradby, JE., Jagadish, C., Munroe, P., Heo, YW., Pearton, SJ., Norton, DP., Inoue, M., Yano, M., 2005 2. Yadav, BC. Richa Srivastava, and Alok Kumar, 2007. International Journal of Nanotechnology and Applications. ISSN 0973-631, 1-11. 3. Bouvy, C., Marine, W., Sporken, R., Su, BL., 2007. Colloids and Surfaces A: Physicochem. Eng Aspects 300, 145 149. 4. Pivin, J.C., Socol, G., Mihailescu, I., Berthet, P., Singh, F., Patel, M.K.: Thin Solid Films 517, 916–922 (2008). 5. Sato, K., Yoshida, H.K.: Jpn. J. Appl. Phys. 39, L555–L558 (2000). 6. Avinash K, Saritha D, Joginder S, Mayank K, Vishnu dev G. Synthesis and characterization of ZnO nanoparticles. Ori-ental Journal of Chemistry. 2014; 30. 7. Alessio B. Maximilian Du Pierandrea Lo Nostro Æ Pie-ro Baglioni. Synthesis and characterization of zinc oxide nanoparticles: Application to textiles as UV-absorbers. J Nanopart Res; 2008; 10:679- 89. DOI 10.1007/s11051-007- 9318-3 8. H. Serier, M. Gaudon, M. Ménétrier, Al-doped ZnO powdered materials: Al solubility limit and IR absorption properties, Solid State Sci.11 (7) (2009) 1192–1197. 9. B. Zhang, N. Binh, K. Wakatsuki, Y. Segawa, Y. Yamada, N. Usami, M. Kawasaki, H .Koinuma, Formation of highly aligned ZnO tubes on sapphire (0 0 0 1 ) substrates, Appl. Phys. Lett. 84 (20) (2004) 4098–4100. 10. E. Hammarberg, A .Prodi-Schwab, C. Feldmann, Microwave-assisted polyol synthesis of aluminium-and indium-doped ZnO nanocrystals, J. Colloid Interface Sci.334 (1) (2009)29–36. 11. L. Ji, W. Shih, T. Fang, C. Wu, S. Peng, T. Meen, Preparation and characteristics of hybrid ZnO-polymer solar cells, J. Mater. Sci. 45(12) (2010) 3266–3269. 12. Boshra Ghanbari Shohany and Ali Khorsand Zak, Ceramics International, https://doi.org/10.1016/j.ceramint.2019.11.051 13. T. Ogi, D. Hidayat, F. Iskandar, A. Purwanto, K. Okuyama, Direct synthesis of highly crystalline transparent conducting oxide nanoparticles by low pressure spray pyrolysis, Adv. Powder Technol. 20(2)(2009) 203–209. 14 14. H. Zhang, D. Yang, S.Li, X. Ma, Y. Ji, J. Xu, D. Que, Controllable growth of ZnO nanostructures by citric acid assisted hydrothermal process, Mater. Lett .59 (13) (2005) 1696–1700. 15. Zhou H M, Yi D Q, Yu Z M, Xiao L R and Li J 2007 Thin Solid Films 515 6909. 16. Chen K J, Fang T H, Hung F Y, Ji LW, Chang S J, Young S J and Hsiao Y J 2008 Appl. Surf. Sci. 254 5791 17. Li QH, Zhu D, Liu W, Liu Y, Ma XC (2008) Optical properties of Al-doped ZnO thin films by ellipsometry. 4. References Appl Surf Sci 254 (10):2922–2926 18. Ehrmann N, Reineke-Koch R (2010) Ellipsometric studies on ZnO:Al thin films: refinement of dispersion theories. Thin Solid Films 519(4):1475–1485 19. L.H. Van, M.H. Hong, J. Ding, Structural and magnetic property of Co-doped- ZnO thin films prepared by pulsed laser deposition, J. Alloys Comp. 449 (1) (2008) 207–209. 20. K. Gherab, Y. Al-Douri, C.H. Voon, U. Hashim, M. Ameri, A. Bouhemadou. Result in Physics 7 (2017) 1190-1197 21. P. Swapna and S. Venkatramana Reddy*. Synthesis and Characterization of Al Doped and (Co, Al) co-doped ZnO Nanoparticles via Chemical co-precipitation Method. Asian Journal of Nanoscience and Materials, 2018, 2(1), 111-119. 22. Mallika AN, Ramachandra Reddy A, Sowribabu K, Venugopal Reddy K. Structural and optical characterization of Zn0.95_xMg0.05AlxO nanoparticles. Ceram Int 2015; 41:9276–84 23. Kadam P, Agashe C, Mahamuni S (2008) J Appl Phys 104: 103501. doi: 10.1063/1.3020527 24. Park KC, Ma DY, Kim KH (1997) Thin Solid Films 305:201. 24. Park KC, Ma DY, Kim KH (1997) Thin Solid Films 305:201. 25. Navale SC, Ravi V, Mulla IS, Gosavi SW, Kulkarni SK (2007) Sens Actuators B 126:382. doi: 10.1016/ j.snb. 2007.03.019 26. Reddy AJ, Kokila MK, Nagabhushana H, Chakradhar RPS, Shivakumara C, Rao JL, et al. Structural, optical and EPR studies on ZnO: Cu nanopowders prepared via low temperature solution combustion synthesis. J Alloys Compd 2011; 509:5349–55. 27. R. Bhargava, P.K. Sharma, S. Kumar, A.C. Pandey, N. Kumar, Consequence of doping mediated strain and the activation energy on the structural and optical properties of ZnO:Cr nanoparticles, Journal of Solid State Chemistry 183 (2010) 1400. 15 ari S.V. Bhat r., Deshpande R., Venkataraman A., Recent Res. Sci. Tech., 4 (2012), 50. 29. Zahraa M. AL-Asady, Ali H. AL-Hamdani, and Mohammed A. Hussein, AIP Conference Proceedings 2213, 020061 (2020); https://doi.org/10.1063/5.0000259 . 30. Zohra N. Kayani, Fareeha Naz, Saira Riaz, Shahzad Naseem, Journal of Saudi Chemical Society (2017) 21, 425-433. 31. Hossam Mohamed Gomaa, I. S. Ali, Ammar S. Morsy and M. I. Sayyed, J. of Applied Physics A, (2020), 126:384, https://doi.org/10.1007/s00339-020-03552-z 32. M. Y. Hassaan, H. A. Saudi, Hossam M. Gomaa and Ammar S. Morsy, J. of Materials and Applications 2020;9(1):46-54, https://doi.org/10.32732/jma.2020.9.1.46 33. Hossam M. Gomaa, M. Y. Hassaanm H. A. Soudi and Ammar S. Morsy, J. of Applied Physics A, (2020), 126:391, https://doi.org/10.1007/s00339-020-03582-7 34. S.M. Elkatlawy, et al., Structural properties, linear, and non-linear optical parameters of ternary Se80Te(20−x) Inxchalcogenide glass systems, Bol. Soc. Esp. Cerám. Vidr. 4. References (2020), https://doi.org/10.1016/j.bsecv.2020.09.007 35. M. Y. Hassaan et al., J. of Materials and Applications (2020) ;9(1):46-54 36. El-Mansy MK, Gomaa H. M, Hendawy N, Morsy AS. Effect of exchange of Bi+3 by Nb+5 on the structural and optical properties of some (BBiNb)2 O7 CaO oxide glasses. J. of Non- Crystalline Solids. 2018;485:42-46 37. Ahmed Saeed Hassanien and Ishu Sharma, J. of Optik, 200 (2020), 163415 38. V. Ganesh, I. Yahia, S. Alfaify, M. Shkir, Sn-doped Zno nanocrystalline thin flms with enhanced linear and nonlinear optical properties for optoelectronic applications. J. Phys. Chem. Solids 100, 115–125 (2017). https://doi.org/10.1016/J.Jpcs.2016.09.022 39. Ahmed Saeed Hassanien, J. of Alloys Compd. 671, (2016) 566-578. 16
https://openalex.org/W2791282328	https://strathprints.strath.ac.uk/64649/1/Ottoboni_etal_CGD_2018_Impact_of_paracetamol_impurities_on_face_properties.pdf	English	null	Impact of Paracetamol Impurities on Face Properties: Investigating the Surface of Single Crystals Using TOF-SIMS	Crystal growth & design	2,018	cc-by	8,834	This is an open access article published under a Creative Commons Attribution (CC-BY) License, which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited. Time of ﬂight secondary ion mass spectrometry (TOF-SIMS) is valuable in determining molecular identity and distribution. By combining TOF-SIMS, SEM, and optical (OM) and Raman microscopies, we can evaluate the usefulness of TOF-SIMS as a surface characterization technique for pharmaceutical crystals. 4-Nitrophenol has been selected as an impurity that can be incorporated during crystallization of acetaminophen (paracetamol). This study explores the distribution of impurity and its concentration on the diﬀerent crystal faces of samples obtained by crystallization over a range of impurity loadings and supersaturation conditions. Raman maps of paracetamol single crystal faces were analyzed using the characteristic Raman peak intensity of 4-nitrophenol to identify regions where it accumulated; Raman maps of three single crystals produced in the presence of 4-nitrophenol using diﬀerent crystallization procedures highlight how it can be diﬃcult to detect very low concentrations of similar chemical species. In contrast, the 4-nitrophenol monoisotopic mass obtained via TOF-SIMS was shown to be detectable in all the three single crystals produced. This indicates that TOF-SIMS can be a valuable technique for single crystal impurity distribution mapping even when the impurity concentration is very low. This is an open access article published under a Creative Commons Attribution (CC-BY) License, which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited. This is an open access article published under a Creative Commons Attribution (CC-BY) License, which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited. This is an open access article published under a Creative Commons Attribution (CC-BY) License, which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited. pubs.acs.org/crystal Cite This: Cryst. Growth Des. 2018, 18, 2750−2758 Impact of Paracetamol Impurities on Face Properties: Investigating the Surface of Single Crystals Using TOF-SIMS Sara Ottoboni,,†,⊥ Michael Chrubasik,†,∥,⊥Layla Mir Bruce,‡,⊥Thai Thu Hien Nguyen,‡,⊥ Murray Robertson,§,⊥ Blair Johnston,†,⊥ Iain D. H. Oswald,§,⊥ Alastair Florence,†,⊥ and Chris Price†,⊥ Impact of Paracetamol Impurities on Face Properties: Investigating the Surface of Single Crystals Using TOF-SIMS Sara Ottoboni,,†,⊥ Michael Chrubasik,†,∥,⊥Layla Mir Bruce,‡,⊥Thai Thu Hien Nguyen,‡,⊥ Impact of Paracetamol Impurities on Face Properties: Investigating the Surface of Single Crystals Using TOF-SIMS †EPSRC Centre for Innovative Manufacturing in Continuous Manufacturing and Crystallisation, University Technology and Innovation Centre, Level 6, CMAC, 99 George Street, G1 1RD Glasgow, U.K. §Strathclyde Institute of Pharmacy & Biomedical Sciences (SIPBS), University of Strathclyde, 161 Cathedral Street, G4 0RE, Glasgow, U.K. §Strathclyde Institute of Pharmacy & Biomedical Sciences (SIPBS), University of Strathclyde, 161 Cathedral Street, G4 0RE, Glasgow, U.K. Downloaded via UNIV OF STRATHCLYDE on June 29, 2018 at 13:51:13 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published artic ∥National Physical Laboratory Scottish Hub, University of Strathclyde, G1 1RD Glasgow, U.K. ∥National Physical Laboratory Scottish Hub, University of Strathclyde, G1 1RD Glasgow, U.K. ABSTRACT: Understanding the mechanism of interaction between pharmaceutical molecules (APIs) and impurities on crystal surfaces is a key concept in understanding puriﬁcation and for the design of pharmaceutical crystallization processes. Several techniques may be used to study crystal surface properties, such as scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS), which provide detailed imaging and elemental surface characterization. Time of ﬂight secondary ion mass spectrometry (TOF-SIMS) is valuable in determining molecular identity and distribution. By combining TOF-SIMS, SEM, and optical (OM) and Raman microscopies, we can evaluate the usefulness of TOF-SIMS as a surface characterization technique for pharmaceutical crystals. 4-Nitrophenol has been selected as an impurity that can be incorporated during crystallization of acetaminophen (paracetamol). This study explores the distribution of impurity and its concentration on the diﬀerent crystal faces of samples obtained by crystallization over a range of impurity loadings and supersaturation conditions. © 2018 American Chemical Society This is an open access article published under a Creative Commons Attribution (CC-BY) License, which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited. Raman maps of paracetamol single crystal faces were analyzed using the characteristic Raman peak intensity of 4-nitrophenol to identify regions where it accumulated; Raman maps of three single crystals produced in the presence of 4-nitrophenol using diﬀerent crystallization procedures highlight how it can be diﬃcult to detect very low concentrations of similar chemical species. In contrast, the 4-nitrophenol monoisotopic mass obtained via TOF-SIMS was shown to be detectable in all the three single crystals produced. This indicates that TOF-SIMS can be a valuable technique for single crystal impurity distribution mapping even when the impurity concentration is very low. ABSTRACT: Understanding the mechanism of interaction between pharmaceutical molecules (APIs) and impurities on crystal surfaces is a key concept in understanding puriﬁcation and for the design of pharmaceutical crystallization processes. Several techniques may be used to study crystal surface properties, such as scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS), which provide detailed imaging and elemental surface characterization. Time of ﬂight secondary ion mass spectrometry (TOF-SIMS) is valuable in determining molecular identity and distribution. By combining TOF-SIMS, SEM, and optical (OM) and Raman microscopies, we can evaluate the usefulness of TOF-SIMS as a surface characterization technique for pharmaceutical crystals. 4-Nitrophenol has been selected as an impurity that can be incorporated during crystallization of acetaminophen (paracetamol). This study explores the distribution of impurity and its concentration on the diﬀerent crystal faces of samples obtained by crystallization over a range of impurity loadings and supersaturation conditions. Raman maps of paracetamol single crystal faces were analyzed using the characteristic Raman peak intensity of 4-nitrophenol to identify regions where it accumulated; Raman maps of three single crystals produced in the presence of 4-nitrophenol using diﬀerent crystallization procedures highlight how it can be diﬃcult to detect very low concentrations of similar chemical species. In contrast, the 4-nitrophenol monoisotopic mass obtained via TOF-SIMS was shown to be detectable in all the three single crystals produced. This indicates that TOF-SIMS can be a valuable technique for single crystal impurity distribution mapping even when the impurity concentration is very low. ABSTRACT: Understanding the mechanism of interaction between pharmaceutical molecules (APIs) and impurities on crystal surfaces is a key concept in understanding puriﬁcation and for the design of pharmaceutical crystallization processes. Several techniques may be used to study crystal surface properties, such as scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS), which provide detailed imaging and elemental surface characterization. Received: October 5, 2017 Revised: February 14, 2018 Published: March 26, 2018 preparation raw material: supersaturated solution of paracetamol with 4 mol % of 4- nitrophenol temperature proﬁle: dissolution = 50 °C cooling = 50 °C down to 5 °C cooling = 50 °C down to 5 °C isolation = 5 °C raw material: paracetamol single crystal grown by the method above, 4- nitrophenol solution (1500 mg/g ethanol) procedure: dropping 4-nitrophenol solution on a ﬂat dominant surface of PP and allowing the solvent to evaporate very ﬁne needle-like 4-nitrophenol crystals were crystallized on the surface of paracetamol crystal and in the solution (PEN) crystal with 4- nitrophenol deposit grown epitaxially temperature proﬁle: immersion = room temperature temperature proﬁle: immersion = room temperature cooling = room temperature down to 5 °C cooling = room temperature down to 5 °C isolation = room temperature procedure: immersing the PP crystal in the saturated solution of 4- nitrophenol and allowing 4-nitrophenol crystals to grow on the paracetamol crystal surface procedure: immersing the PP crystal in the saturated solution of 4- nitrophenol and allowing 4-nitrophenol crystals to grow on the paracetamol crystal surface may be diﬃcult. Furthermore, these impurity molecules have the capability to modify API solubility, nucleation, and crystal growth resulting in changes in crystal morphology and crystallization kinetics. crystal morphology are reported in the literature. Optical (OM) and scanning electron microscopy (SEM) are useful to investigate crystal morphology, aspect ratio, and, to provide some information about surface texture. Saleemi17 reported the inﬂuence of metacetamol on paracetamol crystal morphology, whereby the habit was altered from a tabular to columnar with increasing metacetamol content during crystal- lization. The authors also analyzed the variation of induction time, crystal size distribution (PSD), and solution concen- tration during paracetamol crystallization; these were enabled by the use of in situ UV spectroscopy and focused beam reﬂectance measurements (FBRM) 16 15 y Paracetamol, also known as acetaminophen, is a molecular organic compound and an API that shows analgesic and antipyretic eﬀects.2 Paracetamol consists of a benzene ring core, substituted by one hydroxyl group and a methylamide group in the para position.3,4 The amide and hydroxyl groups act as hydrogen bond donors, whereas the carbonyl and hydroxyl groups act as acceptors. preparation Paracetamol has three diﬀerent polymorphic forms: form I, the stable form, crystallizes in a monoclinic structure; metastable form II adopts an ortho- rhombic structure; and form III is the most metastable form.5−8 The habit of paracetamol form I crystals can vary from being plate-like to needle-like depending on the crystallization conditions. The calculated principal facets of paracetamol (BFDH modeling) and paracetamol functional groups exposed on each facet are evaluated in the Supporting Information.9,10 11 Prasad et al.16 and Chow et al.15 examined the role of 4- acetoxyacetanilide solution concentrations and supersaturation levels on the transition of paracetamol crystals from columnar to plate-like habits. In addition to this they were able, using scanning and optical microscopy, to investigate shape and strain/defect content, as well as quantiﬁcation of 4- acetoxyacetanilide incorporation into paracetamol crystals. g Hendriksen and Grant11 showed that some substances, which are structurally related to paracetamol, such as acetanilide and metacetamol inhibit the primary nucleation of paracetamol in aqueous solution. They also suggested that the nucleation rate is modiﬁed through a mechanism similar to that previously reported for crystal growth modiﬁcation by structurally related additives. Hendriksen et al.10 and then Kuvadia and Doherty12 have proposed that structurally related additives may inﬂuence the nucleation and growth of crystals in three principal ways: Optical and electron microscopy and atomic force microscopy (AFM) allow surface roughness and crystal growth mechanism to be explored. For example, Thompson et al.13 investigated how the morphology and chemical properties of the (001) face of paracetamol can be aﬀected by the presence of diﬀerent structurally related impurities. One of the challenges faced in previous investigations was extracting information about both the chemical and topo- graphic character of individual crystal faces using a single technique. In this work, we have explored alternative analytical tools such as Raman mapping and time of ﬂight secondary ion mass spectrometry (TOF-SIMS) in order to evaluate their eﬀectiveness and sensitivity when used to simultaneously detect impurity distribution on the crystal surface and surface modiﬁcation caused by the structurally related impurities. • Blocking adsorption of solute molecules onto the crystal surface and therefore inducing morphological changes;10,13 for example, 4-acetoxyacetanilide acts as a blocking impurity inhibiting crystal growth on the (110) facet. • Docking onto the surface and become incorporated into the crystal lattice without signiﬁcantly aﬀecting the arrival of further API molecules;10 metacetamol operates in this way. ■INTRODUCTION are produced during the diﬀerent synthesis steps, all of which have chemical structures that are, to some degree, similar to the API, as are any degradation products.1 Active pharmaceutical ingredients (API) are typically produced by chemical synthesis. Various components, including residual solvents and trace amounts of inorganic and organic compounds including unreacted starting materials and by- products, are typically present at the end of the synthesis and are removed to a large extent by crystallization. All of these substances that remain in the ﬁnal API product are deﬁned as impurities. Impurities related to the synthesis can be classiﬁed as intermediate, penultimate intermediate, and byproducts that Structurally related substances can change API crystallization behavior. When the solubility of these substances is similar to or less than the API solubility, elimination of these substances Received: October 5, 2017 Revised: February 14, 2018 Published: March 26, 2018 2750 DOI: 10.1021/acs.cgd.7b01411 Cryst. Growth Des. 2018, 18, 2750−2758 2750 Article DOI: 10.1021/acs.cgd.7b01411 Cryst. Growth Des. 2018, 18, 2750−2758 Table 1. Crystallization Procedure for Single Crystals Prepared in This Work Table 1. Crystallization Procedure for Single Crystals Prepared in This Work d preparation single crystals obtained s raw material: supersaturated solution of paracetamol with 4 mol % of 4- nitrophenol pure paracetamol (PP) temperature proﬁle: dissolution = 50 °C pure 4-nitrophenol (PN) cooling = 50 °C down to 5 °C paracetamol crystal with the presence of 4 mol % 4- nitrophenol (P4%N) isolation = 5 °C - raw material: paracetamol single crystal grown by the method above, 4- nitrophenol solution (1500 mg/g ethanol) layer of 4-nitrophenol crystals deposited on the surface of the paracetamol crystal surface (PDN) procedure: dropping 4-nitrophenol solution on a ﬂat dominant surface of PP and allowing the solvent to evaporate raw material: paracetamol single crystal grown by the method above, saturated solution of 4-nitrophenol in hexane very ﬁne needle-like 4-nitrophenol crystals were crystallized on the surface of paracetamol crystal and in the solution (PEN) temperature proﬁle: immersion = room temperature crystallization method preparation single crystals obtained cooling crystallizations raw material: supersaturated solution of paracetamol with 4 mol % of 4- nitrophenol pure paracetamol (PP) temperature proﬁle: dissolution = 50 °C pure 4-nitrophenol (PN) cooling = 50 °C down to 5 °C paracetamol crystal with the presence of 4 mol % 4- nitrophenol (P4%N) isolation = 5 °C crystal with drop of 4- nitrophenol raw material: paracetamol single crystal grown by the method above, 4- nitrophenol solution (1500 mg/g ethanol) layer of 4-nitrophenol crystals deposited on the surface of the paracetamol crystal surface (PDN) procedure: dropping 4-nitrophenol solution on a ﬂat dominant surface of PP and allowing the solvent to evaporate crystal with 4- nitrophenol deposit grown epitaxially raw material: paracetamol single crystal grown by the method above, saturated solution of 4-nitrophenol in hexane very ﬁne needle-like 4-nitrophenol crystals were crystallized on the surface of paracetamol crystal and in the solution (PEN) temperature proﬁle: immersion = room temperature cooling = room temperature down to 5 °C isolation = room temperature procedure: immersing the PP crystal in the saturated solution of 4- nitrophenol and allowing 4-nitrophenol crystals to grow on the paracetamol crystal surface crystal with drop of 4- nitrophenol preparation crystallization method cooling crystallizations Crystal Growth & Design Article across the sample surface enables elemental and molecular surveying of the sample surface. across the sample surface enables elemental and molecular surveying of the sample surface. solvent to be used in this preparation, it was considered prudent to use an alternative solvent. Very ﬁne needle-like 4-nitrophenol crystals were formed on the surface of paracetamol crystal and in the surrounding solution. This study focuses on the API paracetamol and 4- nitrophenol as a paracetamol related impurity. 4-Nitrophenol was chosen as the representative impurity as it is a precursor in the synthesis of paracetamol and hence could be present as an impurity during the crystallization process. We investigate the limit of detection for this impurity on crystal surfaces by preparing samples using three diﬀerent crystallization techni- ques: cooling crystallization; surface deposition via solvent drop evaporation; and epitaxial growth. The solubility of both compounds in the diﬀerent solvents (ethanol and hexane) was investigated to control the supersaturation through prediction methods using COSMOTherm and by experimental measure- ment by isothermal equilibration. To provide a comparator to allow chemical and physical changes to be identiﬁed, pure crystals of paracetamol and 4-nitrophenol were prepared from an ethanol solution using the method described earlier. Solubility Experiments. The solubility of paracetamol in ethanol was taken from the literature;31 its solubility and that of 4-nitrophenol in ethanol and hexane were compared with predictions made using COSMOTherm and with experimental measurements made by isothermal equilibration. Preweighed suspensions of paracetamol and 4-nitrophenol in ethanol and hexane were prepared and placed in an incubator (Incubator S160D, Stuart) on a multiposition stirrer plate and held isothermally at the selected equilibration temperatures of 25, 40, and 55 °C, and equilibrated with agitation for 24 h. Once the 24 h equilibration period was competed, 1 mL samples were taken and the mass recorded. The samples were then left without lids in a fume hood for 24 h to allow the solvent to evaporate. After 24 h, the sample vials were weighed and their mass recorded. Finally, the sample vials were placed in a vacuum oven (Gallenamp) for 24 h at room temperature and (20 mbar) prior to recording the ﬁnal dry residue mass, which was used to calculate the solubility of each compound. Optical microscopy (OM) and scanning electron microscopy (SEM) were used in combination to evaluate crystal morphology, surface character, and aspect ratio. ■EXPERIMENTAL SECTION Materials. Paracetamol (4-actamidophenol, Bioxtra, ≥99%), 4- nitrophenol (≥99%), absolute ethanol (GC grade ≥99.8%), and n- hexane (≥95%) were all purchased by Sigma-Aldrich. 4-Nitrophenol is an intermediate in the synthesis of paracetamol;2 if present at the end of the synthesis, it could aﬀect the crystallization process, and if not rejected during the crystallization, it could be incorporated into the crystal lattice. 4-Nitrophenol shows oral, respiratory, and skin toxicity. 4-Nitrophenol shows speciﬁc target organ toxicity for repeated exposure. n-Hexane and ethanol are ﬂammable solvents. n-Hexane shows oral, respiratory, and skin toxicity. n-Hexane is suspect of carcinogenic eﬀects. Materials. Paracetamol (4-actamidophenol, Bioxtra, ≥99%), 4- nitrophenol (≥99%), absolute ethanol (GC grade ≥99.8%), and n- hexane (≥95%) were all purchased by Sigma-Aldrich. 4-Nitrophenol is an intermediate in the synthesis of paracetamol;2 if present at the end of the synthesis, it could aﬀect the crystallization process, and if not rejected during the crystallization, it could be incorporated into the crystal lattice. 4-Nitrophenol shows oral, respiratory, and skin toxicity. 4-Nitrophenol shows speciﬁc target organ toxicity for repeated exposure. n-Hexane and ethanol are ﬂammable solvents. n-Hexane shows oral, respiratory, and skin toxicity. n-Hexane is suspect of carcinogenic eﬀects. X-ray diﬀraction intensities were collected on a Bruker APEX 2 diﬀractometer coupled with an Incoatec IuS Mo microsource (0.71073 Å) operating at room temperature (293 K). A total of 780 frames was collected using three angular settings with a scan width of 0.5° in omega. Two crystals from each crystallization experiment were face- indexed to assign the Miller indices. g Methods. To test the sensitivity limit for impurity detection on crystal faces, three diﬀerent crystallization methods were implemented g Methods. To test the sensitivity limit for impurity detection on crystal faces, three diﬀerent crystallization methods were implemented (Table 1). Cooling Crystallization from Impure Solution. A single crystal of paracetamol containing 4-nitrophenol as an impurity was obtained by preparing a supersaturated solution of paracetamol (0.88 g) in absolute ethanol (5 g) by the addition of 4% mol of 4-nitrophenol (0.029 g). The solution was heated to 50 °C and allowed to cool to room temperature and then further to 5 °C in a refrigerator. A suitable single crystal of paracetamol with 4-nitrophenol (P4%N) of size exceeding 1 mm was then isolated from the solution. Chemical Character and Surface Texture Evaluation. preparation TOF-SIMS is a surface-sensitive analytical technique that is extensively used in material science18−22 and has begun to be applied in pharmaceutical applications in recent years.23−28 The technique utilizes a pulsed ion beam to release molecules from the outermost layers of a sample surface resulting in the emission of both neutral and charged species (atoms and molecules); the charged species are termed secondary ions.29,30 These ions, in turn, are extracted into a time-of-ﬂight mass analyzer for detection and quantiﬁcation. Moving the ion beam • Disrupting the emerging nucleus to inhibit the nucleation process;10 metacetamol is also described as a disrupting impurity. Various experimental methodologies to evaluate the role of structurally related impurities on crystal surface texture and These ions, in turn, are extracted into a time-of-ﬂight mass analyzer for detection and quantiﬁcation. Moving the ion beam DOI: 10.1021/acs.cgd.7b01411 Cryst. Growth Des. 2018, 18, 2750−2758 2751 Crystal Growth & Design Crystal Growth & Design AFM was used to study crystal surface topography; the solid state and chemical composition were determined by single crystal X-ray diﬀraction (SC-XRD) and HPLC, respectively. Solid State and Composition Analysis. The chemical composition of single crystal samples of paracetamol with nitrophenol were analyzed using an Agilent 1290 UPLC, 6530 Q-TOF using an internal standard method. An Agilent Poroshell 120 EC-C18 (3.0 × 50 mm, 2.7 μm) column was used. The analysis was performed at 25 °C, using a 1.5 mL/min ﬂow rate recording the absorbance at 230 nm. The mobile phase was 20:80 methanol/water. The samples were prepared by dissolving the selected crystals in 100 mL of a 5% w/w methanol/water solution. A calibration curve for the 4-nitrophenol impurity was prepared using 5 μL solutions containing 0.14 mg/mL paracetamol spiked with 5−15% (w/w) of 4-nitrophenol. Samples were analyzed in triplicate. Chemical composition analysis is a destructive analysis that was performed as last analytical technique. ■EXPERIMENTAL SECTION A stereomicroscope (Brunel Microscopes Ltd., BMDZ zoom stereo- microscope) was used to evaluate single crystal morphology and identify the main macroscopic features. A Leica DM6000M micro- scope was used in diﬀerential interference contrast (DIC) mode to analyze the surface texture of the crystals. A Hitachi TM-1000 version 02−11 scanning electron microscope was used to evaluate the microsurface texture and features of the crystals. The operating conditions for the SEM experiments were back scattered detector, accelerating voltage 15000 V, magniﬁcation 200×, working distance 6700 μm, emission current 57.4 mA, scan speed slow, and vacuum conditions 15.0 kV. g Paracetamol Crystal with 4-Nitrophenol Deposited by Evaporation. Paracetamol single crystals with a surface deposit of 4-nitrophenol (PDN) were prepared by growing pure paracetamol single crystals (PP) using the same cooling crystallization procedure outlined above but in the absence of the impurity. After the pure crystals were isolated and dried at room temperature, a room temperature saturated solution of 4-nitrophenol in ethanol was prepared (1500 mg/g ethanol). A drop of this solution was dispensed onto the dominant face of the PP crystal using a microsyringe. The solvent was allowed to evaporate at ambient temperature producing a layer of 4-nitrophenol crystals on the paracetamol crystal surface. Raman microscopy in mapping mode and TOF-SIMS were used to investigate the distribution of impurities on the crystal faces. g p y Raman microscopy may be operated in mapping mode to analyze localized regions of samples by vibrational microspectroscopy.32−34 g p y p py Mapping involves the sequential measurement of the Raman spectra of adjacent regions of a sample by moving each region of the surface of the sample into the focal point of a Raman microscope. A Horiba Raman Xplora microscope was used to perform the Raman mapping. A 532 nm laser source coupled with a 50× objective (1.23 μm spatial beam width), 50 μm of slit, and 100 μm aperture was used to collect the Raman spectra over the range 50 to 3500 cm−1 at room temperature. Data acquisition time was 2 s with averaging over two spectra with a delay of 2 s. A 25% ﬁlter was used to reduce the eﬀects of interaction of the laser with the sample. The characteristic Raman peaks for paracetamol and 4-nitrophenol were evaluated from published sources35,36 coupled with our own measurements of the Crystal with 4-Nitrophenol Epitaxially Deposited. DOI: 10.1021/acs.cgd.7b01411 Cryst. Growth Des. 2018, 18, 2750−2758 Crystal Growth & Design Figure 1. Raman spectra of paracetamol (PP) (top) and 4-nitrophenol (PN) (bottom). Figure 1. Raman spectra of paracetamol (PP) (top) and 4-nitrophenol (PN) (bottom). Atomic force microscopy (AFM) was carried out using a Bruker AFM with Icon Scanner system and Scan Asyst in air experiment mode, equipped with a MPP-12120-10 tip. The dimensions of the scan area were 50 × 50 μm for each of the samples studied. individual Raman spectra of paracetamol (PP) and 4-nitrophenol (PN) crystals. The main 4-nitrophenol Raman peak has a characteristic frequency of 1111 cm−1, which is related to −CN stretching of the nitro group.36 Based on this characteristic peak, 2D maps were created for 20 × 20 μm or 10 × 10 μm sampling areas by calculation of the intensity ratio between peak and baseline intensity; this was performed using Origin software. Measurements were conducted on crystals obtained from the three diﬀerent crystallization conditions. individual Raman spectra of paracetamol (PP) and 4-nitrophenol (PN) crystals. The main 4-nitrophenol Raman peak has a characteristic frequency of 1111 cm−1, which is related to −CN stretching of the nitro group.36 Based on this characteristic peak, 2D maps were created for 20 × 20 μm or 10 × 10 μm sampling areas by calculation of the intensity ratio between peak and baseline intensity; this was performed using Origin software. Measurements were conducted on crystals obtained from the three diﬀerent crystallization conditions. ■EXPERIMENTAL SECTION Epitaxial deposition of 4-nitrophenol (PEN) onto the surface of a paracetamol crystal was achieved by growing a pure paracetamol single crystal (PP) using the procedure described earlier followed by immersion in a saturated solution of 4-nitrophenol in hexane at room temperature (the composition was in accordance with solubility data reported in the Supporting Information). The crystal and solution was then cooled to 5 °C. Hexane was selected as solvent for the 4-nitrophenol solution due to the relatively high solubility of paracetamol in ethanol, which may have caused slight dissolution of the single crystal during the preparation of the earlier sample. Given the much larger quantity of Crystal with 4-Nitrophenol Epitaxially Deposited. Epitaxial deposition of 4-nitrophenol (PEN) onto the surface of a paracetamol crystal was achieved by growing a pure paracetamol single crystal (PP) using the procedure described earlier followed by immersion in a saturated solution of 4-nitrophenol in hexane at room temperature (the composition was in accordance with solubility data reported in the Supporting Information). The crystal and solution was then cooled to 5 °C. Hexane was selected as solvent for the 4-nitrophenol solution due to the relatively high solubility of paracetamol in ethanol, which may have caused slight dissolution of the single crystal during the preparation of the earlier sample. Given the much larger quantity of 2752 DOI: 10.1021/acs.cgd.7b01411 Cryst. Growth Des. 2018, 18, 2750−2758 Figure 1. Raman spectra of paracetamol (PP) (top) and 4-nitrophenol (PN) (bottom). Crystal Growth & Design Artic Article Article Crystal Growth & Design Crystal Growth & Design The surface feature of the crystal analyzed in the perimeter area where a drop of 4-nitrophenol solution had evaporated. Area of detection 10 × 10 μm. The black dots on the map correspond to the position of the laser detection. Scale is related to the intensity of the 4-nitrophenol peak with respect to the baseline. Raman and TOF-SIMS Analysis. Raman microscopy in mapping mode and TOF-SIMS were evaluated as comple- mentary techniques, both have the potential to allow the distribution of 4-nitrophenol on the crystal surface to be determined. As described previously three diﬀerent crystal- lization techniques were used to prepare samples containing both paracetamol and 4-nitrophenol with which to evaluate the sensitivity of these two analytical tools to investigate concentration and distribution. Raman spectra of the pure PP and PN crystals were collected to establish the diﬀerences in the spectra between the two compounds to identify character- istic Raman bonds so that the local composition could be mapped. 42 43 Figure 3. Raman map of PDN crystal. The surface feature of the crystal analyzed in the perimeter area where a drop of 4-nitrophenol solution had evaporated. Area of detection 10 × 10 μm. The black dots on the map correspond to the position of the laser detection. Scale is related to the intensity of the 4-nitrophenol peak with respect to the baseline. Anitha and co-workers42,43 reported the assignment of spectral bands for paracetamol, while Vijayalakshmi et al.36 reported the assignment of the Raman bands for 4-nitrophenol. Characteristic 4-nitrophenol Raman peaks were 303, 869, 1110, 1586, and 3085 cm−1. Due to the absence of overlapping features in the spectral region, the CN stretching peak at 1111 cm−1 was selected for evaluating the presence of 4-nitrophenol on the paracetamol crystal surface (Figure 1). The Raman maps presented in Figures 2−4 were obtained by tracking this characteristic peak of 4-nitrophenol with respect to the diﬀerence between the maximum peak intensity and baseline intensity. By processing the spectra in this way, concentration contour maps were generated. nitrophenol, PEN, the map centered on an area rich in needle-like 4-nitrophenol crystals (Figure 4). Comparing Raman maps of P4%N, PDN, and PEN crystals, only the PEN crystal showed a signiﬁcant diﬀerence between peak and baseline intensity, and in some areas, the peak intensity to baseline intensity ratio at the selected characteristic wavelength exceeded 10. ■RESULTS AND DISCUSSION In the case of the epitaxially deposited 4- Figure 2. Raman map of P4%N crystal. Crystal was analyzed in a step area. Area of detection 20 × 20 μm. Black dots on the map correspond to the position of the laser detection. Scale is related to the intensity of the 4-nitrophenol peak with respect to the baseline. Figure 3. Raman map of PDN crystal. The surface feature of the crystal analyzed in the perimeter area where a drop of 4-nitrophenol solution had evaporated. Area of detection 10 × 10 μm. The black dots on the map correspond to the position of the laser detection. Scale is related to the intensity of the 4-nitrophenol peak with respect to the baseline. Article Figure 2. Raman map of P4%N crystal. Crystal was analyzed in a step area. Area of detection 20 × 20 μm. Black dots on the map correspond to the position of the laser detection. Scale is related to the intensity of the 4-nitrophenol peak with respect to the baseline. Article ■RESULTS AND DISCUSSION Chemical Character and Surface Texture Evaluation. The SC-XRD, OM, SEM, and AFM results are reported in the Supporting Information. The SC-XRD of the pure crystals were concordant with the literature values for form I (monoclinic) of paracetamol8,37,38 and the alpha form of 4-nitrophenol.3 The lattice parameters of P4%N suggest that 4-nitrophenol modiﬁes the paracetamol crystal lattice without causing a polymorphic change; however, it is known that the errors calculated from X- ray data are overoptimistic given the area-detector and the systematic errors in data collection.39,40 Observation reveals a new crystal face and changes in face shapes on the paracetamol crystal grown in the presence of 4-nitrophenol compared with the crystals grown from pure solution. The change in morphology associated with the incorporation of 4-nitrophenol in the paracetamol lattice is presumed to be due to the reduced capability of the nitro group to form hydrogen bonds compared with the amide. This indicates that the 4-nitrophenol, though present in small quantities, acted as a crystal face modiﬁer. The TOF-SIMS instrument used in this study was a TOF-SIMS 5 from IONTOF, which has four diﬀerent operational modes; surface spectroscopy, surface imaging, depth proﬁling, and 3D-imaging. Depth proﬁling approach is a destructive analysis. Of these modes, surface spectroscopy and surface imaging were the main approaches used. Data were collected in both positive and negative polarity for all the samples that were prepared. Negative polarity was selected as the preferred mode due to enhanced molecular polarizability of the 4- nitrophenol fragments. All images were collected using the Bi3 +2 ion source with settings adjusted toward high lateral resolution (30 keV base setting, due to double charge eﬀective 60 keV, 100 ns pulse width, 0.05 nA beam current). All crystals were analyzed, in each case, as a sample area of 100 × 100 μm. While the PP and PN crystals were recorded at 256 × 256 px raster size, P4%N was recorded at 1024 × 1024 px raster size. The total dose densities recorded were 4 × 1011 ions/cm2 for the PP and PN crystals and 9 × 1011 ions/cm2 for the P4N crystal. To aid the assignment of all mass fragments, the software tools NIST 08 MS Demo and AMDIS 2.6 were used. 2753 DOI: 10.1021/acs.cgd.7b01411 Cryst. Growth Des. 2018, 18, 2750−2758 nitrophenol solution deposited by evaporation, PDN, the detection area was localized on the perimeter area of the drop (Figure 3). Crystal Growth & Design OM in DIC mode, SEM images, and AFM images were examined to assess surface texture characteristics of pure paracetamol (PP), pure 4-nitrophenol (PN), and the three diﬀerent impure crystals P4%N, PDN, and PEN (the images are available in the Supporting Information). Comparing pure paracetamol and pure 4-nitrophenol single crystals surface texture diﬀerences and diﬀerent edge shapes were observed: PP crystal shows characteristic edge steps, while rounded and shorter steps are visible on the PN crystal. Examination of the OM and SEM images indicates that 4-nitrophenol added as impurity during growth acted as a texture modiﬁer, increasing the number of steps and other surface defects; this is consistent with the observation of Prasad and Thompson.13,16 PDN crystal were examined within the perimeter area of the evaporated 4-nitrophenol saturated drop. Needle-like crystals of 4-nitrophenol were randomly distributed in the core area of the drop, while at the perimeter they were aligned in the direction of spread. Areas not covered by the spreading drops of 4-nitrophenol solution showed characteristic paracetamol surface texture. The 4-nitrophenol crystals grown on the paracetamol crystal surface by rapid solvent evaporation exhibit a needle-like morphology that is quite diﬀerent from the slowly grown 4-nitrophenol crystal for SCXRD. This may be due to rapid growth by evaporation or, alternatively, could be due to the formation of the beta polymorph of 4-nitrophenol,41 which have a needle-like morphology (the 4-nitrophenol crystals were too small to determine their crystal form). The epitaxially grown crystals including both micrometer and nanometer size crystals of 4-nitrophenol on the paracetamol crystal surface are seen in the OM image. These crystals showed the same needle- like shape observed when 4-nitrophenol was deposited by evaporation of a drop of ethanol saturated solution. Figure 2. Raman map of P4%N crystal. Crystal was analyzed in a step area. Area of detection 20 × 20 μm. Black dots on the map correspond to the position of the laser detection. Scale is related to the intensity of the 4-nitrophenol peak with respect to the baseline. nitrophenol solution deposited by evaporation, PDN, the detection area was localized on the perimeter area of the drop (Figure 3). In the case of the epitaxially deposited 4- nitrophenol solution deposited by evaporation, PDN, the detection area was localized on the perimeter area of the drop (Figure 3). In the case of the epitaxially deposited 4- Figure 3. Raman map of PDN crystal. Crystal Growth & Design This indicates that, under favorable conditions, Raman microscopy may be used in mapping mode to evaluate the distribution of chemical species. The TOF-SIMS mass spectra and maps of the three diﬀerent crystals were obtained in a similar way to the Raman maps. The pure PP and PN crystals were analyzed to obtain mass spectra of these two pure crystals in order to identify the characteristic mass fragments ion. p g Regions of the crystal surfaces were selected to maximize the probability of detecting the 4-nitrophenol. In the case of the crystal grown from paracetamol solution containing 4 mol % 4- nitrophenol, P4%N, the Raman map was constructed across a growth step (Figure 2), while in the case of the crystal with 4- To verify the mass spectra obtained and to conﬁrm the fragmentation ion identities, two software tools were used; 2754 DOI: 10.1021/acs.cgd.7b01411 Cryst. Growth Des. 2018, 18, 2750−2758 Figure 4. Raman map of PEN crystal. The area of the crystal analyzed was in an area where 4-nitrophenol needle-like crystals were concentrated. Area of detection 10 × 10 μm. The black dots on the map correspond to the centers of the spectrum collections. Scale is related to the intensity of the 4-nitrophenol peak with respect to the baseline. Crystal Growth & Design Crystal Growth & Design Crystal Growth & Design Crystal Growth & Design Article nitrophenol behind, the image does not, however, reveal the shape of the individual impurity crystals. Figure 7 shows the TOF-SIMS mass ion fragment images of the PEN crystal. A needle-shaped 4-nitrophenol crystal is clearly seen at 138 m/z. The corresponding image for the molecular ion fragment of paracetamol (150 m/z) shows the uniform spread of paracetamol; the area covered by the 4- nitrophenol appears darker as the surface of the paracetamol crystal is not accessed by the ionization beam. This sequence of ﬁgures (Figures 5−7) demonstrates the capability of TOF- SIMS to evaluate the distribution of diﬀerent chemical species on crystal surfaces. ■CONCLUSIONS Three diﬀerent approaches, epitaxy, droplet evaporation, and cooling crystallization were used to create paracetamol crystals with molecules of 4-nitrophenol present on the crystal surface in order to study eﬀectiveness of diﬀerent analytical techniques to investigate the quantity and spatial distribution of the impurity on crystal surface and to identify any associated morphological and surface texture changes. Figure 4. Raman map of PEN crystal. The area of the crystal analyzed was in an area where 4-nitrophenol needle-like crystals were concentrated. Area of detection 10 × 10 μm. The black dots on the map correspond to the centers of the spectrum collections. Scale is related to the intensity of the 4-nitrophenol peak with respect to the baseline. p g g TOF-SIMS is a relatively new technique for pharmaceutical material characterization and allows the presence of 4- nitrophenol to be veriﬁed by the distribution of the secondary-ion fragments. NIST 08 MS Demo and AMDIS 2.6 from NIST. The 10 largest molecular fragment peaks from paracetamol and 4-nitrophenol are listed and their mass ion identiﬁed in the Supporting Information. y g The presence and spatial distribution of 4-nitrophenol crystals generated epitaxially are observed. y g p y In the future, TOF-SIMS can be used to build 3D images showing the spatial distribution of speciﬁc molecular species deposited during crystal growth. y g p y In the future, TOF-SIMS can be used to build 3D images showing the spatial distribution of speciﬁc molecular species deposited during crystal growth. The distinctive molecular fragments for 4-nitrophenol were 38, 63, 65, and 139 m/z. Analyzing fragment mass images at 139 m/z, which corresponds to the 4-nitrophenol mono- isotopic mass, allowed the distribution of 4-nitrophenol to be determined. p g y g SEM, AFM, Raman microscopy, and TOF-SIMS were evaluated to assess the level at which the impurity 4- nitrophenol can be detected. TOF-SIMS mass fragment images for the P4%N crystal suggest a homogeneous distribution of 4-nitrophenol (138 m/ z) across the crystal surface. The step surface texture of the crystal can be seen in Figure 5, which is consistent with SEM and OM images also obtained (see Supporting Information). TOF-SIMS mass fragment images of the PDN crystal (Figure 6) allow the evaporated 4-nitrophenol solution drop on top of the paracetamol crystal to be investigated. ■CONCLUSIONS The images show a layer of 4-nitrophenol (138 m/z) covering a large area of the crystal with only small areas of paracetamol (150 m/z) still remaining exposed. TOF-SIMS mass fragment images for the P4%N crystal suggest a homogeneous distribution of 4-nitrophenol (138 m/ z) across the crystal surface. The step surface texture of the crystal can be seen in Figure 5, which is consistent with SEM and OM images also obtained (see Supporting Information). Raman microscopy mapping reveals the impurity location when the main peak intensity ratio is more intense than the baseline; for example, the intensity diﬀerence in Figure 4 is substantially greater than in Figures 2 and 3. g g OM, SEM, and AFM show crystal epitaxial growth on pure paracetamol crystal surface. They reveal surface texture changes and allow the distribution of small crystal agglomerates to be observed. These techniques can be used as complementary analytical tools to address changes in surface texture due to the presence of impurity in the crystal lattice and on the crystal surface. TOF-SIMS mass fragment images of the PDN crystal (Figure 6) allow the evaporated 4-nitrophenol solution drop on top of the paracetamol crystal to be investigated. The images show a layer of 4-nitrophenol (138 m/z) covering a large area of the crystal with only small areas of paracetamol (150 m/z) still remaining exposed. g From these images, it appears that the 4-nitrophenol drop spread across the surface leaving a covering layer of 4- This work demonstrates that TOF-SIMS analysis allowed investigation of both chemical and topographic character of Figure 5. Paracetamol with 4% (mol/mol) of 4-nitrophenol. From left to right, 4-nitrophenol molecular ion image at 138 m/z (maximum counts = 16), paracetamol molecular ion image at 150 m/z (maximum counts = 83), and RGB overlay of paracetamol (red) with 4-nitrophenol (green). Figure 5. Paracetamol with 4% (mol/mol) of 4-nitrophenol. From left to right, 4-nitrophenol molecular ion image at 138 m/z (maximum counts = 16), paracetamol molecular ion image at 150 m/z (maximum counts = 83), and RGB overlay of paracetamol (red) with 4-nitrophenol (green). 2755 DOI: 10.1021/acs.cgd.7b01411 Cryst. Growth Des. 2018, 18, 2750−2758 DOI: 10.1021/acs.cgd.7b01411 Cryst. Growth Des. 2018, 18, 2750−2758 Crystal Growth & Design Article Figure 6. Paracetamol crystal with drop of 4-nitrophenol applied. ■ACKNOWLEDGMENTS The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.cgd.7b01411. The authors wish to acknowledge the contributions of colleagues in each of their organizations. We are grateful to Melissa K. Passarelli, National Physical Laboratory Teddington for TOF-SIMS consultancy; Lauren O’Connor, Strathclyde Institute of Pharmacy & Biomedical Sciences, University of Strathclyde (SIPBS), for consultancy with Raman microscopy; Bruce Wareham, SIPBS, for performing COSMOTherm calculations; Davide Erbogasto, Department of Biomedical Engineering, University of Strathclyde, for SEM analysis; Eleonora Paladino, SIPBS/CMAC for consultancy with TOF- SIMS; Laura Harvey, SIPBS, for LC analysis; Monika Warzecha, SIPBS, for consultancy with AFM. The authors wish to acknowledge our funders: Chris Price, EPSRC Manufacturing Fellowship and the Centre for Innovative Manufacturing in Continuous Manufacturing and Crystalliza- tion; Sara Ottoboni and Michael Chrubasik, EPSRC Doctoral Training Centre for Innovative Manufacturing in Continuous Manufacturing and Crystallization; Michael Chrubasik, Na- tional Physical Laboratory Scottish Hub, University of Strathclyde, Glasgow, UK. The authors would like to thank the Wolfson Foundation. The authors gratefully acknowledge the ﬁnancial support of the University of Strathclyde and the UK Engineering and Physical Sciences Research Council Paracetamol main face form I prediction, molecular packing diagrams based on crystallographic structure, solubility curve of paracetamol in ethanol and hexane, crystal lattice parameters of PP, PN, and P4%N, SC-XRD images of PP and P4%N crystals, stereomicroscopic images of PP, PN, P4%N, PDN, and PEN crystals, OM images in DIC mode of PP, PN, P4%N, PDN, and PEN, AFM maps of PP, P4%N, PDN, and PEN, characteristic neutral fragment of paracetamol and 4-nitrophenol from NIST 08 Demo and AMDIS 2.6 (PDF) ■CONCLUSIONS From left to right, 4-nitrophenol molecular ion image at 138 m/z (maximum counts = 19), paracetamol molecular ion image at 150 m/z (maximum counts = 30), and RGB overlay of paracetamol (red) with 4-nitrophenol (green). Figure 6. Paracetamol crystal with drop of 4-nitrophenol applied. From left to right, 4-nitrophenol molecular ion image at 138 m/z (maximum counts = 19), paracetamol molecular ion image at 150 m/z (maximum counts = 30), and RGB overlay of paracetamol (red) with 4-nitrophenol (green). Figure 7. Suggested paracetamol with 4-nitrophenol epitaxy. From left to right, 4-nitrophenol molecular ion image at 138 m/z (maximum counts = 52), paracetamol molecular ion image at 150 m/z (maximum counts = 32), and RGB overlay of paracetamol (red) with 4-nitrophenol (green). Figure 7. Suggested paracetamol with 4-nitrophenol epitaxy. From left to right, 4-nitrophenol molecular ion image at 138 m/z (maximum counts = 52), paracetamol molecular ion image at 150 m/z (maximum counts = 32), and RGB overlay of paracetamol (red) with 4-nitrophenol (green). amol with 4-nitrophenol epitaxy. From left to right, 4-nitrophenol molecular ion image at 138 m/z (maximum counts = ion image at 150 m/z (maximum counts = 32), and RGB overlay of paracetamol (red) with 4-nitrophenol (green). Figure 7. Suggested paracetamol with 4-nitrophenol epitaxy. From left to right, 4-nitrophenol molecular ion image at 13 52), paracetamol molecular ion image at 150 m/z (maximum counts = 32), and RGB overlay of paracetamol (red) w crystal faces and, in combination with other complementary techniques, proves to be a valuable addition to the analysis of crystals and their impurities. ■ASSOCIATED CONTENT The authors declare no competing ﬁnancial interest. Corresponding Author *Phone: 01414447109. E-mail: sara.ottoboni@strath.ac.uk. ORCID ⊥All authors contributed equally. ⊥All authors contributed equally. ■REFERENCES (22) Marques, A. F. A.; Scott, S. D.; Sodhi, R. N. S. Determining major and trace element compositions of exposed melt inclusions in minerals using ToF-SIMS. Surf. Interface Anal. 2011, 43, 436. (1) Prabu, S. L.; Suriyaprakash, T. N. K. Impurities and its importance in pharmacy. Int. J. Pharm. Sci. Rev. Res. 2010, 3, 66. (2) Ellis, F. Paracetamol: A Curriculum Resource; Royal Society of Chemistry, 2002. g f f (23) Belu, A. M.; Davies, M. C.; Newton, J. M.; Patel, N. TOF-SIMS Characterization and Imaging of Controlled-Release Drug Delivery Systems. Anal. Chem. 2000, 72, 5625. (3) Finnie, S.; Prasad, K. V. R.; Sheen, D. B. Sherwood, Microhardness and Dislocation Identification Studies on Paracetamol Single Crystals. J. N. Pharm. Res. 2001, 18, 674. y (24) Muster, T. H.; Prestidge, C. A. Face specific surface properties of pharmaceutical crystals. J. Pharm. Sci. 2002, 91, 1432. (4) Sudha, C.; Srinivasan, K. Understanding the effect of solvent polarity on the habit modification of monoclinic paracetamol in terms of molecular recognition at the solvent crystal/interface. Cryst. Res. Technol. 2014, 49, 865. (25) Prestidge, C. A.; Barnes, T. J.; Skinner, W. Time-of-flight secondary-ion mass spectrometry for the surface characterization of solid-state pharmaceuticals. J. Pharm. Pharmacol. 2007, 59, 251. p (26) Malmberg, P.; Jennische, E.; Nilsson, D.; Nygren, H. High- resolution, imaging TOF-SIMS: novel applications in medical research. Anal. Bioanal. Chem. 2011, 399, 2711. (5) Haisa, M.; Kashino, S.; Kawai, R.; Maeda, H. The Monoclinic Form of p-Hydroxyacetanilide. Acta Crystallogr., Sect. B: Struct. Crystallogr. Cryst. Chem. 1976, 32, 1283. (6) Di Martino, P.; Conflant, P.; Drache, M.; Huvenne, J.-P.; Guyot- Hermann, A.-M. A new pure paracetamol for direct compression: The orthorhombic form. J. Therm. Anal. 1997, 48, 447. (27) Koch, S.; Ziegler, G.; Hutter, H. ToF-SIMS measurements with topographic information in combined images. Anal. Bioanal. Chem. 2013, 405, 7161. (7) Naumov, D. Y.; Vasilchenko, M. A.; Howard, J. a. K. The Monoclinic Form of Acetaminophen at 150K. Acta Crystallogr., Sect. C: Cryst. Struct. Commun. 1998, 54, 653. (28) Iuraş, A.; Scurr, D. J.; Boissier, C.; Nicholas, M. L.; Roberts, C. J.; Alexander, M. R. Imaging of Crystalline and Amorphous Surface Regions Using Time-of-Flight Secondary-Ion Mass Spectrometry (ToF-SIMS): Application to Pharmaceutical Materials. Anal. Chem. 2016, 88, 3481. (8) Perrin, M.-A.; Neumann, M. A.; Elmaleh, H.; Zaske, L. Crystal structure determination of the elusive paracetamol Form III. Chem. Commun. 2009, 22, 3181. (29) Barnes, T. J.; Kempson, I. ■ABBREVIATIONS J (16) Prasad, K. V. R.; Ristic, R. I.; Sheen, D. B.; Sherwood, J. N. Crystallization of paracetamol from solution in the presence and absence of impurity. Int. J. Pharm. 2001, 215, 29. SIPBS, Strathclyde Institute of Pharmacy & Biomedical Sciences; SEM, scanning electron microscopy; XPS, X-ray photoelectron spectroscopy; TOF-SIMS, time of ﬂight secondary ion mass spectrometry; OM, optical microscopy; HPLC, high performance liquid chromatography; XRD, X-ray diﬀractometry; API, active pharmaceutical ingredient; EDX, energy-dispersive X-ray spectroscopy; PSD, particle size distribution; FBRM, focused beam reﬂectance measurement; AFM, atomic force microscopy; SC-XRD, single crystal X-ray diﬀractometry; P4%N, paracetamol crystal with the presence of 4 mol % 4-nitrophenol made by cooling crystallization; PDN, paracetamol crystal with drop of 4-nitrophenol; PEN, para- cetamol crystal with epitaxially grown 4-nitrophenol; PP, pure paracetamol crystal made by cooling crystallization; PN, pure 4- nitrophenol made by cooling crystallization; WD, working distance; RTD, real time data p y (17) Saleemi, A.; Onyemelukwe, I. I.; Nagy, Z. Effects of a structurally related substance on the crystallization of paracetamol. Front. Chem. Sci. Eng. 2013, 7, 79. g , , (18) Winograd, N. Prospects for imaging TOF-SIMS: from fundamentals to biotechnology. Applied Surface Science, Secondary ion mass spectrometry SIMS. Appl. Surf. Sci. 2003, 203−204, 13. (19) Verlinden, G.; Gijbels, R.; Geuens, I.; De Keyzer, R. Surface analysis of halide distributions in complex AgX microcrystals by imaging time-of-flight SIMS (TOF-SIMS). J. Anal. At. Spectrom. 1999, 14, 429. (20) Rutten, F. J. M.; Roe, M. J.; Henderson, J.; Briggs, D. Surface analysis of ancient glass artefacts with ToF-SIMS: A novel tool for provenancing? Appl. Surf. Sci. 2006, 252, 7124. p g pp f (21) Park, J. S.; Kim, H. Surface characterization of plasma-modified resist patterns by ToF-SIMS analysis. Applied Surface Science, Proceedings of the Symposium on Surface Science. Appl. Surf. Sci. 2009, 256, 1604. ORCID Sara Ottoboni: 0000-0002-2792-3011 Murray Robertson: 0000-0001-9543-7667 Blair Johnston: 0000-0001-9785-6822 Iain D. H. Oswald: 0000-0003-4339-9392 Alastair Florence: 0000-0002-9706-8364 2756 DOI: 10.1021/acs.cgd.7b01411 Cryst. Growth Des. 2018, 18, 2750−2758 Crystal Growth & Design Crystal Growth & Design Article morphology of paracetamol (acetaminophen) crystals. Int. J. Pharm. 2004, 280, 137. (EPSRC) research grants (Grant ref. EP/L014971/1 awarded to C.J.P., L.M.B., and T.T.H.N.; EP/P006965/1 awarded to B.J. and M.R.; EP/N015401/1 awarded to I.D.H.O.) and Doctoral Training Centre for Innovative Manufacturing in Continuous Manufacturing and Crystallization grant (Grant ref. EPK503289) awarded to S.O. All data underpinning this publication are openly available from the University of Strathclyde Knowledge Base at http://dx.doi.org/10.15129/ 7e48f205-e591-465c-89e2-2fc44055a1a7. morphology of paracetamol (acetaminophen) crystals. Int. J. Pharm. 2004, 280, 137. (14) Shekunov, B. Y.; Grant, D. J. W.; Latham, R. J.; Sherwood, J. N. In Situ Optical Interferometric Studies of the Growth and Dissolution Behavior of Paracetamol (Acetaminophen) Crystals. 3. Influence of Growth in the Presence of p-Acetoxyacetanilide. J. Phys. Chem. B 1997, 101, 9107. (15) Chow, A. H.-L.; Chow, P. K. K.; Zhongshan, W.; Grant, D. J. W. Modification of acetaminophen crystals: influence of growth in acqueous solutions containing p-acetoxyacetanilide on crystal proper- ties. Int. J. Pharm. 1985, 24, 239. ■REFERENCES M.; Prestidge, C. A. Surface analysis for compositional, chemical and structural imaging in pharmaceutics with mass spectrometry: A ToF-SIMS perspective. Int. J. Pharm. 2011, 417, 61. (9) Shekunov, B. Y.; Aulton, M. E.; Adama-Acquah, R. W.; Grant, D. J. W. Effect of temperature on crystal growth and crystal properties of paracetamol. J. Chem. Soc., Faraday Trans. 1996, 92, 439. ( ) (10) Hendriksen, B. A.; Grant, D. J. W.; Meenan, P.; Green, D. A. Crystallisation of paracetamol (acetaminophen) in the presence of structurally related substances. J. Cryst. Growth 1998, 183, 629. (30) Leng, Y., Ed.Materials Characterization: Introduction to Micro- scopic and Spectroscopic Methods, 2nd ed.; Wiley, 2013. (31) Granberg, R. A.; Rasmuson, Å. C. Solubility of Paracetamol in Pure Solvents. J. Chem. Eng. Data 1999, 44, 1391. (11) Hendriksen, B. A.; Grant, D. J. W. The effect of structurally related substances on the nucleation kinetics of paracetamol (acetaminophen). J. Cryst. Growth 1995, 156, 252. J g (32) Morris, D. M. In Emerging Raman Applications and Techniques in Biomedical and Pharmaceutical Fields; Matousek, P., Morris, M. D., p y (12) Kuvadia, Z. B.; Doherty, M. F. Effect of Structurally Similar Additives on Crystal Habit of Organic Molecular Crystals at Low Supersaturation. Cryst. Growth Des. 2013, 13, 1412. Eds.; Biological and Medical Physics, Biomedical Engineering; Springer: Berlin Heidelberg, 2010; pp 347−364. p g g pp (33) Gordon, K. C.; McGoverin, C. M. Raman mapping of pharmaceuticals. Int. J. Pharm. 2011, 417, 151. y (13) Thompson, C.; Davies, M. C.; Roberts, C. J.; Tendler, S. J. B.; Wilkinson, M. J. The effects of additives on the growth and 2757 DOI: 10.1021/acs.cgd.7b01411 Cryst. Growth Des. 2018, 18, 2750−2758 Article DOI: 10.1021/acs.cgd.7b01411 Cryst. Growth Des. 2018, 18, 2750−2758 Crystal Growth & Design (34) Pyatski, Y.; Zhang, Q.; Mendelsohn, R.; Flach, C. R. Effects of permeation enhancers on flufenamic acid delivery in Ex vivo human skin by confocal Raman microscopy. Int. J. Pharm. 2016, 505, 319. (35) Al-Zoubi, N.; Koundourellis, J. E.; Malamataris, S. FT-IR and Raman spectroscopic methods for identification and quantitation of orthorhombic and monoclinic paracetamol in powder mixes. J. Pharm. Biomed. Anal. 2002, 29, 459. (36) Vijayalakshmi, S.; Kalyanaraman, S.; Ravindran, T. R. Raman investigation with group theoretical method on structural poly- morphism of the nonlinear optical hexamine: p −nitrophenol cocrystals. Phys. Scr. 2014, 89, 095501. (37) Nichols, G.; Frampton, C. S. Physicochemical Characterization of the Orthorhombic Polymorph of Paracetamol Crystallized from Solution. J. Pharm. Sci. 1998, 87, 684. (38) Druzhbin, D. A.; Drebushchak, T. N.; Min’kov, V. S.; Boldyreva, E. V. Crystal structure of two paracetamol polymorphs at 20K: a search for the “structure-property” relationship. J. Struct. Chem. 2015, 56, 317. (39) Kulkarni, G. U.; Kumaradhas, P.; Rao, C. N. R. Charge Density Study of the Polymorphs of p-Nitrophenol. Chem. Mater. 1998, 10, 3498. (40) Wójcik, G.; Mossakowska, I. Polymorphs of p-nitrophenol as studied by variable-temperature X-ray diffraction and calorimetry: comparison with m-nitrophenol. Acta Crystallogr., Sect. B: Struct. Sci. 2006, 62, 143. (41) Coppens, P.; Schmidt, G. M. The crystal structure of the metastable (β) modification of p-nitrophenol. Acta Crystallogr. 1965, 18, 654. (42) Gandhimathi, R.; Dhanasekaran, R. Third order nonlinear studies and other characterization of 4-nitrophenol (4-NP) single crystals. IOP Conf. Ser.: Mater. Sci. Eng. 2013, 43, 012004. (43) Anitha, R.; Gunasekaran, M.; Kumar, S. S.; Athimoolam, S.; Sridhar, B. Single crystal XRD, vibrational and quantum chemical calculation of pharmaceutical drug paracetamol: A new synthesis form. Spectrochim. Acta, Part A 2015, 150, 488. 2758 DOI: 10.1021/acs.cgd.7b01411 Cryst. Growth Des. 2018, 18, 2750−2758 DOI: 10.1021/acs.cgd.7b01411 Cryst. Growth Des. 2018, 18, 2750−2758
https://openalex.org/W2607736440	https://escholarship.org/content/qt84x839hv/qt84x839hv.pdf?t=p17byg	English	null	Microbial communities in sediment from <i>Zostera marina</i> patches, but not the <i>Z. marina</i> leaf or root microbiomes, vary in relation to distance from patch edge	PeerJ	2,017	cc-by	11,299	Permalink https://escholarship.org/uc/item/84x839hv Journal PeerJ, 5(4) ISSN 2167-8359 Authors Ettinger, Cassandra L Voerman, Sofie E Lang, Jenna M et al. Publication Date 2017 DOI 10.7717/peerj.3246 Peer reviewed Journal PeerJ, 5(4) ISSN 2167-8359 Authors Ettinger, Cassandra L Voerman, Sofie E Lang, Jenna M et al. Publication Date 2017 DOI 10.7717/peerj.3246 Peer reviewed UC Davis UC Davis Previously Published Works Title Title Microbial communities in sediment from Zostera marina patches, but not the Z. marina leaf or root microbiomes, vary in relation to distance from patch edge. Microbial communities in sediment from Zostera marina patches, but not the Z. marina leaf or root microbiomes, vary in relation to distance from patch edge. Powered by the California Digital Library University of California eScholarship.org eScholarship.org How to cite this article Ettinger et al. (2017), Microbial communities in sediment from Zostera marina patches, but not the Z. marina leaf or root microbiomes, vary in relation to distance from patch edge . PeerJ 5:e3246; DOI 10.7717/peerj.3246 Microbial communities in sediment from Zostera marina patches, but not the Z. marina leaf or root microbiomes, vary in relation to distance from patch edge Cassandra L. Ettinger1, Sofie E. Voerman2,3, Jenna M. Lang1,4, John J. Stachowicz5 and Jonathan A. Eisen1,5,6 1 Genome Center, University of California, Davis, CA, United States 2 Climate Change Cluster, University of Technology Sydney, Sydney, Australia 3 School of Life Sciences, University of Technology Sydney, Sydney, Australia 4 Trace Genomics, San Francisco, CA, United States 5 Department of Evolution and Ecology, University of California, Davis, CA, United States 6 Department of Medical Microbiology and Immunology, University of California, Davis, Davis, CA, United States 1 Genome Center, University of California, Davis, CA, United States 2 Climate Change Cluster, University of Technology Sydney, Sydney, Australia 3 School of Life Sciences, University of Technology Sydney, Sydney, Australia 4 Trace Genomics, San Francisco, CA, United States , , , 5 Department of Evolution and Ecology, University of California, Davis, CA, United States 6 Department of Medical Microbiology and Immunology, University of California, Davis, Davis, CA, United States INTRODUCTION The seagrass, Zostera marina, is a foundation species in protected bays and estuaries throughout the temperate northern hemisphere. Seagrasses are fully submerged marine angiosperms and are a paraphyletic group comprised of three lineages in the order Alismatales that convergently adapted to the marine environment between 70 and 100 million years ago (Les, Cleland & Waycott, 1997). There are only approximately 60 species of seagrass compared to the 250,000 species of terrestrial angiosperms, a testament to the strict selective pressure posed by re-entry to the marine environment (Orth et al., 2006). Seagrass patches serve as habitat and nursery grounds for many marine species, play key roles in nutrient cycling and carbon sequestration, and serve to protect the coastline from erosion (Williams & Heck Jr, 2001). Z. marina populations, like those of many seagrass species, are negatively affected by climate change, pollution and habitat destruction and so far, restoration efforts have been costly and ineffective (Orth et al., 2006). As a result, Z. marina is vulnerable to habitat fragmentation and loss. The work described here was originally focused on a phenomenon known as ‘‘edge effects’’ in which the border between habitats is intermediate in abiotic conditions from the center of either adjacent habitat and thus the biotic composition of the border habitat, or edge, may differ from that of interior, intact habitat. Edges often support a mixture of organisms from two adjacent habitats (Fox et al., 1997; Davies-Colley, Payne & Elswijk, 2000), but may be abiotically unsuitable for species found in the center of either habitat. Increased predation and invasion by non-native species can also be a common feature of edges (Paton, 1994; Fox et al., 1997; Harrison & Bruna, 1999; Flaspohler, Temple & Rosenfield, 2001). Prior work on seagrasses have shown edge effects on species abundances (Smith et al., 2008; Smith et al., 2011; Tanner, 2005) and abiotic conditions such as turbulence (Granata et al., 2001; Folkard, 2005), carbon stocks (Ricart et al., 2015) and organic matter deposition (Duarte & Sand-Jensen, 1990; Cebrián et al., 2000). Here we investigate whether such edge effects are evident in the microbiota found in, on and near Z. marina plants. Our interest in the microbiota for this study is driven by our overarching goal of developing Z. marina as a model for studies of microbial communities associated with marine plants. ABSTRACT Background. Zostera marina (also known as eelgrass) is a foundation species in coastal and marine ecosystems worldwide and is a model for studies of seagrasses (a paraphyletic group in the order Alismatales) that include all the known fully submerged marine angiosperms. In recent years, there has been a growing appreciation of the potential importance of the microbial communities (i.e., microbiomes) associated with various plant species. Here we report a study of variation in Z. marina microbiomes from a field site in Bodega Bay, CA. Methods. We characterized and then compared the microbial communities of root, leaf and sediment samples (using 16S ribosomal RNA gene PCR and sequencing) and associated environmental parameters from the inside, edge and outside of a single subtidal Z. marina patch. Multiple comparative approaches were used to examine associations between microbiome features (e.g., diversity, taxonomic composition) and environmental parameters and to compare sample types and sites. Submitted 4 January 2017 Accepted 29 March 2017 Published 27 April 2017 Corresponding author Jonathan A. Eisen, jaeisen@ucdavis.edu, jonathan.eisen@gmail.com Academic editor Gerrit Beemster Additional Information and Declarations can be found on page 16 DOI 10.7717/peerj.3246 Copyright 2017 Ettinger et al. Distributed under Creative Commons CC-BY 4 0 Submitted 4 January 2017 Accepted 29 March 2017 Published 27 April 2017 Corresponding author Jonathan A. Eisen, jaeisen@ucdavis.edu, jonathan.eisen@gmail.com Academic editor Gerrit Beemster Additional Information and Declarations can be found on page 16 DOI 10.7717/peerj.3246 Copyright 2017 Ettinger et al. Distributed under Creative Commons CC-BY 4.0 Results. Microbial communities differed significantly between sample types (root, leaf and sediment) and in sediments from different sites (inside, edge, outside). Carbon:Nitrogen ratio and eelgrass density were both significantly correlated to sediment community composition. Enrichment of certain taxonomic groups in each sample type was detected and analyzed in regard to possible functional implications (especially regarding sulfur metabolism). Discussion. Our results are mostly consistent with prior work on seagrass associated microbiomes with a few differences and additional findings. From a functional point of view, the most significant finding is that many of the taxa that differ significantly between sample types and sites are closely related to ones commonly associated with various aspects of sulfur and nitrogen metabolism. Though not a traditional model organism, we believe that Z. marina can become a model for studies of marine plant- microbiome interactions. Distributed under Creative Commons CC-BY 4.0 OPEN ACCESS Subjects Marine Biology, Microbiology, Plant Science Ettinger et al. (2017), PeerJ, DOI 10.7717/peerj.3246 INTRODUCTION Recently a few culture- independent surveys of seagrass microbiomes have been published and these provide good initial reference points for our work here (Jiang et al., 2015; Sun et al., 2015; Cúcio et al., 2016; Mejia et al., 2016). Although, these studies have similar big picture findings, there are small differences in microbiome composition between them and thus further teasing apart of the factors that shape seagrass microbiomes is necessary and important work. In this study, we characterized and then compared the epiphytic and rhizospheric bacterial communities of eelgrass using root, leaf and sediment samples obtained from the inside, edge and outside of a single subtidal Z. marina patch. We focused on characterizing the bacterial and archaeal members of the microbiome in each of these samples using high throughput sequencing of 16S ribosomal gene PCR libraries. We focus in particular on the following questions: What is the general taxonomic composition of the Z. marina microbiome? Are there changes in sediment microbial community composition or in biodiversity at the patch edge and, if so, what factors are driving observed differences, environmental abiotic factors or presence/absence of Z. marina? This analysis reveals multiple novel insights into the general structure of the Z. marina microbiome and lays the groundwork for further studies. INTRODUCTION Although we speculate that plant microbe interactions are important for seagrasses, little is known about how the roles of microbial communities associated with marine plants may affect plant health and what abiotic and biotic factors affect the composition of these communities. Terrestrial plants, like Arabidopsis (Lundberg et al., 2012), corn (Peiffer et al., 2013; Bouffaud et al., 2014), rice (Peiffer et al., 2013; Edwards et al., 2015) and poplar (Beckers et al., 2016) have been shown to have distinct microbial communities on the inside (endophytes) and on the surface (epiphytes) of plant leaves and roots, as well as in the surrounding soil or sediment (rhizosphere) (Lundberg et al., 2012). 2/25 Ettinger et al. (2017), PeerJ, DOI 10.7717/peerj.3246 These communities can vary across different stages of plant development (Chaparro, Badri & Vivanco, 2014) and with local environmental conditions. In terrestrial systems the main drivers of plant associated microbial community composition are considered to be environmental factors, like soil particle size, pH and moisture content, as well as host plant species (Aleklett et al., 2015; Lakshmanan, 2015). Thus, examining eelgrass microbiota across a known environmental gradient from the center to the outside of a patch has the potential to provide insights into factors that shape the eelgrass microbiome, the full community of microorganisms associated with eelgrass. Recently a few culture- independent surveys of seagrass microbiomes have been published and these provide good initial reference points for our work here (Jiang et al., 2015; Sun et al., 2015; Cúcio et al., 2016; Mejia et al., 2016). Although, these studies have similar big picture findings, there are small differences in microbiome composition between them and thus further teasing apart These communities can vary across different stages of plant development (Chaparro, Badri & Vivanco, 2014) and with local environmental conditions. In terrestrial systems the main drivers of plant associated microbial community composition are considered to be environmental factors, like soil particle size, pH and moisture content, as well as host plant species (Aleklett et al., 2015; Lakshmanan, 2015). Thus, examining eelgrass microbiota across a known environmental gradient from the center to the outside of a patch has the potential to provide insights into factors that shape the eelgrass microbiome, the full community of microorganisms associated with eelgrass. Ettinger et al. (2017), PeerJ, DOI 10.7717/peerj.3246 Molecular methods DNA was extracted from leaf (n = 8), root (n = 8) and both shallow (n = 12) and deep sediment (n = 12) samples as well as from a kit control (n = 1) with the PowerSoil DNA Isolation kit (MO BIO Laboratories, Inc., Carlsbad, CA, USA) according to the manufacturer’s protocol. For the DNA extractions, root and leaf tissues were placed directly into PowerBead tubes from the freezer without grinding. Microbial 16S rRNA genes were amplified using a two-step protocol targeting the V4 region using the ‘‘universal’’ 515F and 806R primers (Caporaso et al., 2012). The primer set was modified to include Illumina adapters and barcode sequences using a dual indexing approach as in Lang, Eisen & Zivkovic (2014). The 41 samples described in this paper were multiplexed with 103 samples from other experiments, for a total of 144 samples on the run. Libraries were sequenced by the UC Davis Genome Center Core Facilities on an Illumina MiSeq (Illumina, Inc., San Diego, CA, USA) to generate 250 bp paired-end reads. Sample collection We collected leaf, root and sediment samples for microbiome analysis from 0.25 m2 quadrats (n = 4) located in the interior (2.5 m from the edge), on the edge (but within the eelgrass habitat) and outside (2.5 m from the edge) of a single shallow subtidal eelgrass patch in Bodega Bay, CA (GPS: 38.319435, −123.053838) during the summer of 2013. Quadrats were positioned 2.5 m from each other parallel to the patch’ edge. Samples were collected during low tide (±0.5 m water depth) at night (11 PM). For quadrats located at the center or edge of the eelgrass patch, one eelgrass shoot was sampled and directly separated into root and shoot tissue. The root tissue consisted of one entire root bundle sampled, the leaf tissue consisted of a clipped leaf of ± 3 cm in length positioned at about half way along the shoot length (±20 cm from the base). For each quadrat, sediment samples were collected at two sediment depths, 0.5 cm or less and 3 cm deep, from randomly selected locations within the quadrats. Microbial samples were directly stored on ice and transported to the laboratory within one hour where samples were frozen at −20 ◦C until further analysis. Environmental data and the samples used for microbiome analysis were collected simultaneously. For each quadrat, eelgrass density was estimated by direct count. Ettinger et al. (2017), PeerJ, DOI 10.7717/peerj.3246 3/25 Temperature, pH, salinity and dissolved oxygen were measured at 20 cm above the sediment with a YSI 556 handheld multimeter (YSI Inc., Yellow Springs, OH, USA), at a similar height as the shoot tissue was sampled. Sediment chemical and physical properties were assessed by separately coring the top 4 cm of sediment (10 cm diameter, taken twice within a quadrat and combined for analysis), to correspond with the sediment layer most influenced by the eelgrass roots. Sediment was dried (three days at 40 ◦C), mixed, sieved (sieve sizes: 710, 500, 355, 250, 180, 90 and 30 µm) and particle size fractions were weighed to investigate particle size distribution. A portion of the mixed sediment samples (±50 g) was separately analyzed for total organic carbon (TOC), total inorganic carbon (TIC) and Carbon:Nitrogen (C:N) ratio by the UC Davis Analytical Laboratory. Ettinger et al. (2017), PeerJ, DOI 10.7717/peerj.3246 Sequence processing A custom in-house script was used to demultiplex, quality check and merge paired reads (https://github.com/gjospin/scripts/blob/master/Demul_trim_prep.pl). The resulting sequences were analyzed using the Quantitative Insights Into Microbial Ecology (QIIME) v. 1.9.0 workflow (Caporaso et al., 2010). For a detailed walkthrough of the following analysis using QIIME, see the IPython notebook (http://nbviewer.jupyter.org/gist/casett/86da7fc8749d27574f183498df65134a). The sequencing run for this project included samples from other projects. In total, for the entire run, 14,163,470 reads passed quality filtering (Q20). Of these reads, 4,573,318 were associated with the 41 samples for this project. Of the 4,573,318 reads for this project, 4,212,549 merged successfully (92.11%). The sample with the lowest number of sequences after merging was the negative control with 444 sequences, the next lowest sample, BB039, had 22,897 sequences, approximately a fifty-fold increase. The most abundant sequence in the negative control was chloroplast DNA, and thus, we conclude that these 444 sequences were likely the result of contamination from other samples during sequencing or molecular analyses. We considered removing shared operational taxonomic units (OTUs) or 100 percent identical DNA sequences between the negative control and our environmental samples, but determined both of these actions to be too stringent on the dataset when Ettinger et al. (2017), PeerJ, DOI 10.7717/peerj.3246 4/25 taking into account the abundance of the OTU’s/DNA sequences in the samples and the number of reads in the negative control. Instead the negative control was simply removed from downstream analysis. A total of 4,976 chimeras were identified using USEARCH v. 6.1 and were filtered out. The remaining sequences were clustered using the open reference approach into OTUs at 97 percent similarity using UCLUST (Edgar, 2010). Taxonomy was assigned using the assign_taxonomy.py QIIME script with the GreenGenes database (v.13_8) (DeSantis et al., 2006) using UCLUST. Further filtering was performed using the QIIME scripts, filter_taxa_from_otu_table.py and filter_otus_from_otu_table.py, to remove chloroplast DNA, mitochondrial DNA and singletons. Reads classified as ‘‘Unassigned’’ at the domain level were also removed from downstream analysis. After these filtering steps, the lowest number of sequences in a sample dropped to 3,277. This reduction in the number of sequence reads can be largely attributed to the removal of Z. marina chloroplast DNA from the leaf and root samples. To aid in statistical comparison between different sample types (leaf, root, sediment), we subset our 16S rDNA sequences to a minimum sequence count of 3,277 to retain the maximum number of samples. Sequence processing However, when comparing only sediment samples, the 16S rDNA sequences were randomly subset to 20,000 sequences using the single_rarefaction.py QIIME script. Ettinger et al. (2017), PeerJ, DOI 10.7717/peerj.3246 Data visualization and statistical analyses Data visualization was performed exclusively in R and statistical analyses were performed using a combination of QIIME scripts and R (R Core Team, 2016). For analysis done in R, the rarefied OTU tables were converted to json format and exported for analysis using the ggplot2 (Wickham, 2009), vegan (Dixon, 2003) and phyloseq (McMurdie & Holmes, 2013) packages. Initial analysis indicated no significant differences between the microbial communities associated with shallow (0.5 cm or less) and deep (3 cm) sediment samples, thus sediment depth was not considered further here. We describe the different types of analyses below. • Intra-sample (alpha) diversity. We were interested in if significant differences existed between the intra-sample (alpha) diversities (richness, evenness) of the microbial communities associated with different sample types (leaf, root, sediment) and different sediment locations (inside, edge, outside). We calculated the following diversity metrics: Chao1 (Chao, 1984), Observed OTUs, Shannon (Shannon & Weaver, 1949) and Simpson Indices (Simpson, 1949) in R. To determine if there were significant differences between the alpha diversities of different sample types and different sediment locations, we first performed Kruskal–Wallis tests. We then implemented Bonferroni corrected post-hoc Dunn tests to identify which pairwise comparisons were driving differences. • Inter-sample (beta) diversity. We assessed the inter-sample (beta) diversities of the microbial communities associated with different sample groupings (sample type, location, etc) and if there were any significant correlations between environmental variables and community dissimilarity. We used both Unifrac (weighted and unweighted) (Lozupone et al., 2007; Hamady, Lozupone & Knight, 2010) and Bray–Curtis (Bray Ettinger et al. (2017), PeerJ, DOI 10.7717/peerj.3246 5/25 5/25 & Curtis, 1957) dissimilarities calculated in R using phyloseq. These dissimilarities were then plotted using principal coordinate analysis (PCoA) and non-metric multidimensional scaling (NMDS) methods. Multiple tests were then performed on these beta-diversity results. To test for significant differences in centroids between different sample groupings (sample type, location, etc.) PERMANOVA tests were performed using the adonis function from the vegan package in R with 9,999 permutations (Anderson, 2001). PERMANOVA tests can be sensitive to differences in dispersion when using abundance-based distance matrices (Warton, Wright & Wang, 2012), but are more robust than other tests, especially for balanced designs (Anderson & Walsh, 2013). To test for differences in mean dispersions between different groupings, the betadisper and permutest functions from the vegan package in R were used with 999 permutations. Data visualization and statistical analyses To test for correlations between the Bray Curtis dissimilarities of our samples and the environmental factors (C:N ratio, pH, etc) measured, euclidean distances were calculated in R using vegan and Mantel tests were performed using 9,999 permutations. The supervised_learning.py QIIME script was used to see if a random forest classifier could differentiate between sample type or sediment location using leave-one-out cross validation and 1,000 trees. • Taxonomic variation. To determine if the mean relative abundance of taxonomic orders varied significantly between different sample types and sediment locations, we first used the summarize_taxa.py QIIME script to remove rare OTUs (less than one percent of total abundance) and to collapse OTUs at the Order level. We then used the group_significance.py QIIME script on the resulting OTU table to test for differences using Bonferroni corrected Kruskal–Wallis tests with 1000 permutations. We removed the rare OTUs, as suggested in the documentation for the groups_significance.py QIIME script, to avoid spurious significance from very low abundance OTUS, to simplify analyses and to focus on abundant organisms and overall patterns. • Environmental variation. To determine if environmental factors varied significantly between different locations in the eelgrass patch (inside, edge, outside), ANOVA tests were performed in R for each factor. The post-hoc Tukey’s Honest Significant Difference (HSD) test was performed in R for factors found significantly different by the ANOVA (Tukey, 1953; Kramer, 1956; Kramer, 1957). Ettinger et al. (2017), PeerJ, DOI 10.7717/peerj.3246 Diversity metrics I: intra-sample variation between sample types and locations Diversity metrics I: intra-sample variation between sample types and locations Alpha diversity is greater in the sediment than in the leaves and roots (p < 0.001) for a variety of metrics including observed number of OTUs, Chao1, Shannon and Simpson (Fig. 1). However, there is no difference in alpha diversity between leaf and root samples (p > 0.05) (Table S1 ). This is not altogether unexpected as in terrestrial systems soil has been observed to have increased diversity compared to host associated sample types (Edwards et al., 2015). There is conflict between the diversity metrics when determining if the intra-sample diversity of sediment at different locations (inside, edge, outside) varies Ettinger et al. (2017), PeerJ, DOI 10.7717/peerj.3246 6/25 Figure 1 Alpha diversity across samples. Four alpha diversity metrics, observed number of OTUs, Chao1, the Shannon and Simpson diversity indices, are shown as boxplots for (A) different sample types (leaf, root, sediment) and for (B) sediment from different locations (inside, edge, outside). Figure 1 Alpha diversity across samples. Four alpha diversity metrics, observed number of OTUs, Chao1, the Shannon and Simpson diversity indices, are shown as boxplots for (A) different sample types (leaf, root, sediment) and for (B) sediment from different locations (inside, edge, outside). (Table S2). Two of the metrics, observed number of OTUs and Chao1, indicate greater diversity outside compared to inside the patch (p < 0.05). The non-significant metrics, the Shannon and Simpson indices, account for both richness and evenness and are less sensitive to rare taxa than richness only metrics (Bent & Forney, 2008). Thus, one possible explanation for the difference in diversity between the inside and outside sediment is an increased number of rare taxa in sediment from outside the patch. No significant differences were found between the alpha diversity of leaves and roots between the inside and edge of the eelgrass patch. Diversity metrics II: inter-sample variation between sample types and locations Distinct microbial communities were detected in association with Z. marina leaves, roots and sediment (Fig. 2). PERMANOVA tests performed on three different beta diversity metrics, weighted UniFrac, unweighted UniFrac and Bray–Curtis Dissimilarity, found these communities to be significantly different from each other with p = 0.0001 (Table 1). Root and leaf associated communities were found to have more with-in group variance, or dispersion, than sediment communities (p = 0.001), which could indicate that stabilizing Ettinger et al. (2017), PeerJ, DOI 10.7717/peerj.3246 7/25 Figure 2 Principal coordinates analysis (PCoA) of microbial communities based on weighted Unifrac distances. Samples are colored by sample type (leaf, root, sediment) with different shapes for location (in- side, edge, outside). Figure 2 Principal coordinates analysis (PCoA) of microbial communities based on weighted Unifrac distances. Samples are colored by sample type (leaf, root, sediment) with different shapes for location (in- side, edge, outside). Table 1 Permanova results. Comparing microbial community composition between different sample types (leaf, root, sediment) and locations (inside, edge, outside) using multiple beta diversity metrics. Category Diversity metric Pseudo-F R2 P (perm) Location Weighted UniFrac 2.22 0.107 0.0213 Unweighted UniFrac 1.91 0.0938 0.0043 Bray Curtis 2.82 0.133 0.0009 Sample type Weighted UniFrac 13.75 0.426 0.0001 Unweighted UniFrac 6.16 0.249 0.0001 Bray Curtis 9.53 0.34 0.0001 LocXType Weighted UniFrac 1.98 0.0541 0.0426 Unweighted UniFrac 1.19 0.0455 0.1586 Bray Curtis 1.482 0.0458 0.0795 selection is acting on these sediment communities. Random forest analysis further validated selection is acting on these sediment communities. Random forest analysis further validated the observed differences between leaves, roots and sediment microbial communities (Table S3). The classifier had an estimated error of 5% (versus a baseline error of 40%) and correctly identified all leaf samples (n = 8) and all sediment samples (n = 24). The classifier did misclassify two of the root samples (n = 8) as leaves, but this is not unexpected as these two samples also appear to cluster more closely with the leaf samples when visualized using Principal Coordinates Analysis (PCoA) (Fig. 2). The reason that these root samples cluster more closely with the leaf samples may be due to which root bundles were sampled; preliminary results indicate that the microbiota associated with the roots can vary depending on the proximity of the root to the base of the leaf, with roots closer to the base appearing more ‘‘leaf-like’’ (HE Holland-Moritz et al., 2017, unpublished data). Ettinger et al. (2017), PeerJ, DOI 10.7717/peerj.3246 Ettinger et al. (2017), PeerJ, DOI 10.7717/peerj.3246 Diversity metrics II: inter-sample variation between sample types and locations Figure 4 Average relative abundance of taxonomic groups associated with each sample type (leaf, root, sediment). OTUs are shown grouped by taxonomic order and colored by taxonomic class. Only orders with a mean abundance of at least one percent are shown here. The bars represent the standard error of the mean. Random forest analysis confirmed differences between sediment microbial communities taken from the inside of the patch, the edge and unvegetated sediment from outside the patch (Table S4). The classifier had an estimated error of 12.5% (versus a baseline error of 66.7%) and correctly identified all of the unvegetated sediment (n = 8). The classifier did mistakenly classify one sample from the edge (n = 8) as being from the inside of the patch and two samples from the inside of the patch (n = 8) as being from the edge. In Fig. 3, there is some overlap in the clustering of sediment from the inside and edges of patches which might account for these misclassifications. Diversity metrics II: inter-sample variation between sample types and locations Ettinger et al. (2017), PeerJ, DOI 10.7717/peerj.3246 8/25 Figure 3 Principal coordinates analysis (PCoA) of microbial communities in sediment based on weighted Unifrac distances. Samples are colored by location (inside, edge, outside). Figure 3 Principal coordinates analysis (PCoA) of microbial communities in sediment based on weighted Unifrac distances. Samples are colored by location (inside, edge, outside). Table 2 Sediment PERMANOVA results. Comparing sediment microbial community composition be- tween different locations (inside, edge, outside) and eelgrass densities using multiple beta diversity met- rics. Table 2 Sediment PERMANOVA results. Comparing sediment microbial community composition be- tween different locations (inside, edge, outside) and eelgrass densities using multiple beta diversity met- rics Table 2 Sediment PERMANOVA results. Comparing sediment microbial community composition be- tween different locations (inside, edge, outside) and eelgrass densities using multiple beta diversity met- rics. Category Diversity metric Pseudo-F R2 P (perm) Location Weighted UniFrac 8.69 0.453 0.0001 Unweighted UniFrac 2.92 0.217 0.0001 Bray Curtis 8.01 0.433 0.0001 Density Weighted UniFrac 2.81 0.551 0.0002 Unweighted UniFrac 1.51 0.398 0.0001 Bray Curtis 2.86 0.555 0.0001 To determine if there was a difference in community composition at the eelgrass patch edge relative to the inside or outside of the patch, beta diversity metrics were calculated for the sediment microbial communities. As can be seen in Fig. 3, these diversity metrics show the communities clustering by sampling location (inside, edge, outside). PERMANOVA tests indicate that these clusters are significantly different between locations (p = 0.0001) and also for eelgrass shoot densities (p < 0.0002) (Table 2). However, leaf and root microbial communities do not differ significantly based on sampling location, possibly indicating that these plant tissue associated communities are more stable than the sediment communities in regards to location. Whereas sediment communities, although distinct when associated with eelgrass, may be under less selection from the host plant. One possible explanation for the correlation between the sediment communities and eelgrass shoot density may be the release of exudates and oxygen by the roots of the eelgrass, which would increase in concentration with eelgrass density. Ettinger et al. (2017), PeerJ, DOI 10.7717/peerj.3246 9/25 Figure 4 Average relative abundance of taxonomic groups associated with each sample type (leaf, root, sediment). OTUs are shown grouped by taxonomic order and colored by taxonomic class. Only orders with a mean abundance of at least one percent are shown here. The bars represent the standard error of the mean. Ettinger et al. (2017), PeerJ, DOI 10.7717/peerj.3246 Major patterns in community composition of the leaves, roots and rhizosphere sediment The analysis of diversity metrics presented above shows that there are distinct communities associated with leaves and roots, and these both differ from the sediment, whereas location effects are weaker. We therefore examined in more detail the taxonomic composition and possible functional roles of the microbes on Z. marina leaves, roots and rhizosphere sediment (sediment from the inside and edge of the eelgrass patch). We summarize our findings regarding this here. Figure 4 shows the average relative abundance of different orders of bacteria for leaves, roots and sediment. On leaves, the most abundant orders were Clostridiales, Bacteroidales, Rhodobacterales, Flavobacterales, Saprospirales, Thiotrichales and Unidentified Gammaproteobacteria. On roots, the most abundant orders were Campylobacterales, Bacteroidales, Clostridiales, Desulfobacterales, Flavobacteriales and Desulfuromonadales. In the rhizosphere sediment, the most abundant orders were Bacteroidales, Flavobacteriales, Desulfobacterales, Thiotrichales, Clostridiales and Alteromonadales. Ettinger et al. (2017), PeerJ, DOI 10.7717/peerj.3246 10/25 We also examined the overall patterns in our results at the class level (Table S5). For leaves, the most abundant class of epiphytes observed was Gammaproteobacteria (20.5 ± 7.3%). Other abundant classes included Clostridia (16.5 ± 12%), Bacteriodia (12.6 ± 8.6%), Alphaproteobacteria (11.4 ± 8.5%), Flavobacteria (7.8 ± 4.7%) and Saprospirae (7.7 ± 4.2%). For roots, the dominant class associated with the roots were Epsilonproteobacteria (17.9 ±15.5%). Other abundant classes observed on the roots include Deltaproteobacteria (13.4 ± 11.3%), Bacteriodia (12.8 ± 8.8%), Gammaproteobacteria (12.6 ± 11%), Clostridia (8 ± 8.4%), Flavobacteriia (6.1 ± 6.1%) and Alphaproteobacteria (4.8 ± 6.3%). In the rhizosphere sediment, the dominant class was Gammaproteobacteria (18.2 ± 3.4%), as it was on the leaves. Other abundant classes found in the rhizosphere sediment include Deltaproteobacteria (14.9 ± 2.6%), Bacteriodia (13.3 ± 2.3%), Flavobacteriia (9.3 ± 3.4%), Clostridia (5.1 ± 3.3%) and Anaerolineae (3.9 ± 1.6%). The summary results above allow a comparison to findings from a recent study on the rhizosphere sediment microbiomes of three seagrass species, including Z. marina, Cúcio et al., 2016. We chose to focus our comparison on the Cúcio et al. study because it is one of the more comprehensive culture independent studies of seagrasses. Overall, there are general similarities and differences when comparing the class-level patterns between the studies. The authors reported that the most abundant classes were Gammaproteobacteria (32– 38% depending on the species sampled), Deltaproteobacteria (23–26%), and Bacteroidia (6–7%). Major patterns in community composition of the leaves, roots and rhizosphere sediment These were the three most abundant classes in our sediment samples as well, but at different relative abundances (see above). These differences could be due to true differences in microbiomes in the sediments sampled, or due to the use of different primer sets, extraction methods, and sample collection strategies (among many other differences). When examined at higher taxonomic ranks, the microbiome of the leaves of Z. marina shares some similarities with the microbiomes of various marine algae (e.g., kelp and seaweeds), with Gammaproteobacteria being the most abundant class in both cases (Hollants et al., 2013). However, these similarities are not seen at lower ranks (e.g., order, family, genus). This finding is similar to what has been observed between different marine algal microbiomes, with similarities observed at higher, but not lower taxonomic levels (Hollants et al., 2013; Egan et al., 2013). This is further supported by a recent study, which focused on surface-associated communities, that observed that the microbiomes of seagrass and seaweed species were host specific, but had broad-scale functional similarities (Roth-Schulze et al., 2016). Diﬀerences in microbial communities between sample types (leaves, roots and rhizosphere sediment) and possible functional implications We used a Bonferroni corrected Kruskall–Wallis test to test for differences in relative abundance of the orders between sample types. This showed that Saprospirales, Thiotrichales, Rhodobacterales, Desulfobacterales, Desulfuromonadales, Marinicellales, Spirochaetales, Chromatiales and Campylobacterales are significantly different between sample types (p < 0.05). Campylobacterales, Desulfobacterales, Spirochaetales and Desulfuromonadales were enriched on Z. marina roots. Thiotrichales, Rhodobacterales and Saprospirales were enriched on the leaves. Thiotrichales, Marinicellales, Chromatiales, Desulfobacterales and Ettinger et al. (2017), PeerJ, DOI 10.7717/peerj.3246 Spirochaetales were enriched in the rhizosphere. We note that many of the taxa that differ significantly between communities are closely related to ones commonly associated with various aspects of sulfur and nitrogen metabolism. This is interesting because prior studies have suggested that nitrogen and sulfur metabolism are critical functions for the seagrass associated microbiome (Lovell, 2002). For example, acquisition of nitrogen (in its many forms) is frequently a limiting factor for the health of plants, including seagrasses (Short, 1987; Elser et al., 2007) and associations with microbes are frequently critical for such acquisition (Welsh, 2000; Nielsen et al., 2001). In addition, since the reduced sulfur compounds that accumulate in aquatic sediments are known phytotoxins (Lamers et al., 2013), it is thought that sulfur metabolizing microbes could play important roles in aiding seagrass survival in such sediments (Barber & Carlson, 1993; Terrados et al., 1999; Erskine & Koch, 2000; Van der Heide et al., 2012). Sulfur and nitrogen metabolism are not necessarily independent—it has been postulated that sulfate-reducing bacteria may be responsible for most of the nitrogen fixation that occurs in seagrass sediments (Capone, 1982). Given this context, we discuss several of the specific taxa that differ between samples and their possible connection to nitrogen and/or sulfur metabolism below. For example, Campylobacterales, specifically Sulfurimonas species, from the class Epsilonproteobacteria, were enriched on Z. marina roots. Previous studies of Spartina (Thomas et al., 2014) and Z. marina (Jensen, Kühl & Priemé, 2007) also found enrichment of Epsilonproteobacteria on roots relative to the surrounding sediment. All known Sulfurimonas species are sulfur- oxidizing chemolithoautotrophs, can perform denitrification and are postulated to play significant roles in biogeochemical cycling in marine sediments (Campbell et al., 2006). Members of Campylobacterales have previously been identified as nitrogen fixers when isolated from Spartina roots (McClung & Patriquin, 1980). Additionally, Campylobacterales and Desulfobacterales, known sulfur-reducing bacteria, have been previously found to be abundant in association with plants from brackish habitats (e.g., mangroves—Gomes et al., 2010). Diﬀerences in microbial communities between sample types (leaves, roots and rhizosphere sediment) and possible functional implications Rhodobacterales which are enriched on the Z. marina leaves in our study, are purple nonsulfur bacteria, that have been identified as primary surface colonizers in marine habitats and have been shown to have the ability to fix nitrogen (Palacios & Newton, 2005; Dang et al., 2008). Desulfobacterales and Rhodobacterales species have been previously found in association with the tropical seagrass Thalassia hemprichii (Jiang et al., 2015). Thiotrichales, which are enriched in the sediment, are generally filamentous sulfur-oxidizing bacteria (Garrity, Bell & Lilburn, 2005) and are postulated to be dominant sulfur-oxiders in salt marsh sediments (Thomas et al., 2014). Variation in sediment microbial communities between locations Variation in sediment microbial communities between locations The analysis of diversity metrics reported above also showed that there are significant differences in the sediment microbial communities from different locations (inside a eelgrass patch, the edge of a patch and outside of a patch). We therefore examined in more detail the taxonomic groups that differ significantly between sediment locations and their potential functional roles (Fig. 5). Bacteroidales, Myxococcales, Thiotrichales and Chromatiales are significantly different between locations with a Bonferroni corrected Kruskall–Wallis test (p < 0.01). Thiotrichales 12/25 Ettinger et al. (2017), PeerJ, DOI 10.7717/peerj.3246 Figure 5 Average relative abundance of taxonomic groups associated with sediment from each loca- tion (inside, edge, outside). Operational taxonomic units (OTUs) are shown grouped by taxonomic order and are colored by taxonomic class. Only orders with a mean relative abundance of at least one percent are shown. Bars represent the standard error of the mean. Figure 5 Average relative abundance of taxonomic groups associated with sediment from each loca- tion (inside, edge, outside). Operational taxonomic units (OTUs) are shown grouped by taxonomic order and are colored by taxonomic class. Only orders with a mean relative abundance of at least one percent are shown. Bars represent the standard error of the mean. and Chromatiales are enriched outside of Z. marina patches in the unvegetated sediment compared to the inside or edge of patches. In contrast, Bacteroidales and Myxococcales are enriched in the rhizosphere sediment inside and at the edge of eelgrass patches compared to the outside. The functional significance of these differences is unclear but we note a few things here. First, Thiotrichales and Chromatiales are common taxa in other marine and brackish sediments including those associated with various plants (e.g., Thomas et al., 2014). This is thought be reflective of a role in sulfur oxidation (see discussion above). Some studies have indicated that these taxa are associated with plants (e.g., seagrasses in Portugal Cúcio et al., 2016). However, other studies have indicated that these are found more in the sediment near plants but not specifically associated with plants (Thomas et al., 2014). Myxococcales, commonly found in freshwater and marsh sediments, includes microorganisms known to be involved in organic matter degradation (Bowen et al., 2012; Kou et al., 2016; Cleary et al., 2016). Variation in sediment microbial communities between locations The abundance of Myxococcales inside the eelgrass patch aligns with the expectation of higher prevalence of organic matter degradation inside the patch as opposed to surrounding unvegetated sediment. Environmental drivers of sediment communities In addition to investigating the taxonomic composition of the microbial communities of sediment collected from the inside, edge and outside of eelgrass patches, we decided to test for correlations between observed community differences and environmental factors to elucidate key factors that may be driving the microbial communities in eelgrass patches. In addition to investigating the taxonomic composition of the microbial communities of sediment collected from the inside, edge and outside of eelgrass patches, we decided to test for correlations between observed community differences and environmental factors to elucidate key factors that may be driving the microbial communities in eelgrass patches. A variety of abiotic factors were significantly different between locations including C:N ratio, TIC, dissolved oxygen, pH and sediment size fractions 710 µm and 63 µm (ANOVA, p < 0.05) (Tables S6 and S7). Unsurprisingly, eelgrass shoot density was significantly A variety of abiotic factors were significantly different between locations including C:N ratio, TIC, dissolved oxygen, pH and sediment size fractions 710 µm and 63 µm (ANOVA, p < 0.05) (Tables S6 and S7). Unsurprisingly, eelgrass shoot density was significantly 13/25 Ettinger et al. (2017), PeerJ, DOI 10.7717/peerj.3246 Figure 6 Relationship between environmental data and microbial communities. Non-metric multi- dimensional scaling (NMDS) of Bray Curtis dissimilarities of microbial communities found in sediment samples are shown here colored by location (inside, edge, outside). Environmental factors (p < 0.055, ANOVA) were overlaid as vectors onto the NMDS using the envfit function in vegan. Figure 6 Relationship between environmental data and microbial communities. Non-metric multi- dimensional scaling (NMDS) of Bray Curtis dissimilarities of microbial communities found in sediment samples are shown here colored by location (inside, edge, outside). Environmental factors (p < 0.055, ANOVA) were overlaid as vectors onto the NMDS using the envfit function in vegan. different between locations (ANOVA, p < 0.05). To determine which pair-wise locations were driving the significant differences between location overall, we performed Tukey’s HSD tests (Tables S8 and S9). We also performed Tukey’s HSD tests on percent TOC which was marginally non significantly different (ANOVA, p = 0.0519). All pair-wise location comparisons of eelgrass density and dissolved oxygen were significantly different (Tukey’s HSD, p < 0.05). The C:N ratio and sediment fraction 63 µm were significantly different for the outside-inside comparison (Tukey’s HSD, p < 0.05). Ettinger et al. (2017), PeerJ, DOI 10.7717/peerj.3246 Environmental drivers of sediment communities Percent TIC and TOC as well as sediment fraction 710 µm were significantly different for the outside-edge comparison and pH was significantly different for the inside-edge comparisons (Tukey’s HSD, p < 0.05). To test if there was a correlation between environmental measures and microbial community composition, Mantel tests were performed on Euclidean distances of environmental measures and the Bray–Curtis dissimilarities of sediment communities. A combined dataset including C:N ratio, TIC, TOC, dissolved oxygen, pH and eelgrass density was found to be significantly positively correlated with the sediment microbial community data (r = 0.1122, p = 0.0474) (Fig. 6). However, when measures were tested individually only the C:N ratio (r = 0.1701, p = 0.016) and eelgrass density (r = 0.1292, p = 0.0381) were significantly correlated with microbial community composition. The significant correlation between the sediment communities and the C:N ratio may indicate a change in ecosystem nutrient cycling at the patch edge. As Carbon (TIC and TOC) was not correlated with the sediment microbial communities, the correlation with the C:N ratio may hint at the importance of nitrogen, which was not measured in this study, to sediment community composition. Nitrogen is often a limiting terrestrial plant nutrient Ettinger et al. (2017), PeerJ, DOI 10.7717/peerj.3246 14/25 and N-limitation has also been observed in several seagrass studies, more frequently in temperate habitats (De Boer, 2007). Terrestrial plants overcome N-limitation by having beneficial interactions with nitrogen fixing bacteria and these bacteria have previously been observed to form associations with eelgrasses (Capone & Budin, 1982; Welsh, 2000; Bagwell et al., 2002; Adhitya, Thomas & Ward, 2007; Sun et al., 2015). Nitrogen fixation can account for 5–10% of plant nitrogen requirements in temperate eelgrass patch sediments, and up to 50% in tropical patch sediments, indicating an important role for nitrogen fixation in overall patch health (Welsh, 2000). A previous study looking at forest soil microbial communities found that microbial biomass and activity were significantly lower at forest edges due to decreased litter decomposition inthe edgehabitat andthus, changesin nutrientcycling (Malmivaara-Lämsä et al., 2008). In seagrass patches, on average, vegetated sediments are significantly enriched in organic matter compared to unvegetated sediments, with carbon stocks generally higher on the inside of patches (Duarte, Holmer & Marbà, 2005; Ricart et al., 2015). Ettinger et al. (2017), PeerJ, DOI 10.7717/peerj.3246 Environmental drivers of sediment communities It is possible that the observed community structure changes in the sediment between locations and the correlations with C:N ratio and eelgrass density here are indicative of a similar trend of location based nutrient cycling resulting from differing nutrient deposition and decomposition rates. Eelgrass density may have direct or indirect effects on sediment microbial communities as a result of the role eelgrass plays in its environment as a foundation species and an ecosystem engineer (Koch, 2001) Seagrasses are known to modify their surrounding habitat in a variety of ways including enhancing the input and retention of carbon and other nutrients in the sediment (Gacia et al., 2002; Duarte et al., 2005; Duarte & Cebrián, 1996), altering flow velocity and turbulence in the water column above patches (Fonseca et al., 1982; Granata et al., 2001; Folkard, 2005) which can increase sedimentation (Short & Short, 1984; Dauby et al., 1995; Gacia et al., 2002) and oxygenating the sediment using their roots (Caffrey & Kemp, 1991; Pedersen et al., 1998; Connell, Colmer & Walker, 1999). Other factors at play in the observed differences between locations as a result of eelgrass density may be the development stage of the eelgrass at the edge (if the patch is expanding or contracting) and the variable use of eelgrass as a habitat by macroorganisms. From terrestrial systems, it is known that microbial communities can vary across different stages of plant development (Chaparro, Badri & Vivanco, 2014). Seagrasses at earlier stages in development are known to have different carbon deposition rates than later stages, so if seagrass patch was in the process of expanding this may change the abiotic conditions at the patch edge, and thus might be reflected in distinct microbial communities at the edge of a patch compared to the inside (Duarte & Sand-Jensen, 1990; Cebrián et al., 2000). Additionally, seagrass patches are habitats for a large number of macroorganisms with variable abundance across seagrass patch landscapes (seagrass densities) (Tanner, 2005; Smith et al., 2008; Smith et al., 2011). Ultimately, although we see differences between locations in environmental abiotic measurements, we are unable, given the limitations of this study, to decouple these measurements from the eelgrass itself (eelgrass density), which is highly correlated with sediment community composition. 15/25 Ettinger et al. (2017), PeerJ, DOI 10.7717/peerj.3246 CONCLUSIONS This study provides new insights into the composition and assembly of the Z. marina microbiome. Major findings include that distinct microbial communities are associated with the leaves and roots of the plant, sediment associated communities are correlated with host plant density,and specific microbial taxa are found to have high relative abundances on particular tissues. Differences in the rhizosphere sediment community composition at the patch edge were observed and correlated with variation in environmental measurements. However, we were unable to disentangle these measures from eelgrass density, with the strongest correlated factor with community differences being presence/absence of the host plant. This is perhaps not unexpected from a field study, as eelgrass species are ecosystem engineers that actively change the sediment chemistry and landscape (Orth et al., 2006; Bos et al., 2007). Overall, we believe that the results of this study hint at a network of complex interactions between Z. marina, the microbes associated with Z. marina and biogeochemical cycling. Untangling such networks is difficult but increasingly feasible. Although Z. marina is not a model organism in the sense of Arabidopsis or poplar, we believe it can nevertheless become a model for host-microbiome-environment interaction studies. Advantages of working on this species include that there is a genome now available (Olsen et al., 2016), that there is a large network of collaborating labs focusing on this species (Zostera Experimental Network; http://zenscience.org), and that it can be used in common garden and reciprocal transplant experiments. Along these lines we have been building a library of cultured isolates associated with this species and sequencing the genomes of many of these (Lee et al., 2015a; Lee et al., 2015b; Lee et al., 2016a; Lee et al., 2016b; Alexiev et al., 2016a; Alexiev et al., 2016b). There are still areas in need of improvement (e.g., there a limited amount of full length 16S and 18S other reference data; only limited information on the in situ functions of microbes are available, there is a need for more genetic tools for the host), but given the importance of coastal marine systems and seagrasses generally, we believe continued efforts to study the host-microbiome-environment interactions in this and related species are important. ACKNOWLEDGEMENTS Illumina sequencing was performed at the DNA Technologies Core facility in the Genome Center at UC Davis, Davis, California. We thank Qingyi ‘‘John’’ Zhang for his help with the Illumina library preparation and Hannah Holland-Moritz for her help with DNA extractions. We thank Ted Grosholz and Susan Williams for hosting Sofie Voerman during her visit and for help with the sampling design. We thank Daniel Bradley for his help with sample collection. Author Contributions • Cassandra L. Ettinger analyzed the data, wrote the paper, prepared figures and/or tables, reviewed drafts of the paper. • Sofie E. Voerman conceived and designed the experiments, reviewed drafts of the paper, performed sampling. • Jenna M. Lang conceived and designed the experiments, reviewed drafts of the paper. • Jenna M. Lang conceived and designed the experiments, reviewed drafts of the paper. • John J. Stachowicz reviewed drafts of the paper, advised on experimental design, edited drafts of paper. • John J. Stachowicz reviewed drafts of the paper, advised on experimental design, edited drafts of paper. • Jonathan A. Eisen contributed reagents/materials/analysis tools, reviewed drafts of the paper, advised on data analysis, edited drafts of paper. Competing Interests Jonathan A. Eisen is an Academic Editor for PeerJ. Jenna M. Lang is an employee of Trace Genomics, Inc. DNA Deposition The following information was supplied regarding the deposition of DNA sequences: The following information was supplied regarding the deposition of DNA sequences: g g g This 16S rRNA sequencing project has been deposited at GenBank under the accession no. PRJNA350006. This 16S rRNA sequencing project has been deposited at GenBank under the accession no. PRJNA350006. Grant Disclosures Grant Disclosures The following grant information was disclosed by the authors: The following grant information was disclosed by the authors: Gordon and Betty Moore Foundation: GBMF333. Funding g This work was supported by a grant from the Gordon and Betty Moore Foundation (GBMF333) ‘‘Investigating the co-evolutionary relationships between seagrasses and their Ettinger et al. (2017), PeerJ, DOI 10.7717/peerj.3246 microbial symbionts.’’ The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. microbial symbionts.’’ The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Supplemental Information Supplemental Information Supplemental information for this article can be found online at http://dx.doi.org/10.7717/ peerj.3246#supplemental-information. Supplemental information for this article can be found online at http://dx.doi.org/10.7717/ peerj.3246#supplemental-information. Supplemental information for this article can be found online at http://dx.doi.org/10.7717/ peerj 3246#supplemental-information peerj.3246#supplemental-information. Data Availability The following information was supplied regarding data availability: The following information was supplied regarding data availability: Coil, David; Eisen, Jonathan; Stachowicz, Jay; Green, Jessica; Holland-Moritz, Hannah; Lang, Jenna (2014): The Seagrass Microbiome. figshare. https://doi.org/10.6084/m9.figshare.1014334.v1. The following information was supplied regarding data availability: Coil, David; Eisen, Jonathan; Stachowicz, Jay; Green, Jessica; Holland-Moritz, Hannah; Lang, Jenna (2014): The Seagrass Microbiome. figshare. https://doi.org/10.6084/m9.figshare.1014334.v1. Coil, David; Eisen, Jonathan; Stachowicz, Jay; Green, Jessica; Holland-Moritz, Hannah; Lang, Jenna (2014): The Seagrass Microbiome. figshare. https://doi.org/10.6084/m9.figshare.1014334.v1. REFERENCES Adhitya A, Thomas FIM, Ward BB. 2007. Diversity of assimilatory nitrate reductase genes from plankton and epiphytes associated with a seagrass bed. Microbial Ecology 54:587–597 DOI 10.1007/s00248-006-9175-0. Aleklett K, Leff JW, Fierer N, Hart M. 2015. Wild plant species growing closely con- nected in a subalpine meadow host distinct root-associated bacterial communities. PeerJ 3:e804 DOI 10.7717/peerj.804. Aleklett K, Leff JW, Fierer N, Hart M. 2015. Wild plant species growing closely con- nected in a subalpine meadow host distinct root-associated bacterial communities. PeerJ 3:e804 DOI 10.7717/peerj.804. Ettinger et al. (2017), PeerJ, DOI 10.7717/peerj.3246 Alexiev A, Krusor ML, Jospin G, Lang JM, Eisen JA, Coil DA. 2016a. Draft genome sequences of two Pseudoalteromonas strains isolated from roots and leaf blades of the seagrass Zostera marina. Genome Announcements 4(1):e00010-16 DOI 10.1128/genomeA.00010-16. Alexiev A, Krusor ML, Jospin G, Lang JM, Eisen JA, Coil DA. 2016b. Draft genome sequence of Cobetia sp. UCD-24C, isolated from roots and leaves of the seagrass Zostera marina. Genome Announcements 4(2):e00116-16 DOI 10.1128/genomeA.00116-16. Anderson MJ. 2001. A new method for non-parametric multivariate analysis of variance. Austral Ecology 26:32–46. Anderson MJ, Walsh DC. 2013. PERMANOVA, ANOSIM, and the mantel test in the face of heterogeneous dispersions: what null hypothesis are you testing? Ecological Monographs 83:557–574 DOI 10.1890/12-2010.1. Bagwell CE, Rocque JR, Smith GW, Polson SW, Friez MJ, Longshore JW, Lovell CR. 2002. Molecular diversity of diazotrophs in oligotrophic tropical seagrass bed communities. FEMS Microbiology Ecology 39:113–119 OI 10.1111/j.1574-6941.2002.tb00912.x. Barber TR, Carlson PR. 1993. Effects of seagrass die-off on benthic fluxes and porewater concentrations of PCO2, PH2S, and CH4 in Florida Bay Sediments. In: Oremland RS, ed. Biogeochemistry of global change: radiatively active trace gases. New York: Chapman and Hall, 530–550. Beckers B, OP De Beeck M, Weyens N, Van Acker R, Van Montagu M, Boerjan W, Vangronsveld J. 2016. Lignin engineering in field-grown poplar trees affects the endosphere bacterial microbiome. Proceedings of the National Academy of Sciences of the United States of America 113:2312–2317 DOI 10.1073/pnas.1523264113. Bent SJ, Forney LJ. 2008. The tragedy of the uncommon: understanding limi- tations in the analysis of microbial diversity. The ISME Journal 2:689–695 DOI 10.1038/ismej.2008.44. Bos AR, Bouma TJ, De Kort GLJ, Van Katwijk MM. 2007. Ecosystem engineering by annual intertidal seagrass beds: sediment accretion and modification. Estuarine, Coastal and Shelf Science 74:344–348 DOI 10.1016/j.ecss.2007.04.006. Bouffaud ML, Poirier MA, Muller D, Moënne-Loccoz Y. 2014. Ettinger et al. (2017), PeerJ, DOI 10.7717/peerj.3246 REFERENCES Root microbiome relates to plant host evolution in maize and other Poaceae. Environmental Microbiology 16:2804–2814 DOI 10.1111/1462-2920.12442. Bowen JL, Morrison HG, Hobbie JE, Sogin ML. 2012. Salt marsh sediment diversity: a test of the variability of the rare biosphere among environmental replicates. The ISME Journal 6:2014–2023 DOI 10.1038/ismej.2012.47. Bray JR, Curtis JT. 1957. An ordination of the upland forest communities of Southern Wisconsin. Ecological Monographs 27:325–349 DOI 10.2307/1942268. Caffrey JM, Kemp WM. 1991. Seasonal and spatial patterns of oxygen production, respiration and root-rhizome release in Potamogeton perfoliatus L. and Zostera marina L. Aquatic Botany 40:109–128 DOI 10.1016/0304-3770(91)90090-R. 18/25 Ettinger et al. (2017), PeerJ, DOI 10.7717/peerj.3246 18/25 Campbell BJ, Engel AS, Porter ML, Takai K. 2006. The versatile epsilon-proteobacteria: key players in sulphidic habitats. Nature Reviews. Microbiology 4:458–468 DOI 10.1038/nrmicro1414. Capone DG. 1982. Nitrogen fixation (Acetylene Reduction) by rhizosphere sedi- ments of the eelgrass Zostera marina. Marine Ecology Progress Series 10:67–75 DOI 10.3354/meps010067. Capone DG, Budin JM. 1982. Nitrogen fixation associated with rinsed roots and rhizomes of the eelgrass Zostera marina. Plant Physiology 70:1601–1604 DOI 10.1104/pp.70.6.1601. Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD, Costello EK, Fierer N, Peña AG, Goodrich JK, Gordon JI, Huttley GA, Kelley ST, Knights D, Koenig JE, Ley RE, Lozupone CA, McDonald D, Muegge BD, Pirrung M, Reeder J, Sevinsky JR, Turnbaugh PJ, Walters WA, Widmann J, Yatsunenko T, Zaneveld J, Knight R. 2010. QIIME allows analysis of high-throughput community sequencing data. Nature Methods 7:335–336 DOI 10.1038/nmeth.f.303. Caporaso JG, Lauber CL, Walters WA, Berg-Lyons D, Huntley J, Fierer N, Owens SM, Betley J, Fraser L, Bauer M, Gormley N, Gilbert JA, Smith G, Knight R. 2012. Ultra- high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms. The ISME Journal 6:1621–1624. Cebrián J, Pedersen MF, Kroeger KD, Valiela I. 2000. Fate of production of the seagrass Cymodocea nodosa in different stages of meadow formation. Marine Ecology Progress Series 204:119–130 DOI 10.3354/meps204119. Chao A. 1984. Non-parametric estimation of the number of classes in a population. Scandinavian Journal of Statistics, Theory and Applications 11:265–270. Chaparro JM, Badri DV, Vivanco JM. 2014. Rhizosphere microbiome assemblage is affected by plant development. The ISME Journal 8:790–803 DOI 10.1038/ismej.2013.196. Cleary DFR, Polónia ARM, Sousa AI, Lillebø AI, Queiroga H, Gomes NCM. 2016. Tem- poral dynamics of sediment bacterial communities in monospecific stands of Juncus maritimus and Spartina maritima. Plant Biology 18:824–834 DOI 10.1111/plb.12459. Connell EL, Colmer TD, Walker DI. REFERENCES 1999. Radial oxygen loss from intact roots ofHalophila ovalisas a function of distance behind the root tip and shoot illumina- tion. Aquatic Botany 63:219–228 DOI 10.1016/S0304-3770(98)00126-0. Cúcio C, Engelen AH, Costa R, Muyzer G. 2016. Rhizosphere microbiomes of European seagrasses are selected by the plant, but are not species specific. Frontiers in Microbi- ology 7:440 DOI 10.3389/fmicb.2016.00440. Dang H, Li T, Chen M, Huang G. 2008. Cross-ocean distribution of Rhodobacterales bacteria as primary surface colonizers in temperate coastal marine waters. Applied and Environmental Microbiology 74:52–60 DOI 10.1128/AEM.01400-07. Dauby P, Bale AJ, Bloomer N, Canon C, Ling RD, Norro A, Robertson JE, Simon A, Théate JM, Watson AJ, Frankignoulle M. 1995. Particle fluxes over a Mediterranean seagrass bed: a one year case study. Marine Ecology Progress Series 126:233–246 DOI 10.3354/meps126233. Ettinger et al. (2017), PeerJ, DOI 10.7717/peerj.3246 Davies-Colley RJ, Payne GW, Van Elswijk M. 2000. Microclimate gradients across a forest edge. New Zealand Journal of Ecology 11:1–121. De Boer WF. 2007. Seagrass–sediment interactions, positive feedbacks and critical thresholds for occurrence: a review. Hydrobiologia 591:5–24 DOI 10.1007/s10750-007-0780-9. DeSantis TZ, Hugenholtz P, Larsen N, Rojas M, Brodie EL, Keller K, Huber T, Dalevi D, Hu P, Andersen GL. 2006. Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB. Applied and Environmental Micro- biology 72:5069–5072 DOI 10.1128/AEM.03006-05. Dixon P. 2003. VEGAN, a package of R functions for community ecology. Journal of Vegetation Science: Official Organ of the International Association for Vegetation Science 14:927–930 DOI 10.1111/j.1654-1103.2003.tb02228.x. Duarte CM, Borum K, Short FT, Walker DI. 2005. Seagrass ecosystems: their global status and prospects. In: Polunin NVC, ed. Aquatic ecosystems: trends and global prospects. Cambridge Univ., 281–294. Duarte CM, Cebrián J. 1996. The fate of marine autotrophic production. Limnology and Oceanography 41:1758–1766 DOI 10.4319/lo.1996.41.8.1758. Duarte CM, Holmer M, Marbà N. 2005. Plant-microbe interactions in seagrass meadows. Coastal and Estuarine Studies Epub ahead of print Mar 23 2013 DOI 10.1029/CE060p0031. Duarte CM, Sand-Jensen K. 1990. Seagrass colonization: patch formation and patch growth in Cymodocea nodosa. Marine Ecology Progress Series 65:193–200 DOI 10.3354/meps065193. Edgar RC. 2010. Search and clustering orders of magnitude faster than BLAST. Bioinfor- matics 26:2460–2461 DOI 10.1093/bioinformatics/btq461. Edwards J, Johnson C, Santos-Medellín C, Lurie E, Podishetty NK, Bhatnagar S, Eisen JA, Sundaresan V. 2015. Structure, variation, and assembly of the root-associated microbiomes of rice. Proceedings of the National Academy of Sciences of the United States of America 112:E911–E920 DOI 10.1073/pnas.1414592112. Ettinger et al. (2017), PeerJ, DOI 10.7717/peerj.3246 REFERENCES Egan S, Harder T, Burke C, Steinberg P, Kjelleberg S, Thomas T. 2013. The seaweed holobiont: understanding seaweed—bacteria interactions. FEMS Microbiology Reviews 37:462–476 DOI 10.1111/1574-6976.12011. Elser JJ, Bracken MES, Clel EE, Gruner DS, Harpole WS, Hillebr H, Ngai JT, Seabloom EW, Shurin JB, Smith JE. 2007. Global analysis of nitrogen and phosphorus limitation of primary producers in freshwater, marine and terrestrial ecosystems. Ecology Letters 10:1135–1142 DOI 10.1111/j.1461-0248.2007.01113.x. Erskine JM, Koch MS. 2000. Sulfide effects on Thalassia testudinum carbon balance and adenylate energy charge. Aquatic Botany 67:275–285 DOI 10.1016/S0304-3770(00)00099-1. Flaspohler DJ, Temple SA, Rosenfield RN. 2001. Species-specific edge effects on nest success and breeding bird density in a forested landscape. Ecological Applications 11:32–46 DOI 10.2307/3061053. Ettinger et al. (2017), PeerJ, DOI 10.7717/peerj.3246 Folkard AM. 2005. Hydrodynamics of modelPosidonia oceanicapatches in shallow water. Limnology and Oceanography 50:1592–1600 DOI 10.4319/lo.2005.50.5.1592. Fonseca MS, Fisher JS, Zieman JC, Thayer GW. 1982. Influence of the seagrass, Zostera marina L. on current flow. Estuarine, Coastal and Shelf Science 15:351–364 DOI 10.1016/0272-7714(82)90046-4. Fox BJ, Taylor JE, Fox MD, Williams C. 1997. Vegetation changes across edges of rainforest remnants. Biological Conservation 82:1–13 Fox BJ, Taylor JE, Fox MD, Williams C. 1997. Vegetation changes across edges of rainforest remnants. Biological Conservation 82:1–13 DOI 10.1016/S0006-3207(97)00011-6. Gacia E, Esperan¸ca G, Duarte CM, Middelburg JJ. 2002. Carbon and nutrient deposition in a Mediterranean seagrass (Posidonia oceanica) meadow. Limnology and Oceanog- raphy 47:23–32 DOI 10.4319/lo.2002.47.1.0023. Garrity GM, Bell JA, Lilburn T. 2005. Order V. Thiotrichales ord nov. In: Brenner DJ, Krieg NR, Staley JT, Garrity GM, eds. Bergey’s manual of systematic bacteriology (The Proteobacteria), Part B (The Gammaproteobacteria). Vol. 2. New York: Springer, 131. Gomes NCM, Cleary DFR, Pinto FN, Egas C, Almeida A, Cunha A, Mendon¸ca-Hagler LCS, Smalla K. 2010. Taking root: enduring effect of rhizosphere bacterial coloniza- tion in mangroves. PLOS ONE 5(11):e14065 DOI 10.1371/journal.pone.0014065. Granata TC, Serra T, Colomer J, Casamitjana X, Duarte CM, Gacia E. 2001. Flow and particle distributions in a nearshore seagrass meadow before and after a storm. Marine Ecology Progress Series 218:95–106 DOI 10.3354/meps218095. Hamady M, Lozupone C, Knight R. 2010. Fast UniFrac: facilitating high-throughput phylogenetic analyses of microbial communities including analysis of pyrosequenc- ing and PhyloChip data. The ISME Journal 4:17–27 DOI 10.1038/ismej.2009.97. Harrison S, Bruna E. 1999. Habitat fragmentation and large-scale conservation: what do we know for sure? Ecography 22:225–232 DOI 10.1111/j.1600-0587.1999.tb00496.x. Hollants J, Leliaert F, De Clerck O, Willems A. 2013. REFERENCES What we can learn from sushi: a review on seaweed–bacterial associations. FEMS Microbiology Ecology 83:1–16 DOI 10.1111/j.1574-6941.2012.01446.x. Jensen SI, Kühl M, Priemé A. 2007. Different bacterial communities associated with the roots and bulk sediment of the seagrass Zostera marina. FEMS Microbiology Ecology 6:108–117. Jiang YF, Ling J, Dong JD, Chen B, Zhang YY, Zhang YZ, Wang YS. 2015. Illumina- based analysis the microbial diversity associated with Thalassia hemprichii in Xincun Bay, South China Sea. Ecotoxicology 24:1548–1556 DOI 10.1007/s10646-015-1511-z. Koch EW. 2001. Beyond light: physical, geological, and geochemical parameters as possible submersed aquatic vegetation habitat requirements. Estuaries 24:1–17 DOI 10.2307/1352808. Kou W, Zhang J, Lu X, Ma Y, Mou X, Wu L. 2016. Identification of bacterial communi- ties in sediments of Poyang Lake, the largest freshwater lake in China. SpringerPlus 5:401 DOI 10.1186/s40064-016-2026-7. Kramer CY. 1956. Extension of multiple range tests to group means with unequal numbers of replications. Biometrics 12:307–310 DOI 10.2307/3001469. 21/25 Ettinger et al. (2017), PeerJ, DOI 10.7717/peerj.3246 Kramer CY. 1957. Extension of multiple range tests to group correlated adjusted means. Biometrics 13:13–18 DOI 10.2307/3001898. Lakshmanan V. 2015. Root microbiome assemblage is modulated by plant host factors. In: Bais H, Sherrier J, eds. Plant microbe interactions. Advances in botanical research. Vol. 75, 57–79. Lamers LPM, Govers LL, Janssen ICJM, Geurts JJM, Van der Welle MRW, Van Katwijk MM, Van der Heide T, Roelofs JGM, Smolders AJP. 2013. Sulfide as a soil phytotoxin-a review. Frontiers in Plant Science 4:Article 268 DOI 10.3389/fpls.2013.00268. Lang JM, Eisen JA, Zivkovic AM. 2014. The microbes we eat: abundance and taxonomy of microbes consumed in a day’s worth of meals for three diet types. PeerJ 2:e659 DOI 10.7717/peerj.659. Lee RD, Jospin G, Lang JM, Eisen JA, Coil DA. 2015a. Draft genome sequence of Pseudoalteromonas tetraodonis Strain UCD-SED8 (Phylum Gammaproteobacteria). Genome Announcements 3(6):e01276-15 DOI 10.1128/genomeA.01276-15. Lee RD, Jospin G, Lang JM, Eisen JA, Coil DA. 2015b. Draft genome sequence of Bacillus vietnamensis Strain UCD-SED5 (Phylum Firmicutes). Genome Announcements 3(6):e01376-15 DOI 10.1128/genomeA.01376-15. Lee RD, Jospin G, Lang JM, Eisen JA, Coil DA. 2016a. Draft genome sequences of two Vibrio splendidus strains, isolated from seagrass sediment. Genome Announcements 4(1):e01769-15 DOI 10.1128/genomeA.01769-15. Lee RD, Jospin G, Lang JM, Eisen JA, Coil DA. 2016b. Draft genome sequences of two Pseudoalteromonas porphyrae strains isolated from seagrass sediment. Genome Announcements 4(2):e00092-16 DOI 10.1128/genomeA.00092-16. Les DH, Cleland MA, Waycott M. 1997. Ettinger et al. (2017), PeerJ, DOI 10.7717/peerj.3246 REFERENCES Phylogenetic studies in alismatidae, II: evolution of marine angiosperms (seagrasses) and hydrophily. Systematic Botany 22:443–463 DOI 10.2307/2419820. Lovell CR. 2002. Plant-microbe interactions in the marine environment. In: Bitton G, ed. Encyclopedia of environmental microbiology. Vol. 5. Hoboken: Wiley, 2539–2554. Lozupone CA, Hamady M, Kelley ST, Knight R. 2007. Quantitative and qualita- tive diversity measures lead to different insights into factors that structure mi- crobial communities. Applied and Environmental Microbiology 73:1576–1585 DOI 10.1128/AEM.01996-06. Lundberg DS, Lebeis SL, Paredes SH, Yourstone S, Gehring J, Malfatti S, Tremblay J, Engelbrektson A, Kunin V, Del Rio TG, Edgar RC, Eickhorst T, Ley RE, Hugen- holtz P, Tringe SG, Dangl JL. 2012. Defining the core Arabidopsis thaliana root microbiome. Nature 488:86–90 DOI 10.1038/nature11237. Malmivaara-Lämsä M, Hamberg L, Haapamäki E, Liski J, Kotze DJ, Lehvävirta S, Fritze H. 2008. Edge effects and trampling in boreal urban forest fragments– impacts on the soil microbial community. Soil Biology & Biochemistry 40:1612–1621 DOI 10.1016/j.soilbio.2008.01.013. Ettinger et al. (2017), PeerJ, DOI 10.7717/peerj.3246 22/25 McClung CR, Patriquin DG. 1980. Isolation of a nitrogen-fixing Campylobacter species from the roots of Spartina alterniflora Loisel. Canadian Journal of Microbiology 26:881–886 DOI 10.1139/m80-153. McMurdie PJ, Holmes S. 2013. phyloseq: an R package for reproducible interac- tive analysis and graphics of microbiome census data. PLOS ONE 8(4):e61217 Mejia AY, Rotini A, Lacasella F, Bookman R, Thaller MC, Shem-Tov R, Winters G, Migliore L. 2016. Assessing the ecological status of seagrasses using morphology, biochemical descriptors and microbial community analyses. A study in Halophila Stipulacea (Forsk.) Aschers meadows in the northern Red Sea. Ecological Indicators 60:1150–1163. Nielsen LB, Finster K, Welsh DT, Donelly A, Herbert RA, De Wit R, Lomstein BA. 2001. Sulphate reduction and nitrogen fixation rates associated with roots, rhizomes and sediments from Zostera noltii and Spartina maritima meadows. Environmental Microbiology 3:63–71 DOI 10.1046/j.1462-2920.2001.00160.x. Olsen JL, Rouzé P, Verhelst B, Lin Y-C, Bayer T, Collen J, Dattolo E, De Paoli E, Dit- tami S, Maumus F, Michel G, Kersting A, Lauritano C, Lohaus R, Töpel M, Tonon T, Vanneste K, Amirebrahimi M, Brakel J, Boström C, Chovatia M, Grimwood J, Jenkins JW, Jueterbock A, Mraz A, Stam WT, Tice H, Bornberg-Bauer E, Green PJ, Pearson GA, Procaccini G, Duarte CM, Schmutz J, Reusch TBH, Van de Peer Y. 2016. The genome of the seagrass Zostera marina reveals angiosperm adaptation to the sea. Nature 530:331–335 DOI 10.1038/nature16548. Ettinger et al. (2017), PeerJ, DOI 10.7717/peerj.3246 REFERENCES Orth RJ, Carruthers TJB, Dennison WC, Duarte CM, Fourqurean JW, Heck KL, Hughes RA, Kendrick GA, Kenworthy WJ, Olyarnik S, Short FT, Waycott M, Williams SL. 2006. A global crisis for seagrass ecosystems. Bioscience 56:987–996 DOI 10.1641/0006-3568(2006)56[987:AGCFSE]2.0.CO;2. Palacios R, Newton WE. 2005. Genomes and genomics of nitrogen-fixing organisms. Dordrecht: Springer. Paton PWC. 1994. The effect of edge on avian nest success: how strong is the evidence? Conservation Biology: the Journal of the Society for Conservation Biology 8:17–26 DOI 10.1046/j.1523-1739.1994.08010017.x. Pedersen O, Borum J, Duarte CM, Fortes MD. 1998. Oxygen dynamics in the rhi- zosphere of Cymodocea rotundata. Marine Ecology Progress Series 169:283–288 DOI 10.3354/meps169283. Peiffer JA, Spor A, Koren O, Jin Z, Tringe SG, Dangl JL, Buckler ES, Ley RE. 2013. Diversity and heritability of the maize rhizosphere microbiome under field condi- tions. Proceedings of the National Academy of Sciences of the United States of America 110:6548–6553 DOI 10.1073/pnas.1302837110. R Core Team. 2016. R: a language and environment for statistical computing. Vienna: R Foundation for Statistical Computing. Available at https://www.r-project.org. Ricart AM, York PH, Rasheed MA, Pérez M, Romero J, Bryant CV, Macreadie PI. 2015. Variability of sedimentary organic carbon in patchy seagrass landscapes. Marine Pollution Bulletin 100:476–482 DOI 10.1016/j.marpolbul.2015.09.032. Ettinger et al. (2017), PeerJ, DOI 10.7717/peerj.3246 23/25 Roth-Schulze AJ, Zozaya-Valdés E, Steinberg PD, Thomas T. 2016. Partitioning of functional and taxonomic diversity in surfaceêassociated microbial communities. Environmental Microbiology 18:4391–4402 DOI 10.1111/1462-2920.13325. Shannon CE, Weaver W. 1949. The mathematical theory of communication. Urbana: University of Illinois Press, 29. Short FT. 1987. Effects of sediment nutrients on seagrasses: literature review and meso- cosm experiment. Aquatic Botany 27:41–57 DOI 10.1016/0304-3770(87)90085-4. Short FT, Short CA. 1984. The seagrass filter: purification of estuarine and coastal waters. In: Kennedy VS, ed. The estuary as a filter. Orlando: Acadamic Press, 395–413. Simpson EH. 1949. Measurement of diversity. Nature 163:688–688 DOI 10.1038/163688a0. Smith TM, Hindell JS, Jenkins GP, Connolly RM. 2008. Edge effects on fish associated with seagrass and sand patches. Marine Ecology Progress Series 359:203–213 DOI 10.3354/meps07348. Smith TM, Hindell JS, Jenkins GP, Connolly RM, Keough MJ. 2011. Edge ef- fects in patchy seagrass landscapes: the role of predation in determining fish distribution. Journal of Experimental Marine Biology and Ecology 399:8–16 DOI 10.1016/j.jembe.2011.01.010. Sun F, Zhang X, Zhang Q, Liu F, Zhang J, Gong J. 2015. Ettinger et al. (2017), PeerJ, DOI 10.7717/peerj.3246 Welsh DT. 2000. Nitrogen fixation in seagrass meadows: regulation, plant-bacteria interactions and significance to primary productivity. Ecology Letters 3:58–71 DOI 10.1046/j.1461-0248.2000.00111.x. REFERENCES Seagrass (Zostera marina) colonization promotes the accumulation of diazotrophic bacteria and alters the relative abundances of specific bacterial lineages involved in benthic car- bon and sulfur cycling. Applied and Environmental Microbiology 81:6901–6914 DOI 10.1128/AEM.01382-15. Tanner JE. 2005. Edge effects on fauna in fragmented seagrass meadows. Austral Ecology 30:210–218 DOI 10.1111/j.1442-9993.2005.01438.x. Terrados J, Duarte CM, Kamp-Nielsen L, Agawin NSR, Gacia E, Lacap D, Fortes MD, Borum J, Lubanski M, Greve T. 1999. Are seagrass growth and survival constrained by the reducing conditions of the sediment? Aquatic Botany 65:175–197 DOI 10.1016/S0304-3770(99)00039-X. Thomas F, Giblin AE, Cardon ZG, Sievert SM. 2014. Rhizosphere heterogeneity shapes abundance and activity of sulfur-oxidizing bacteria in vegetated salt marsh sediments. Frontiers in Microbiology 5:Article 309. Tukey JW. 1953. The problem of multiple comparisons. The collected works of John W. Tukey, vol. 8. New York: Chapman & Hall. Van der Heide T, Govers LL, De Fouw J, Olff H, Van Der Geest M, Van Katwijk MM Piersma T, Van de Koppel J, Silliman BR, Smolders AJP, Van Gils JA. 2012. A three-stage symbiosis forms the foundation of seagrass ecosystems. Science 336:1432–1434 DOI 10.1126/science.1219973. Warton DI, Wright ST, Wang Y. 2012. Distance-based multivariate analyses con- found location and dispersion effects. Methods in Ecology and Evolution 3:89–101 DOI 10.1111/j.2041-210X.2011.00127.x. Ettinger et al. (2017), PeerJ, DOI 10.7717/peerj.3246 24/25 Welsh DT. 2000. Nitrogen fixation in seagrass meadows: regulation, plant-bacteria interactions and significance to primary productivity. Ecology Letters 3:58–71 DOI 10.1046/j.1461-0248.2000.00111.x. j Wickham H. 2009. ggplot2: elegant graphics for data analysis. New York: Springer-Verlag. Wickham H. 2009. ggplot2: elegant graphics for data analysis. New York: Springer-Verlag. Williams SL, Heck Jr KL. 2001. Seagrass communities ecology. In: Bertness MD, Gaines SD, Hay ME, eds. Marine community ecology. Sunderland: Sinauer Associates, 317–337. 25/25 25/25
https://openalex.org/W3110854217	https://www.biorxiv.org/content/biorxiv/early/2020/12/07/2020.12.06.411850.full.pdf	English	null	The Importance of Individual Beliefs in Assessing Treatment Efficacy: Insights from Neurostimulation Studies	bioRxiv (Cold Spring Harbor Laboratory)	2,020	cc-by	10,884	. CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 7, 2020. ; https://doi.org/10.1101/2020.12.06.411850 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 7, 2020. ; https://doi.org/10.1101/2020.12.06.411850 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 7, 2020. ; https://doi.org/10.1101/2020.12.06.411850 doi: bioRxiv preprint Is it all in our head? When subjective beliefs about receiving an intervention are better predictors of experimental results than the intervention itself Luisa Fassi & Roi Cohen Kadosh* Luisa Fassi & Roi Cohen Kadosh* Wellcome Centre for Integrative Neuroimaging, Department of Experimental Psychology, University of Oxford, UK Correspondence: Luisa Fassi (luisafassi@live.com) Roi Cohen Kadosh (roi.cohenkadosh@psy.ox.ac.uk) The effect of subjective intervention 2 . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 7, 2020. ; https://doi.org/10.1101/2020.12.06.411850 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 7, 2020. ; https://doi.org/10.1101/2020.12.06.411850 doi: bioRxiv preprint 2 The effect of subjective intervention The effect of subjective intervention 2 Abstract In recent years, there has been debate about the effectiveness of interventions from different fields (e.g., non-invasive brain stimulation (NIBS), neurofeedback, cognitive training programs) due to contradictory and nuanced experimental findings. Up to date, studies are focused on comparing the effects of an active form of the intervention to a placebo/control condition. However, a neglected question is how to consider individual differences in response to blinding procedures, and their effect on behavioural outcomes, rather than merely compare the efficacy of blinding using a group-based approach. To address this gap in the literature, we here suggest using subjective intervention—the participants’ subjective beliefs about receiving or not receiving an intervention—as a factor. Specifically, we examined whether subjective intervention and subjective dosage (i.e. participants’ subjective beliefs about the intensity of the intervention they received) affected performance scores independently, or interacting with, the active experimental condition. We carried out data analysis on an open-access dataset that has shown the efficacy of active NIBS in altering mind wandering. We show that subjective intervention and subjective dosage successfully explained alteration in mind wandering scores, over and beyond the objective intervention. These findings highlight the importance of accounting for the participants’ beliefs about receiving interventions at the individual level by demonstrating their effect on human behaviour independently of the actual intervention. Altogether, our approach allows more rigorous and improved experimental design and analysis, which will strengthen the conclusions coming from basic and clinical research, for both NIBS and non-NIBS interventions . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 7, 2020. ; https://doi.org/10.1101/2020.12.06.411850 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 7, 2020. ; https://doi.org/10.1101/2020.12.06.411850 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 7, 2020. ; https://doi.org/10.1101/2020.12.06.411850 doi: bioRxiv preprint The effect of subjective intervention 3 3 predictors of experimental results than the intervention itself A substantial amount of research from the fields of medicine, neuroscience, psychology and education is aimed at establishing the effectiveness of different interventions on both clinical and non-clinical populations. In recent years, a great deal of resources has been invested in the experimental assessment of drugs, vaccines, cognitive training programs, neurofeedback, and brain stimulation. In many cases, research findings have failed to provide definite and convincing results. The nuanced and often contradictory picture emerging from the published literature has led to increasing scepticism concerning the effectiveness of the examined interventions (Lampit et al., 2014; López-Alonso et al., 2014; Sitaram et al., 2017). Several factors have been pointed at as the plausible reasons underlying the heterogeneity in research findings that arises from different studies. Literature suggests that a big part of the variability in experimental results may be due to the intra-individual and inter-individual variation in the investigated outcome measures, the differences in the spectrum of features that each intervention entails, poor experimental designs, as well as the complex interaction between those factors (Filmer et al., 2020; Gruzelier, 2014; Guerra et al., 2020). Arguably, experimental design is the factor that falls under researchers’ control to a more considerable extent. Therefore, it is essential to thoroughly consider its effects on the results of a given experiment. Two crucial, yet often neglected, aspects of experimental design are 1) the way effective blinding is assessed and, 2) the lack of modelling individual responses associated with blinding in explaining experimental data. In the present study, we focus on the latter, while also providing an advancement on the former by analysing open-access data from the field of non-invasive brain stimulation (NIBS). . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 7, 2020. ; https://doi.org/10.1101/2020.12.06.411850 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 7, 2020. ; https://doi.org/10.1101/2020.12.06.411850 doi: bioRxiv preprint The effect of subjective intervention The effect of subjective intervention The effect of subjective intervention 5 The effect of subjective intervention 5 The effect of subjective intervention 4 4 In recent years, NIBS has been highlighted as a promising intervention to treat psychiatric, neurological, and neurodevelopmental disorders (Brunoni et al., 2019; Krause & Cohen Kadosh, 2013; Stefaniak et al., 2020; Vicario et al., 2019). Among the latest research developments, an increasing number of studies have also investigated the use of NIBS for cognitive enhancement (Cohen Kadosh, 2014; Santarnecchi et al., 2015). In this context, converging evidence shows that NIBS has the potential to improve various cognitive abilities - such as attention, memory, and intelligence - in typical and atypical populations from different age groups (Cohen Kadosh et al., 2012; Hamilton et al., 2011; Santarnecchi et al., 2015). One of the most popular NIBS techniques, transcranial direct current stimulation (tDCS), involves delivering either a positive (anodal) or negative (cathodal) electric constant current at a fixed amplitude over a selected cortical region (Filmer et al., 2014; Polania et al., 2018; Santarnecchi et al., 2015). Studies that employ this technique have shown promising evidence concerning the benefits brought by a single tDCS session to various cognitive domains, including notable findings on working memory, short-term and long-term memory, as well as speech, language, and mathematical cognition (André et al., 2016; Fregni et al., 2005; Javadi & Cheng, 2013; Monti et al., 2013; Sarkar et al., 2014). Despite the encouraging results on the cognitive effects of tDCS, scepticism has been raised concerning the effectiveness of this technique (Horvath et al., 2015; Medina & Cason, 2017; Wang et al., 2018; Westwood et al., 2017). Different factors have been hypothesised to underly the disparity in research findings that emerges across tDCS experiments. Here, we focus on two factors, namely: blinding and subjective intervention. . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 7, 2020. ; https://doi.org/10.1101/2020.12.06.411850 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 7, 2020. ; https://doi.org/10.1101/2020.12.06.411850 doi: bioRxiv preprint The effect of subjective intervention The effect of subjective intervention 6 6 (Fonteneau et al., 2019; O’connell et al., 2012; Turi et al., 2019; Wallace et al., 2016). Notably, ineffective blinding is likely to mask or inflate the observed findings. Therefore, it becomes of crucial importance to thoroughly account for its emergence. Thus far, literature has shown that the experimental manipulation of participants’ beliefs about a given intervention can moderate the intervention’s efficacy at both the physiological and behavioural level (Benedetti et al., 2003; Boot et al., 2013; Finniss et al., 2010). However, a gap in the current knowledge is not whether blinding was successful or not, but whether the participant’s subjective experience of receiving an intervention, whether it is true or not, can impact results over and beyond the actual intervention that participants receive. We term the former subjective intervention and the latter objective intervention. Blinding and Subjective Intervention Blinding refers to the practice of withholding information that may undesirably influence participants’ responses. Participants that take part in NIBS studies consistently report various perceptual sensations, such as audible clicks, visual disturbances, and cutaneous feelings (Davis et al., 2013; May et al., 2007). As a result of these sensations, participants can become aware of having received stimulation, and they are, therefore, unblinded to their experimental condition. This, in turn, makes it more likely that expectations about the effect of NIBS and demand characteristics about the aim of the experiment might influence participants’ performance (Polania et al., 2018). In order to tackle the issue of unblinding and the consequent emergence of any bias associated with it, studies have developed experimental designs that, at first sight, seem to control successfully for the sensations produced by active stimulation. In this regard, NIBS experiments most commonly employ sham (placebo) control stimulation (henceforth, sham stimulation). During sham stimulation, a minimal amount or no stimulation is administered to the subject while keeping the experiment otherwise identical. For example, in tDCS, the most prevalent way of administering sham is the fade in- short stimulation- fade out approach. Following this procedure, the stimulation intensity is slowly ramped up and, after a few seconds of actual stimulation (e.g., 30 seconds), ramped down promptly. Consequently, no stimulation is given for a total duration that is equal to the duration of the active stimulation (e.g., 20 min). Hence, sham stimulation aims to mimic the perceptual sensations associated with active stimulation without substantially affecting cortical excitability (Fritsch et al., 2010; Nitsche & Paulus, 2000). Although previous tDCS studies have suggested that sham stimulation delivers sensations that are comparable to the active stimulation, and therefore allows adequate blinding (Ambrus et al., 2010, Ambrus et al., 2012; Gandiga et al., 2006), unblinding has also been reported . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 7, 2020. ; https://doi.org/10.1101/2020.12.06.411850 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 7, 2020. Blinding and Subjective Intervention ; https://doi.org/10.1101/2020.12.06.411850 doi: bioRxiv preprint The effect of subjective intervention The effect of subjective intervention The effect of subjective intervention 7 towards the task at hand (Smallwood & Schooler, 2006, but see McVay & Kane, 2010). (). Noteworthy, mind wandering emerges as a critical cognitive process across the literature and is associated with multiple mental disorders, such as anxiety, depression and ADHD (Deng et al., 2014; Figueiredo et al., 2020). In Filmer et al.’s experiment (2019), the authors showed that active 2mA cathodal tDCS reliably led to an increase in mind wandering scores compared to sham tDCS. In contrast, 1mA anodal, 1mA cathodal tDCS, and 1.5mA cathodal tDCS did not show a robust effect on mind wandering. Filmer et al.’s (2019) results are important, as they provide a potential explanation for the discrepancy in the literature between studies that did and did not find an effect of cathodal tDCS on mind wandering using a lower stimulation threshold (Axelrod et al., 2015; Axelrod et al., 2018; Boayue et al., 2020). Here, we show that two aspects of the study merit further attention. Firstly, the procedure to assess participants’ blinding. Secondly, the contribution of adding subjective intervention as an explanatory variable to test whether the examined intervention, in this case, tDCS, has a genuine effect on mind wandering. Participants and design One hundred fifty healthy participants (age M=23, SD=5, 96 females) took part in this study. Participants were tested as part of a between-subject design. Each participant was randomly assigned to either one of the following five conditions: anodal 1 mA, cathodal 1 mA, 1.5 mA, 2 mA, or sham tDCS. The original study’s analysis plan, sample size and methodology were all pre-registered on the OSF by the authors (https://osf.io/j6mqa/). The present study Based on the above literature, we aimed to answer two research questions: (1) Does subjective intervention or subjective dosage (participants’ subjective beliefs about the dose of the intervention, in this case, the strength of NIBS) affect performance scores over and beyond what is explained by objective (active/sham) intervention?; (2) Is there an interaction between subjective intervention/dosage and objective intervention in predicting performance scores?. To answer these questions, we conducted secondary data analysis on an open-access dataset of 150 individuals retrieved from a published paper by Filmer et al. (2019). In the study that we examined, Filmer et al. (2019) tested the effect of tDCS over the prefrontal cortex on mind wandering. Mind wandering has been conceptualised as an executive function that occurs when attention shifts away from the primary task, and it can, therefore, lead to failures in performance and superficial representations of the external environment. Differently from most controlled processes, mind wandering occurs outside of awareness. Therefore, this mental capacity can be defined as a goal-driven process, albeit one that might not be directed . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 7, 2020. ; https://doi.org/10.1101/2020.12.06.411850 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 7, 2020. ; https://doi.org/10.1101/2020.12.06.411850 doi: bioRxiv preprint The effect of subjective intervention The effect of subjective intervention 7 Experimental task Participants completed a sustained attention task (SART) in which they were asked to respond via a keypress (space bar) to non-target stimuli (single digits excluding the number 3) and withholding responses to target stimuli (the number 3), see Figure 1a. Half of the trials ended in a target stimulus; the other half ended in a task unrelated thought (TUT) probe. The TUT probe asked: “To what extent have you experienced task unrelated thoughts prior to the thought probe? 1 (minimal) – 4 (maximal)”. Participants average response to the probe across trails was taken as a measure of mind wandering performance, with higher scores indicating higher mind wandering. The effect of subjective intervention 8 8 open and stimulation was applied offline to the left prefrontal cortex for 20 min. Lastly, participants performed a sustained attention task for 40 minutes, during which mind wandering, which was the main outcome of this research, was measured. Overall, each participant completed a single session, lasting approximately 1.5 hours. open and stimulation was applied offline to the left prefrontal cortex for 20 min. Lastly, participants performed a sustained attention task for 40 minutes, during which mind wandering, which was the main outcome of this research, was measured. Overall, each participant completed a single session, lasting approximately 1.5 hours. Procedure The experiment consisted of three main parts. Firstly, participants were familiarised with the experimental paradigm. Secondly, participants were instructed to sit quietly with their eyes . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 7, 2020. ; https://doi.org/10.1101/2020.12.06.411850 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 7, 2020. ; https://doi.org/10.1101/2020.12.06.411850 doi: bioRxiv preprint The effect of subjective intervention The effect of subjective intervention Objective intervention Stimulation was delivered with a NeuroConn stimulator (neuroConn GmbH, Ilmenau, Germany). The target was placed over F3 (EEG 10–20 system), and the reference over the right orbitofrontal region (e.g., Figure 2b). For the four groups who received active stimulation (e.g., Figure 2c), tDCS lasted 20 minutes (including 30 s ramping up and down). During stimulation, participants were asked to sit quietly and keep their eyes open. The group who received sham stimulation had the same instructions but only received 15 s of constant current. The current was ramped up for 30 s up to 1.5 mA, and then ramped down for 30 s. Stimulation was single- blinded, meaning that while the participants were blind to the stimulation they received, the experimenters were aware of the participant’s stimulation group. . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 7, 2020. ; https://doi.org/10.1101/2020.12.06.411850 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 7, 2020. ; https://doi.org/10.1101/2020.12.06.411850 doi: bioRxiv preprint 9 The effect of subjective intervention 9 Figure 1. Experimental design, montage, and modelling of current distribution. (a) Graphical representation of the sustained attention to response task (SART) for the target and thought probe trials; (b) electrode montage showed here for anodal stimulation targeting the left prefrontal cortex; (c) imaging of the amount of current induced using a standard head model, in the image, modelled current distribution for the 2 mA cathodal stimulation condition. Figure adapted from Filmer et al. (2019) with permission. g p g , g , g ( ) Graphical representation of the sustained attention to response task (SART) for the target and thought probe trials; (b) electrode montage showed here for anodal stimulation targeting the left prefrontal cortex; (c) imaging of the amount of current induced using a standard head model, in the image, modelled current distribution for the 2 mA cathodal stimulation condition. Figure adapted from Filmer et al. (2019) with permission. Objective intervention It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 7, 2020. ; https://doi.org/10.1101/2020.12.06.411850 doi: bioRxiv preprint The effect of subjective intervention 10 Objective intervention Subjective intervention At the end of the experiment, participants were asked whether they thought they received active or sham stimulation (presented as a binary choice) via a short questionnaire. We here refer to the subject’s judgment of whether they received active or sham stimulation as subjective intervention, in opposition to objective intervention, which indicates the actual type of stimulation that each subject received during the experiment. Moreover, at the end of the study, participants were also asked to guess which stimulation dosage they received, having to choose between the following options: none, weak, moderate, or strong. We here refer to the participants’ judgment of stimulation dosage as subjective dosage. Subjective intervention Subjective intervention Subjective intervention At the end of the experiment, participants were asked whether they thought they received active or sham stimulation (presented as a binary choice) via a short questionnaire. We here refer to the subject’s judgment of whether they received active or sham stimulation as subjective intervention, in opposition to objective intervention, which indicates the actual type of stimulation that each subject received during the experiment. Moreover, at the end of the study, participants were also asked to guess which stimulation dosage they received, having to choose between the following options: none, weak, moderate, or strong. We here refer to the participants’ judgment of stimulation dosage as subjective dosage. At the end of the experiment, participants were asked whether they thought they received active or sham stimulation (presented as a binary choice) via a short questionnaire. We here refer to the subject’s judgment of whether they received active or sham stimulation as subjective intervention, in opposition to objective intervention, which indicates the actual type of stimulation that each subject received during the experiment. Moreover, at the end of the study, participants were also asked to guess which stimulation dosage they received, having to choose between the following options: none, weak, moderate, or strong. We here refer to the participants’ judgment of stimulation dosage as subjective dosage. . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 7, 2020. ; https://doi.org/10.1101/2020.12.06.411850 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. The effect of subjective intervention Following the original study by Filmer et al. (2019), we interpreted BF10 of 1–3 as anecdotal, BF10 of 3–10 as moderate, and BF10>10 as strong evidence in favour of the alternate hypothesis (namely, that the examined model provided better fit than a null model). BF10 values ∼1 were interpreted as providing no evidential value. Similarly, for support in favour of the null hypothesis, BF01 1–3 was interpreted as anecdotal, BF01 of 3–10 as moderate, and BF01>10 as strong evidence. Besides Bayesian statistics, we also reported results based on frequentist statistics for our primary analyses. In such cases, we accepted statistical significance for all tests at p<.05. Our choice to report both orthodox statistics, such as F and P values, alongside Bayes factors is based on literature that has highlighted the importance of addressing the relationship between significance testing and Bayesian analysis (Dienes & Mclatchie, 2018). We believe that reporting both statistical approaches will allow wider access to results’ interpretability for researchers that may not be familiar with Bayesian methods. Statistical Analysis Statistical analysis was run using both R (version 4.0.1. for Windows) and JASP (version 0.13.1.0 for Windows) on the open-access dataset made available by Filmer et al. (2019). All the analyses were run on average mind wandering scores calculated over the whole experimental session. While Filmer et al. (2019) also examined the effect of tDCS on the first and second half of their experiment, for brevity we do not report such analyses as they are out of the scope of our research. However, when we ran such analyses, the results yielded a similar conclusion to our primary reported findings. In R, we performed a chi-square test to assess blinding effectiveness by examining the relationship between stimulation guess (subjective intervention) and stimulation group (objective intervention). Given that Filmer et al. (2019) used Bayesian ANOVA in their study, for consistency, we decided to employ the same statistical approach chosen by the authors. Therefore, in JASP with default priors, we performed two tests. Firstly, we reproduced Filmer et al.’s (2019) findings by running Bayesian ANOVAs with objective intervention as a between-subject factor, followed by the relevant post-hoc pairwise comparisons. Consequently, our primary analysis consisted of two Bayesian ANOVAs that were run to examine model fit. In the first analysis, objective intervention and subjective intervention were included as between-subject factors; while, in the second one, we examined model fit for objective intervention and subjective dosage. In both cases, average TUT ratings were taken as the outcome measure. If subjective dosage or subjective intervention led to better model fit, we separately analysed the effect of each of these factors on mind wandering. Moreover, for the subjective dosage factor, we conducted a trend analysis to examine whether the association between an increase in perceived stimulation dosage (from none to strong) and mind wandering scores is linear. . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 7, 2020. ; https://doi.org/10.1101/2020.12.06.411850 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 7, 2020. Statistical Analysis ; https://doi.org/10.1101/2020.12.06.411850 doi: bioRxiv preprint The effect of subjective intervention The effect of subjective intervention 11 Blinding We would like the reader first to judge the following two scenarios. In the first scenario, the correct guess rate (referring to the percentage of participants that successfully guessed their experimental condition) in the active group is 70%, while it is 40% in the sham group. In the second scenario, the correct guess rate is undisguisable between the active and sham group and is 70%. In which scenario was blinding more effective? The second scenario is similar to what occurred in Filmer et al.’s experiment (2019). At the end of the experiment, participants were asked whether they thought they received active or sham stimulation. The reported correct guess rate was similar across all stimulation groups (ranging from 53% to 77%, Table 1). Given that the correct success rates did not appear to increase with higher stimulation intensity and based on their similarity between the active . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 7, 2020. ; https://doi.org/10.1101/2020.12.06.411850 doi: bioRxiv preprint The effect of subjective intervention 12 stimulation conditions and the sham condition (70%), the authors concluded that blinding was successful. stimulation conditions and the sham condition (70%), the authors concluded that blinding was successful. Table 1. Correct guess rate for the judgment of active and sham stimulation as in Filmer et al. (2019) versus active stimulation guess rate per stimulation type. Stimulation type n Correct guess rate Active stimulation guess rate Anodal 1 mA 30 73% 73% Cathodal 1 mA 30 53% 53% Cathodal 1.5 mA 30 73% 73% Cathodal 2 mA 30 57% 57% Sham 30 70% 30% Table 1. Correct guess rate for the judgment of active and sham stimulation as in Filmer et al. (2019) versus active stimulation guess rate per stimulation type. Table 1. Correct guess rate for the judgment of active and sham stimulation as in Filmer et al. (2019) versus active stimulation guess rate per stimulation type. Fallacious reasoning may lead to answering that blinding was effective in this study and in the second scenario we presented. However, we here show that Filmer et al.’s approach (2019) allows a justified conclusion regarding blinding effectiveness only when the guess rate is 50%. The effect of subjective intervention 13 receiving active stimulation was dependent upon the stimulation group to which participants were assigned. To decompose what contributed to the overall association between the investigated chi-square measures, we further examined the standardised residuals. For people in the sham group, significantly more participants than expected by chance guessed to be receiving sham stimulation (z=2.29, p=0.022) and significantly fewer participants than expected guessed to be receiving active stimulation (z=-1.98, p= 0.048). From these findings, we concluded that, in contrast to the claim by Filmer et al. (2019), blinding was ineffective. Blinding When the guess rate deviates from 50%, as in the current study, the fact that the correct guess rates are similar across conditions does not necessarily indicate that all groups are successfully blinded. On the contrary, in this case, the high correct guess rate indicates that the number of people in the sham condition who made an incorrect guess and thought they had received active stimulation was substantially lower (30%) than the number of people in the stimulation conditions that thought they had received active stimulation (53%-73%). We analysed the potential differences in blinding success across all experimental groups using a chi-square test of independence. This test examined the association between the active stimulation guess rate and participants’ experimental group (Table 1). Results showed that the association was significant, χ2(4, N=150)=15.64, p=.004, indicating that the feeling of . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 7, 2020. ; https://doi.org/10.1101/2020.12.06.411850 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 7, 2020. ; https://doi.org/10.1101/2020.12.06.411850 doi: bioRxiv preprint The effect of objective and subjective intervention on mind wandering After examining blinding effectiveness, we tackled the question of whether subjective intervention or subjective dosage explained participants mind wandering scores over and beyond objective intervention. To address such a question, we employed Bayesian ANOVA as in Filmer et al. (2019), and we also report the results based on frequentist statistics. Firstly, we reproduced the results from the original study by performing Bayesian ANOVA with objective intervention only as a between-subject factor. While the frequentist statistics resulted in a positive finding, the Bayesian ANOVA showed no evidential value for overall differences between all tDCS conditions in average TUT ratings over the whole experimental session (BF10=0.984; F(4,145)=2.607, p=0.038). Comparing pairs of groups on average TUT ratings, we found anecdotal evidence for higher mind wandering in the 1.5 mA cathodal tDCS group compared to the sham group (BF10=2.189, t(58)=2.272, p=.027), and moderate evidence for higher mind wandering in the 2 mA cathodal group (BF10=7.436, t(58)=2.875, p=.006) compared to sham. No evidence emerged in support of 1mA anodal or cathodal stimulation, relative to sham, modulating TUTs (anodal stimulation: BF10 < 1, t(58)=1.619, p=.11; cathodal stimulation: BF10 < 1, t(58)=1.313, p=.19). These results successfully reproduced Filmer et al.’s (2019) findings. . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 7, 2020. ; https://doi.org/10.1101/2020.12.06.411850 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 7, 2020. ; https://doi.org/10.1101/2020.12.06.411850 doi: bioRxiv preprint The effect of subjective intervention The effect of subjective intervention 14 Next, we run Bayesian ANOVA to examine the effect of subjective intervention and subjective dosage on mind wandering. Subjective intervention, alone, led to the best model fit in explaining mind wandering (Table 2). Hence, participants’ beliefs about having received active or sham stimulation during the experiment were better at explaining mind wandering performance than objective intervention only, objective and subjective intervention together, or the interaction between objective and subjective intervention. Table 2. tion (BF10=3.374, t(148)=2.55, p=0.012). . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 7, 2020. ; https://doi.org/10.1101/2020.12.06.411850 doi: bioRxiv preprint The effect of objective and subjective intervention on mind wandering Model comparison run using Bayesian ANOVA to predict average mind wandering scores Models P(M) P (M\| data) BF10 Error % Subjective intervention 0.200 0.464 3.374 8.94 e-8 Objective+Subjective intervention 0.200 0.205 1.492 2.201 Null model 0.200 0.137 1.000 Objective 0.200 0.135 0.984 1.40 e-4 ObjectiveSubjective intervention 0.200 0.059 0.429 1.000 As shown in Figure 2, people that thought they had received active stimulation during the experiment had higher mind wandering scores than people that thought they had received sham stimulation (BF10=3.374, t(148)=2.55, p=0.012). Table 2. Model comparison run using Bayesian ANOVA to predict average wandering scores The effect of subjective intervention 15 . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 7, 2020. ; https://doi.org/10.1101/2020.12.06.411850 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 7, 2020. ; https://doi.org/10.1101/2020.12.06.411850 doi: bioRxiv preprint The effect of subjective intervention The effect of subjective intervention 15 Figure 2. A violin plot showing the full distribution of the mind wandering score highlighting the differences as a function of subjective intervention. Solid and dotted black lines indicate the medians and quartiles, respectively. Figure 2. A violin plot showing the full distribution of the mind wandering score highlighting the differences as a function of subjective intervention. Solid and dotted black lines indicate the medians and quartiles, respectively. Given that, at the end of Filmer et al.’s experiment (2019), participants were also asked to guess stimulation dosage (none, weak, moderate or strong), we re-run Bayesian ANOVA replacing subjective intervention with stimulation dosage. Our results showed that subjective dosage, alone, also led to the best model fit in explaining mind wandering (Table 3, BF10=3.708). Therefore, we concluded that participants’ beliefs about stimulation dosage during the experiment were better at explaining mind wandering performance than objective intervention only, objective and subjective intervention together, or the interaction between objective and subjective intervention. . CC-BY 4.0 International license perpetuity. The effect of objective and subjective intervention on mind wandering ; https://doi.org/10.1101/2020.12.06.411850 doi: bioRxiv preprint The effect of subjective intervention The effect of subjective intervention 17 The effect of subjective intervention 17 Figure 3. A violin plot showing the full distribution of the mind wandering score as a function of subjective dosage, showing an increase in mind wandering score as a function of subjective dosage. Solid and dotted black lines indicate the medians and quartiles, respectively. Figure 3. A violin plot showing the full distribution of the mind wandering score as a function of subjective dosage, showing an increase in mind wandering score as a function of subjective dosage. Solid and dotted black lines indicate the medians and quartiles, respectively. The effect of objective and subjective intervention on mind wandering It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 7, 2020. ; https://doi.org/10.1101/2020.12.06.411850 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 7, 2020. ; https://doi.org/10.1101/2020.12.06.411850 doi: bioRxiv preprint The effect of subjective intervention 16 Table 3. Model comparison run using Bayesian ANOVA to predict average mind wandering scores Models P(M) P (M\| data) BF10 Error % Subjective dosage 0.200 0.489 3.708 1.715 e-4 Objective + Subjective dosage 0.200 0.219 1.658 0.587 Null model 0.200 0.132 1.000 Objective 0.200 0.130 0.984 1.402 e-4 Objective *Subjective dosage 0.200 0.031 0.232 0.582 Table 3. Model comparison run using Bayesian ANOVA to predict average mind wandering scores M d l P(M) P (M\| d ) BF E % 3. Model comparison run using Bayesian ANOVA to predict average mind ring scores When running Bayesian ANOVA on the subjective dosage factor only, results showed that people that thought they had received no stimulation, weak, moderate or strong stimulation during the experiment presented significant differences in average mind wandering (BF10=5.911, F(3,146)=4.198, p=0.007). This association between subjective dosage and mind wandering was best explained by a significant linear trend (SE=0.13, t(146)=2.63, p=0.009, Figure 3). Further analysis using a linear regression analysis showed that subjective dosage was a significant predictor of mind wandering (BF10=24.26, β=.263, t(148)=3.13, p=.001). These results indicate that as the subjective dosage increased (from none to strong), mind wandering increased proportionally. The effect of subjective intervention 17 . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 7, 2020. ; https://doi.org/10.1101/2020.12.06.411850 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 7, 2020. . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 7, 2020. ; https://doi.org/10.1101/2020.12.06.411850 doi: bioRxiv preprint Discussion In the present study, we highlighted the importance of an effective procedure to assess blinding, as well as accounting for the individual subjective belief about receiving an intervention, in this case, NIBS. Our results showed that in the experiment by Filmer et al. (2019), blinding of the sham condition was compromised. Notably, in that study participants received NIBS offline, before the task was delivered. The fact that participants could entirely focus on the sensations associated with the delivery of stimulation might have contributed to unblinding. Importantly, ineffective blinding can affect experimental outcomes in two ways. Firstly, it could mask positive or negative effects that the intervention may have on performance. Alternatively, it . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 7, 2020. ; https://doi.org/10.1101/2020.12.06.411850 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 7, 2020. ; https://doi.org/10.1101/2020.12.06.411850 doi: bioRxiv preprint The effect of subjective intervention 18 could inflate such effects. Therefore, it is of crucial importance to thoroughly account for its emergence. could inflate such effects. Therefore, it is of crucial importance to thoroughly account for its emergence. We would like to highlight that the way in which Filmer et al. (2019) assessed the efficacy of their blinding is not uncommon in the literature (for recent clinical trials, e.g., Schecklmann et al., 2020; Sebastian et al., 2020). For instance, in a recent clinical trial that assessed the effect of NIBS on depression, and concluded its inefficacy (Schecklmann et al., 2020), the authors used the same procedure to assess blinding efficacy and wrote: “In the group of patients, 39% (2 sham, 8 real treatment) subjects rated the treatment correctly” (p. 119). Again, the correct analysis of this blinding shows that there is a significant difference between the groups, and that blinding was ineffective (χ2(1, N=36)=4.12, p=.042). While highlighting the fallacy of using such procedure for assessing the success of blinding in interventions, the more notable contribution of our study is demonstrating how subjective beliefs about the type of intervention received explained the participants’ performance better than objective intervention, subjective and objective intervention together, or the interaction between subjective and objective intervention. Hence, in Filmer et al.’s study (2019), the fact that participants’ believed to have or have not received the real intervention affected their performance to a more considerable extent than the actual intervention to which they were assigned. The same pattern of results emerged when we replaced subjective intervention with subjective dosage. In this case, a higher subjective dosage was linearly associated with higher mind wandering. One question that emerges from this study is whether our results that were observed with self- report measures would apply to more objective behavioural outcomes and neural functions. In our view, the answer for this is likely to be positive, given that placebo effects have been shown . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 7, 2020. ; https://doi.org/10.1101/2020.12.06.411850 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. The effect of subjective intervention It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 7, 2020. ; https://doi.org/10.1101/2020.12.06.411850 doi: bioRxiv preprint The effect of subjective intervention 19 The effect of subjective intervention The effect of subjective intervention 19 to impact objective behavioural outcomes and neural activity (Hashmi, 2018; Schmidt et al., 2014; Oken et al., 2008). Independently of this possibility, we argue that the effect of subjective intervention on self-reported outcomes shall not be underestimated. Noteworthy, in randomised controlled trials that investigate the effect of NIBS on clinical and subclinical populations (e.g., depression, chronic pain, ADHD), some of which have also been approved by US Food and Drug Administration (FDA), the evidence is based on self-reported questionnaires. This consideration makes the case of subjective intervention even stronger, hinting to the potential role played by this factor in explaining experimental results across a variety of experiments. In our view, it is a crucial responsibility of the relevant researchers to ensure that results from previous studies, especially those approved by the FDA, are not solely due to subjective intervention. Therefore, we strongly encourage the scientists in these studies to re-examine their data or make it publicly available in a format that will allow testing the effect of subjective intervention as in the present study. Of note, when we approached researchers in the field with a request to access their data, some of them could not share it due to restrictions by their ethical committee, while others reported that the assessment of blinding, aside from side effects, was not made. As a point of improvement for the quality of research in the field of interventions, future experiments that investigate the effect of subjective intervention on performance should strive to record participants’ expectations about the effect of stimulation thoroughly (Boot et al., 2013). Some participants may expect an intervention to improve their capabilities, while others could expect even the opposite, and the level of these expectations may vary as well. Accounting for participant’s expectations would allow to more accurately model the directionality and strength of the effect of subjective intervention on the outcome of interest. . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 7, 2020. ; https://doi.org/10.1101/2020.12.06.411850 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. The effect of subjective intervention It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 7, 2020. ; https://doi.org/10.1101/2020.12.06.411850 doi: bioRxiv preprint The effect of subjective intervention The effect of subjective intervention The effect of subjective intervention 20 In a nutshell, we showed that participants’ subjective beliefs about receiving an intervention affect the primary outcome independently of the actual intervention condition to which participants are assigned. While we focused in this study on NIBS, our approach applies to other types of interventions (e.g., neurofeedback, pharmacological studies) that collect data on participant’s subjective experience concerning whether they received a placebo or active intervention to assess blinding effectiveness. Our finding holds twofold importance. Firstly, it introduces two concepts in the intervention literature, namely: subjective intervention and subjective dosage. Secondly, it highlights the importance of assessing participants’ blinding, independent of side effects. Unfortunately, different studies, including pre-registered reports, that claim for the efficacy or inefficacy of a given intervention do not measure or report data on the success of blinding (for examples in the field of NIBS see: Axelrod et al., 2015; Axelrod et al., 2018; Boayue et al., 2020; Horne et al., 2020). It is therefore impossible to draw strong conclusions from those studies, without knowing whether blinding was effective, and how beliefs and/or expectations played a role in inflating or masking the observed results. In this respect, it should be highlighted that reports on side effects are an unsuitable measure to assess blinding efficacy, as participants might subjectively feel being in the active intervention group, while not reporting side effects. We call for future studies to systematically collect data on participants’ subjective beliefs, as well as previous studies, to examine the potential effect of such beliefs on their results. Aside from estimating the contribution of subjective beliefs about belonging to the active or control condition, future research shall also strive to collect and analyse data on participants’ expectations about the directionality and strength of the effect of subjective intervention on expected outcomes. Such data will allow to thoroughly examine the effect of subjective beliefs, yielding more valid and replicable results in order to progress scientific and clinical studies for Th ff t f bj ti i t ti 21 . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 7, 2020. ; https://doi.org/10.1101/2020.12.06.411850 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 7, 2020. ; https://doi.org/10.1101/2020.12.06.411850 doi: bioRxiv preprint The effect of subjective intervention It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 7, 2020. ; https://doi.org/10.1101/2020.12.06.411850 doi: bioRxiv preprint The effect of subjective intervention The effect of subjective intervention 21 the benefit of human wellbeing and cognition. Otherwise, the effect of those interventions, or the lack of it, might all only be in our head. the benefit of human wellbeing and cognition. Otherwise, the effect of those interventions, or the lack of it, might all only be in our head. the lack of it, might all only be in our head. . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 7, 2020. ; https://doi.org/10.1101/2020.12.06.411850 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 7, 2020. ; https://doi.org/10.1101/2020.12.06.411850 doi: bioRxiv preprint 22 The effect of subjective intervention Acknowledgements We appreciate the transparency that Hannah L. Filmer, Ashleigh Griﬃn, and Paul E. Dux provided by making their data accessible, without that the production of this work and progress of knowledge would not be possible. . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 7, 2020. ; https://doi.org/10.1101/2020.12.06.411850 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 7, 2020. ; https://doi.org/10.1101/2020.12.06.411850 doi: bioRxiv preprint 23 The effect of subjective intervention References Ambrus, G. G., Al-Moyed, H., Chaieb, L., Sarp, L., Antal, A., & Paulus, W. (2012). The fade- in–short stimulation–fade out approach to sham tDCS–reliable at 1 mA for naive and experienced subjects, but not investigators. Brain stimulation, 5(4), 499-504. Ambrus, G. G., Al-Moyed, H., Chaieb, L., Sarp, L., Antal, A., & Paulus, W. (2012). The fade- in–short stimulation–fade out approach to sham tDCS–reliable at 1 mA for naive and experienced subjects, but not investigators. Brain stimulation, 5(4), 499-504. Ambrus, G. G., Paulus, W., & Antal, A. (2010). Cutaneous perception thresholds of electrical stimulation methods: comparison of tDCS and tRNS. Clinical Neurophysiology, 121(11), 1908-1914. André, S., Heinrich, S., Kayser, F., Menzler, K., Kesselring, J., Khader, P. H., ... & Mylius, V. (2016). At-home tDCS of the left dorsolateral prefrontal cortex improves visual short-term memory in mild vascular dementia. Journal of the Neurological Sciences, 369, 185-190. Axelrod, V., Rees, G., Lavidor, M., & Bar, M. (2015). Increasing propensity to mind-wander with transcranial direct current stimulation. Proceedings of the National Academy of Sciences, 112(11), 3314-3319. Axelrod, V., Zhu, X., & Qiu, J. (2018). Transcranial stimulation of the frontal lobes increases propensity of mind-wandering without changing meta-awareness. Scientific reports, 8(1), 1- 14. Benedetti, F., Pollo, A., Lopiano, L., Lanotte, M., Vighetti, S., & Rainero, I. (2003). Conscious expectation and unconscious conditioning in analgesic, motor, and hormonal placebo/nocebo responses. The Journal of Neuroscience: The Official Journal of the Society for Neuroscience, 23(10), 4315–4323. Boayue, N. M., Csifcsák, G., Aslaksen, P., Turi, Z., Antal, A., Groot, J., ... & Mittner, M. (2020). Increasing propensity to mind‐wander by transcranial direct current stimulation? A registered report. European Journal of Neuroscience, 51(3), 755-780. . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 7, 2020. ; https://doi.org/10.1101/2020.12.06.411850 doi: bioRxiv preprint 24 Boot, W. R., Simons, D. J., Stothart, C., & Stutts, C. (2013). The pervasive problem with placebos in psychology: Why active control groups are not sufficient to rule out placebo effects. Perspectives on Psychological Science, 8(4), 445-454. Brunoni, A. R., Sampaio-Junior, B., Moffa, A. H., Aparício, L. V., Gordon, P., Klein, I., ... & Valiengo, L. (2019). Non-invasive brain stimulation in psychiatric disorders: a primer. Brazilian Journal of Psychiatry, 41(1), 70-81. Davis, N. References J., Gold, E., Pascual‐Leone, A., & Bracewell, R. M. (2013). Challenges of proper placebo control for non‐invasive brain stimulation in clinical and experimental applications. European Journal of Neuroscience, 38(7), 2973-2977. Deng, Y. Q., Li, S., & Tang, Y. Y. (2014). The relationship between wandering mind, depression and mindfulness. Mindfulness, 5(2), 124-128. Dienes, Z., & Mclatchie, N. (2018). Four reasons to prefer Bayesian analyses over significance testing. Psychonomic Bulletin & Review, 25(1), 207-218. g , , , , , , , , ç , , , , , ( ) Mind-wandering, depression, anxiety and ADHD: Disentangling the relationship. Psychiatry Research, 285, 112798. Filmer, H. L., Dux, P. E., & Mattingley, J. B. (2014). Applications of transcranial direct current stimulation for understanding brain function. Trends in neurosciences, 37(12), 742-753. Filmer, H. L., Griffin, A., & Dux, P. E. (2019). For a minute there, I lost myself… dosage dependent increases in mind wandering via prefrontal tDCS. Neuropsychologia, 129, 379-384. Filmer, H. L., Mattingley, J. B., & Dux, P. E. (2020). Modulating brain activity and behaviour with tDCS: Rumours of its death have been greatly exaggerated. Cortex, 123, 141- 151. . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 7, 2020. ; https://doi.org/10.1101/2020.12.06.411850 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 7, 2020. ; https://doi.org/10.1101/2020.12.06.411850 doi: bioRxiv preprint The effect of subjective intervention The effect of subjective intervention 25 Filmer, H.L., Griffin, A., Dux, P.E., (2019). Dosage Dependent Increases in Mind Wandering via Prefrontal tDCS. UQ eSpace. https://doi.org/10.14264/uql.2019.295. Finniss, D. G., Kaptchuk, T. J., Miller, F., & Benedetti, F. (2010). Biological, clinical, and ethical advances of placebo effects. The Lancet, 375(9715), 686-695. Fonteneau, C., Mondino, M., Arns, M., Baeken, C., Bikson, M., Brunoni, A. R., ... & Poulet, E. (2019). Sham tDCS: A hidden source of variability? Reflections for further blinded, controlled trials. Brain Stimulation, 12(3), 668-673. Fregni, F., Boggio, P. S., Nitsche, M., Bermpohl, F., Antal, A., Feredoes, E., ... & Pascual- Leone, A. (2005). Anodal transcranial direct current stimulation of prefrontal cortex enhances working memory. Experimental Brain Research, 166(1), 23-30. Fritsch, B., Reis, J., Martinowich, K., Schambra, H. M., Ji, Y., Cohen, L. G., & Lu, B. (2010). Direct current stimulation promotes BDNF-dependent synaptic plasticity: potential implications for motor learning. Neuron, 66(2), 198-204. Gandiga, P. C., Hummel, F. C., & Cohen, L. G. (2006). Transcranial DC stimulation (tDCS): a tool for double-blind sham-controlled clinical studies in brain stimulation. Clinical Neurophysiology, 117(4), 845-850. Gruzelier, J. H. (2014). EEG-neurofeedback for optimising performance. III: a review of methodological and theoretical considerations. Neuroscience & Biobehavioral Reviews, 44, 159-182. Guerra, A., López-Alonso, V., Cheeran, B., & Suppa, A. (2020). Variability in non-invasive brain stimulation studies: reasons and results. Neuroscience Letters, 719, 133330. Hamilton, R., Messing, S., & Chatterjee, A. (2011). Rethinking the thinking cap: ethics of neural enhancement using non-invasive brain stimulation. Neurology, 76(2), 187-193. . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 7, 2020. ; https://doi.org/10.1101/2020.12.06.411850 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 7, 2020. ; https://doi.org/10.1101/2020.12.06.411850 doi: bioRxiv preprint The effect of subjective intervention . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 7, 2020. ; https://doi.org/10.1101/2020.12.06.411850 doi: bioRxiv preprint The effect of subjective intervention 26 Hashmi, J. A. (2018). Placebo effect: theory, mechanisms and teleological roots. International Review of Neurobiology, 139,233-253. Horne, K. S., Filmer, H. L., Nott, Z. E., Hawi, Z., Pugsley, K., Mattingley, J. B., & Dux, P. E. (2020). Evidence against benefits from cognitive training and transcranial direct current stimulation in healthy older adults. Nature Human Behaviour. Horne, K. S., Filmer, H. L., Nott, Z. E., Hawi, Z., Pugsley, K., Mattingley, J. B., & Dux, P. E. (2020). Evidence against benefits from cognitive training and transcranial direct current stimulation in healthy older adults. Nature Human Behaviour. Horvath, J. C., Forte, J. D., & Carter, O. (2015). Quantitative review finds no evidence of cognitive effects in healthy populations from single-session transcranial direct current stimulation (tDCS). Brain Stimulation, 8(3), 535-550. Javadi, A. H., & Cheng, P. (2013). Transcranial direct current stimulation (tDCS) enhances reconsolidation of long-term memory. Brain Stimulation, 6(4), 668-674. Kadosh, R. C. (Ed.). (2014). The stimulated brain: cognitive enhancement using non-invasive brain stimulation. Elsevier. Kadosh, R. C., Levy, N., O’Shea, J., Shea, N., & Savulescu, J. (2012). The neuroethics of non- invasive brain stimulation. Current Biology, 22(4), R108-R111. Kadosh, R. C., Levy, N., O’Shea, J., Shea, N., & Savulescu, J. (2012). The neuroethics of non- invasive brain stimulation. Current Biology, 22(4), R108-R111. Krause, B., & Kadosh, R. C. (2013). Can transcranial electrical stimulation improve learning difficulties in atypical brain development? A future possibility for cognitive training. Developmental Cognitive Neuroscience, 6, 176-194. Krause, B., & Kadosh, R. C. (2013). Can transcranial electrical stimulation improve learning difficulties in atypical brain development? A future possibility for cognitive training. Developmental Cognitive Neuroscience, 6, 176-194. Lampit, A., Hallock, H., & Valenzuela, M. (2014). Computerised cognitive training in cognitively healthy older adults: a systematic review and meta-analysis of effect modifiers. PLoS medicine, 11(11), e1001756. López-Alonso, V., Cheeran, B., Río-Rodríguez, D., & Fernández-del-Olmo, M. (2014). Inter- individual variability in response to non-invasive brain stimulation paradigms. Brain Stimulation, 7(3), 372-380. . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 7, 2020. ; https://doi.org/10.1101/2020.12.06.411850 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. The effect of subjective intervention It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 7, 2020. ; https://doi.org/10.1101/2020.12.06.411850 doi: bioRxiv preprint The effect of subjective intervention 27 May, A., Hajak, G., G€anßbauer, S., Steffens, T., Langguth, B., Kleinjung, T., & Eichhammer, P. (2007). Structural brain alterations following 5 days of intervention: dynamic aspects of neuroplasticity. Cerebral Cortex, 17, 205–210. McVay, J. C., & Kane, M. J. (2010). Does mind wandering reflect executive function or executive failure? Comment on Smallwood and Schooler (2006) and Watkins (2008). Psychological Bulletin, 136(2), 188–197. Medina, J., & Cason, S. (2017). No evidential value in samples of transcranial direct current stimulation (tDCS) studies of cognition and working memory in healthy populations. Cortex, 94, 131-141. Monti, A., Ferrucci, R., Fumagalli, M., Mameli, F., Cogiamanian, F., Ardolino, G., & Priori, A. (2013). Transcranial direct current stimulation (tDCS) and language. Journal of Neurology, Neurosurgery & Psychiatry, 84(8), 832-842. Nitsche, M. A., & Paulus, W. (2000). Excitability changes induced in the human motor cortex by weak transcranial direct current stimulation. The Journal of physiology, 527(Pt 3), 633. O’connell, N. E., Cossar, J., Marston, L., Wand, B. M., Bunce, D., Moseley, G. L., & De Souza, L. H. (2012). Rethinking clinical trials of transcranial direct current stimulation: participant and assessor blinding is inadequate at intensities of 2mA. PloS One, 7(10), e47514. Oken, B. S., Flegal, K., Zajdel, D., Kishiyama, S., Haas, M., & Peters, D. (2008). Expectancy effect: impact of pill administration on cognitive performance in healthy seniors. Journal of Clinical and Experimental Neuropsychology, 30(1), 7-17. Polania, R., Nitsche, M. A., & Ruff, C. C. (2018). Studying and modifying brain function with non-invasive brain stimulation. Nature Neuroscience, 21(2), 174-187. . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 7, 2020. ; https://doi.org/10.1101/2020.12.06.411850 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 7, 2020. ; https://doi.org/10.1101/2020.12.06.411850 doi: bioRxiv preprint The effect of subjective intervention The effect of subjective intervention 28 Santarnecchi, E., Brem, A.-K., Levenbaum, E., Thompson, T., Kadosh, R. C., & Pascual- Leone, A. (2015). Enhancing cognition using transcranial electrical stimulation. Current Opinion in Behavioral Sciences, 4, 171–178. Sarkar, A., Dowker, A., & Kadosh, R. C. (2014). Cognitive enhancement or cognitive cost: trait-specific outcomes of brain stimulation in the case of mathematics anxiety. Journal of Neuroscience, 34(50), 16605-16610. Schecklmann, M., Nejati, V., Poeppl, T. B., Peytard, J., Rupprecht, R., Wetter, T. C., ... & Kreuzer, P. M. (2020). Bifrontal high-frequency transcranial random noise stimulation is not effective as an add-on treatment in depression. Journal of Psychiatric Research, 132, 116-122. Schmidt, L., Braun, E. K., Wager, T. D. & Shohamy, D. (2014). Mind matters: placebo enhances reward learning in parkinson’s disease. Nature Neuroscience 17, 1793. Sitaram, R., Ros, T., Stoeckel, L., Haller, S., Scharnowski, F., Lewis-Peacock, J., ... & Birbaumer, N. (2017). Closed-loop brain training: the science of neurofeedback. Nature Reviews Neuroscience, 18(2), 86-100. Smallwood, J., & Schooler, J. W. (2006). The restless mind. Psychological Bulletin, 132(6), 946. Stefaniak, J. D., Halai, A. D., & Ralph, M. A. L. (2020). The neural and neurocomputational bases of recovery from post-stroke aphasia. Nature Reviews Neurology, 16(1), 43-55. Turi, Z., Bjørkedal, E., Gunkel, L., Antal, A., Paulus, W., & Mittner, M. (2018). Evidence for cognitive placebo and nocebo effects in healthy individuals. Scientific Reports, 8(1), 1-14. Turi, Z., Csifcsák, G., Boayue, N. M., Aslaksen, P., Antal, A., Paulus, W., ... & Thielscher, A. (2019). Blinding is compromised for transcranial direct current stimulation at 1 mA for 20 min in young healthy adults. European Journal of Neuroscience, 50(8), 3261-3268. . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 7, 2020. ; https://doi.org/10.1101/2020.12.06.411850 doi: bioRxiv preprint . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 7, 2020. ; https://doi.org/10.1101/2020.12.06.411850 doi: bioRxiv preprint The effect of subjective intervention The effect of subjective intervention 29 Vicario, C. M., Salehinejad, M. A., Felmingham, K., Martino, G., & Nitsche, M. A. (2019). A systematic review on the therapeutic effectiveness of non-invasive brain stimulation for the treatment of anxiety disorders. Neuroscience & Biobehavioral Reviews, 96, 219-231. Wallace, D., Cooper, N. R., Paulmann, S., Fitzgerald, P. B., & Russo, R. (2016). Perceived comfort and blinding efficacy in randomised sham-controlled transcranial direct current stimulation (tDCS) trials at 2 mA in young and older healthy adults. PloS One, 11(2), e0149703. Wang, J., Wen, J. B., & Li, X. L. (2018). No effect of transcranial direct current stimulation of the dorsolateral prefrontal cortex on short‐term memory. CNS Neuroscience & Therapeutics, 24(1), 58-63. Westwood, S. J., Olson, A., Miall, R. C., Nappo, R., & Romani, C. (2017). Limits to tDCS effects in language: failures to modulate word production in healthy participants with frontal or temporal tDCS. Cortex, 86, 64-82. Westwood, S. J., Olson, A., Miall, R. C., Nappo, R., & Romani, C. (2017). Limits to tDCS effects in language: failures to modulate word production in healthy participants with frontal or temporal tDCS. Cortex, 86, 64-82.
https://openalex.org/W4392604581	https://dergipark.org.tr/tr/download/article-file/3546677	English	null	Teacher-tech creativity fostering behaviour as determinant of Mathematics teacher classroom practices	International online journal of primary education	2,024	cc-by	11,775	Abstract This research investigated teacher technological-creative fostering behaviour as determinant of teacher classroom practices in private primary schools in the Makurdi Local Government Area, Benue State, Nigeria. This study adopted the correlational research design. The population was all teachers in private primary schools in Makurdi. A sample of 70 mathematics teachers was drawn from 50 private primary schools. Two researcher-structured instruments were used for data collection, namely; the Mathematics Teacher Tech-Creativity Inventory (MTTI) and the Mathematics Teacher Classroom Practice Inventory (MTCPI). Correlation, scatterplots, and histograms were used to answer research questions, while analysis of variance was used to test the hypotheses at .05 level of significance. The following were the findings: the top three technological tools among others which mathematics teachers at the primary school level use most often to facilitate their teaching practices are, first interactive whiteboard, second, the calculator and third, internet surfing. Teacher tech-creativity fostering behaviour has a significant impact on teacher clarity, teacher classroom discussions, teacher feedback, teacher formative assessment and, teacher-teacher collaboration as teacher classroom practices. It was recommended that teachers of mathematics consider utilizing technology creatively during lessons as a catalyst to advance classroom teaching practices of teacher classroom clarity, class discussions, teacher feedback, formative assessment, and teacher-teacher collaboration. Keywords: Tech-creativity, teacher clarity, teacher feedback, teacher formative assessment, teacher-teacher collaboration. This is an open access article under the CC BY 4.0 license. This is an open access article under the CC BY 4.0 license. TEACHER TECH-CREATIVITY FOSTERING BEHAVIOUR AS DETERMINANT OF PRIMARY SCHOOL MATHEMATICS TEACHER CLASSROOM PRACTICES Emmanuel Edoja ACHOR Prof.Dr., Department of Science and Mathematics Education, Benue State University, Makurdi, Nigeria ORCID: https://orcid.org/0000-0002-6272-0877 nuelachor@yahoo.com Benjamin ROTT Prof.Dr., Institute of Mathematics Education, University of Cologne, Germany ORCID: https://orcid.org/0000-0002-8113-1584 benjamin.rott@uni-koeln.de Emmanuel Edoja ACHOR Prof.Dr., Department of Science and Mathematics Education, Benue State University, Makurdi, Nigeri ORCID: https://orcid.org/0000-0002-6272-0877 nuelachor@yahoo.com Received: November 20, 2023 Accepted: March 08, 2024 Published: March 31, 2024 Suggested Citation: Chianson-Akaa, M. M., Achor, E. E., & Rott, B. (2024). Teacher tech-creativity fostering behaviour as determinant of primary school mathematics teacher classroom practices. International Online Journal of Primary Education (IOJPE), 13(1), 1-22 https://doi.org/10.55020/iojpe.1392996 IOJPE ISSN: 1300 – 915X www.iojpe.org 2024, volume 13, issue 1 International Online Journal of Primary Education Copyright © International Online Journal of Primary Education IOJPE ISSN: 1300 – 915X www.iojpe.org 2024, volume 13, issue 1 International Online Journal of Primary Education Comparably, digital technology fosters connections, enriches student experiences, simulates scenarios, encourages collaboration, and generates engaging learning settings (UNESCO, 2023). In technologically advanced settings, teachers can employ digital tools and technologies to foster students' creative thinking (Henriksen, Mishra, & Fisser, 2016; Yalcinalp & Avci, 2019). By using technology in teaching and learning, educators can provide students with hands-on learning opportunities that sustain their interest in a subject without diverting focus from it (Haleem, Javaid, Qadri, & Suman, 2022). By assigning assignments that use technologically-based resources like computers, projectors, and other state-of-the-art technological tools, teachers can make their students' learning more dynamic and engaging. This could make their classes incredibly fascinating and interesting for the students (Lopez-Fernandez, 2021). Teachers of mathematics may at some point face challenges while trying to plan lessons on difficult mathematics concepts, solve difficult mathematics problems, or try to incorporate techniques to make mathematics teaching-learning processes simplistic and modify their teaching practices. If this becomes a dilemma, blending technology and teacher creativity may more or less become essential to benefit the teaching-learning process. The innovativeness and ingenuity that comes with creative thinking and technology in the classroom strengthen and thicken the teacher’s expertise and favors excellence in the teaching profession. Theoretical background of the research g Generally in this work, the theoretical underpins of tech-creativity and teacher classroom practices are borne from the premises of models such as the technological pedagogical content knowledge (TPACK) model (Mishra & Koehler, 2006), Substitution, Augmentation, Modification, Redefinition (SAMR) model (Puentedura, 2006), feedback model (Hattie & Timperley, 2007), and on theories of socio- constructivist theory by Vygotsky (1978), formative assessment theory (Black & Williams, 1998) and the cognitive load theory (Sweller, 1988). The TPACK model is a framework developed to address the complex interplay of technological knowledge, pedagogical knowledge, and content knowledge required for effective teaching with technology (Mishra & Koehler, 2006). The model has helped educators to integrate technology into their teaching practices. Consequently, the SAMR model was designed to help educators integrate technology into teaching and learning (Puentedura, 2006). Also, the feedback model was developed to emphasize the importance of feedback in enhancing students’ learning and teacher instructions (Hattie & Timperley, 2007). The theory of socio-constructivism emphasizes that knowledge is actively constructed or developed through social interactions and collaborations with others, which fosters a learning environment where joint activities, discussions, and problem-solving tasks are emphasized (Vygotsky, 1978). Also, the cognitive load theory is an educational theory that focuses on the mental effort involved in learning, and how the cognitive resources of learners are assigned, and it proposes strategies to manage cognitive load effectively for maximum learning outcomes to be achieved (Sweller, 1988). Furthermore, the theory of formative assessment highlights the importance of ongoing classroom-based assessments to enhance students’ learning. The theory emphasizes the pivotal role of feedback, questioning, and self-assessment in formative assessment practices, thus promoting the idea that if such methods are employed, teaching, and learning outcomes will be improved (Black & Williams, 1998). These aforementioned models and theories form the basis for which classroom practices of teacher clarity, class discussions, teacher feedback, formative assessment, and teacher-teacher collaboration are intertwined with teacher tech-creativity to revolutionize the classroom scenario. Teacher clarity is an essential element of effective teaching (Brckalorenz, Cole, Kinzie, & Ribera (2012). Teacher clarity draws on the sync of cognitive load theory and technology integration models such as the SAMR. Teacher clarity is aligned with the extraneous cognitive load which exposes that where additional mental effort imposed by instructional design or presentation of information is poorly designed or where unclear instruction is given, this can contribute to extraneous load (Benton & Li, 2021). Copyright © International Online Journal of Primary Education INTRODUCTION With its innovations and advantages for producing and exchanging ideas and content, new technologies have quickly changed the way that teaching and learning are done. Therefore, it is important to think about the advancement and impact of learning technology in conjunction with chances for creative education rather than in isolation. Technology is used to distribute, interact with, or promote information. It encompasses electronic teaching (e-teaching) and electronic learning (e-learning). Increased access to teaching-learning possibilities, time and place convenience, a wider range of teaching and learning resources available, enhanced opportunities for individual learning, and the development of more cognitive tools are all benefits of e-teaching and e-learning (Ugwuogo, 2011). Copyright © International Online Journal of Primary Education 1 IOJPE ISSN: 1300 – 915X www.iojpe.org 2024, volume 13, issue 1 ISSN: 1300 – 915X www.iojpe.org 2024, volume 13, issue 1 ISSN: 1300 – 915X www.iojpe.org International Online Journal of Primary Education 2024, volume 13, issue 1 integration, the teacher can modify and redesign tasks to enhance new possibilities and capabilities. Therefore, the interface between tech-creativity and teacher clarity involves the teacher utilizing technology creatively to redesign and modify learning experiences to enhance clarity in conveying information, fostering engagement, and providing meaningful learning experiences. The theoretical framework for tech-creativity and class discussion incorporates Vygotsky’s theory of socio-constructivism which highlights the importance of social interaction and collaborative learning. In this framework, teachers are encouraged to use technology to augment students’ interactions and discussions in class. This can be done by employing online platforms, discussion forums, or using collaborative tools to extend class discourse beyond conventional classroom boundaries. The TPACK may be significant in emphasizing the interaction of technological skills, pedagogical knowledge, and subject matter expertise. Incorporating technology creatively can enhance the quality of active participation, class discussions, critical thinking, and meaningful dialogue among students (Sharma, 2023; Eiland & Todd, 2019). Consequently, the theoretical framework of tech-creativity and teacher feedback is founded on the TPACK model, cognitive load theory, and feedback model. It underpins the importance of using technology creatively to facilitate effective feedback mechanisms in the learning process (Deeley, 2018). The framework explores how tools and platforms can be employed to provide timely and personalized feedback. Cognitive load theory guides the design of technology-enhanced feedback that aligns with students’ cognitive capacities while promoting comprehension and retention. The theory of cognitive load contends that learning is prevented when the brain experiences cognitive overload. By enabling pupils to concentrate on the intended instruction, technology can help lessen cognitive overload. Students can use the time saved to study, practice, and get feedback on materials they have learned. In all, the framework encourages teachers should creatively integrate technology in class to improve the quality and efficiency of feedback which can foster students’ learning and teacher instructions. In the same vein, tech-creativity and formative assessment incorporate technological tools and formative assessment theories. It emphasizes the integration of technology to enhance the formative assessment process, which involves gathering information during instruction to inform teaching and improve learning (Deeley, 2018). IOJPE The framework encourages teachers to employ a variety of tech-based formative assessment strategies such as quizzes, interactive simulation, and instructional feedback tools to tailor instructions and support students’ learning. Finally, tech-creativity and teacher-teacher collaboration are founded on socio-constructivist theory of Vygotsky and the technology integration model such as TPACK. Given that Vygotsky's theory places a strong emphasis on social contact and teamwork in the learning process, the framework for teacher-teacher collaboration encourages educators to use technology in innovative ways to promote communication, resource/material sharing, and cooperative lesson planning (Cicconi, 2013). Teachers may interact electronically, discuss creative teaching techniques, and participate in continuous professional development using technology. Theoretical background of the research With technology 2 2 Copyright © International Online Journal of Primary Education IOJPE ISSN: 1300 – 915X www.iojpe.org 2024, volume 13, issue 1 ISSN: 1300 – 915X www.iojpe.org 2024, volume 13, issue 1 ISSN: 1300 – 915X www.iojpe.org International Online Journal of Primary Education 2024, volume 13, issue 1 of a creative process, it is not enough for it to be novel, it must have value or be appropriate to the cognitive demands of the situation. A creative mind is an innovative mind prepared to bring about solutions to identified challenges or hitches faced when such challenges are identified. (Gyuse, Achor, & Chianson, 2014). The imaginable cannot suppress creativity because it can generate ideas and solutions that transcend both reason and the conceivable. Ideas that are qualitatively distinct and not tangibly attributable to any one preceding notion can be produced by creativity (Rott & Liljedahl, 2018). Agogo (2018) avers that the ultimate aim of creativity is the production of responses, ideas or products that are novel, original, and uncommon. In the same vein, Ortese, Yawe, and Akume (2014) define creativity in a different way, which is the ability to see problems in a new way and the ability to escape the bonds of conventional thinking. Creativity is a cognitive activity that is based on human thinking which leads to new, original, and useful ideas and products. By implication, creativity celebrates ingenuity and hard work that yields positive results. This has become so relevant now that Benjamin Bloom and his students (Anderson, Krathwohl, & Bloom, 2001) have modified Bloom’s taxonomy of educational objectives; in an earlier version, evaluation was placed as the higher-level of thinking but it has now been replaced with creating as the highest-level of thinking (Achor, 2020). This makes it evident that creativity is a fundamental skill necessary for knowledge formation. Numerous factors can impact a teacher's creativity, such as their prior work experience in the classroom, their history of interacting with students, the use of various learning environments, tools, and techniques, and their ability to collaborate with other educators and school administrators (Oroujlou & Vahedi, 2011; Blazar & Kraft, 2017). By creating and implementing innovative teaching methods, mediating— using their expertise to interpret and seek support on various improvement plans—and cultivating strong bonds with other educators, teachers can bring creativity into the classroom and support learning by helping students grow and change through the reciprocity of the teaching-learning process. Literature review Literature review In this paper pertinent concepts like teacher creativity, technology conceptualization, tech-creativity, and teacher classroom practice are explained for better understanding. The collection of abilities known as creativity is what makes ideas clever, worthwhile, and concise. The ability to effect change and transition from one outdated paradigm to a more modern one while achieving learning objectives is a component of teacher creativity, a word that is frequently used both within and outside of the classroom (Darma, Notosudjono, & Herfina, 2021). To help students develop a variety of skills, including social, emotional, and cognitive skills, teachers must be creative in their approach to teaching. This involves using innovative approaches and responding nimbly to novel situations. Ultimately, this helps make learning more engaging and effective (Lapeniene & Dumciene, 2014; Rankin & Brown, 2016). In most conceptualizations, teacher creativity also encompasses teaching for creativity, that is enabling students to become creative themselves (Lapeniene & Dumciene, 2014). For something to be the product 3 Copyright © International Online Journal of Primary Education Copyright © International Online Journal of Primary Education IOJPE ISSN: 1300 – 915X www.iojpe.org 2024, volume 13, issue 1 ISSN: 1300 – 915X www.iojpe.org 2024, volume 13, issue 1 International Online Journal of Primary Education International Online Journal of Primary Education 2024, volume 13, issue 1 and class engagement. It can foster better overall comprehension, practical learning, time management, and combined learning methodologies (Bay Atlantic University, 2022). The greatest applications of educational technology, according to Mishra, Koehler, and Henriksen (2011), must be based on innovative mindsets that value taking intellectual risks and being open to new ideas. Any teacher, but especially novice instructors, has a great struggle with this. Technology has altered the way educators find material to use in their classes, organize it, and incorporate it. This may affect their methods of instruction, raising the bar for professionalism. Teachers must make decisions about what and when to use digital technology in their lessons more frequently as a result of the expanding role and availability of this technology in society (Gonscherowski & Rott, 2022). Most likely, this is an attempt to make lessons more interesting and captivating. The rapidly evolving landscape of teaching and learning necessitates that educators become more adaptive and take a creative approach to the digitally advanced classroom. Teachers must thus stay up to date on the use of technology and creativity in the classroom; nonetheless, the effectiveness of their efforts depends on how they represent tech-creativity. Teachers of mathematics who are technologically creative should be able to assess themselves based on a few of the following statements but not restrictively: ‘I enjoy trying out new mathematical ideas using technologically-driven tools in class,’ ‘I am willing to try any new technology supported method even if there is a chance it could fail,’ ‘I love to modify and adapt mathematics lesson routines in line with new technology’, ‘I am continually looking for new technology-driven ideas to make the teaching of mathematics easier’, ‘Once I have developed a technology supported plan, I am prepared to use it during mathematics lessons’, and lastly ‘I continuously look at old problems with a fresh mindset guided by latest technology developments during mathematics lessons’. The aforementioned development may tend to force teachers to independently decide and construct their reality within the context of their personal and work environment. Copyright © International Online Journal of Primary Education IOJPE When teaching methods incorporate creativity, the classroom becomes more of a breeding ground for creative and effective instruction. Imagination flourishes when it is encouraged. Creativity displayed by a teacher depicts a person’s competence as an ideal teacher. Teaching with creative expressions, guiding students with creative approaches and methods, and as well giving students opportunities through instructions to develop their ideas that can break impending barriers and achieve ideas due to thinking ‘out of the box’, serves as a positive pathway to enhancing class lesson productivity and students’ knowledge. Technology is the practical application of knowledge so that something entirely new can be done, or so that something can be done in a completely new way (European Space Agency, 2023). This implies that technology can afford teachers of mathematics the opportunity to revamp their knowledge, skills, and teaching practices when the need arises. Technology has been integrated into some traditional teaching methods and class instructions to foster more engaging and exciting learning experiences. For instance, students now enjoy e-scaffolding, e-simulation, and more. Technology integration is the effective implementation of educational technology to accomplish intended learning outcomes (Achor, 2022). Teachers of mathematics now have a myriad of options via technology at their disposal to select, tryout, and approve the appropriate instructional methods and strategies to utilize in class, which may help to improve their teaching. The term tech-creativity is a derivation from technological creativity. According to Sierotowicz (2015) technological creativity is highly fostered in “knowledge-creating” organizations for innovation of new products, new processes, and services. Creativity can be viewed as the ability to bring in new ideas, while technology encapsulates useful innovations. Hence, tech-creativity can be defined as the ability to come up with new ideas whose consequence is the development of useful innovations. Tech-creativity can be seen as using novel idea-driven applications from the exploitation of creative inventions or innovations in pursuit of solving societal and classroom problems (Rambe, Ndofirepi, & Dzansi, 2016). Technology use in the classroom is exceptional then when blended with creativity, lessons become enriched. Enriched in the sense that in the classroom, technological impact has increased interactivity Copyright © International Online Journal of Primary Education 4 IOJPE ISSN: 1300 – 915X www.iojpe.org 2024, volume 13, issue 1 International Online Journal of Primary Education 2024, volume 13, issue 1 Classroom discussion can help students acquire better communication skills, as they learn to present ideas clearly and briefly; it also provides opportunities to practice listening skills and follow what others are saying (Cashin, 2011; Kosko, 2012). Teachers need to frequently initiate and facilitate entire class discussions with the view of allowing students to learn from each other. It is also a great opportunity for teachers to formatively assess (through observation) how well students comprehend the new content and concepts. Teachers can creatively foster class discussions by using open-ended questions that allow students to think critically, and develop new ideas and opinions. Incorporate multimedia to spark discussions and help students visualize concepts and ideas that will cause discussions to be more interactive. Teachers can use reflective questions and give students room to process, and engage students in discussions that can help them express themselves creatively. Consistent feedback gives students a better understanding of their progress. Teachers should give whole- group feedback on areas of need and patterns they observe in the growth of the classes as a whole in addition to written or verbal input to individuals. For the teacher to modify the teaching strategy, resources, and guidelines as necessary, students must also be given the chance to offer feedback (Zhang & Zheng, 2018). According to Jimin, Chianson-Akaa, and Amua (2023), one environmental element that affects students' self-efficacy is the feedback they receive from their teachers. Feedback has the potential to steer classroom conversations. Teachers who are providing innovative feedback can also urge students to reflect on their work and identify areas that require improvement. This approach empowers students to take responsibility for their learning. Using peer feedback is another way of creatively giving out feedback, students should be allowed to provide feedback to their peers which will help them develop critical thinking skills and further improve communication. Using technology such as online quizzes, surveys, or interactive whiteboards, is another way to make feedback creative and more engaging. Using visual aids such as graphs, charts or diagrams helps students to track and understand their progress better, then identify areas they need improvement. IOJPE It is in the process of personal learning and development that teacher creativity using technology becomes a necessary means to evolve teacher classroom practices and thereby imparting knowledge to the students. Teacher classroom practices range from designing learning experiences for the students, selecting instructional materials, developing lesson objectives, presentation of the lesson, and managing students’ behaviours (Cornelius-Ukpepi & Aglazor, 2019). For this work, some suggested highly effective teaching practices by Alber (2015) were investigated for effective teaching of Mathematics in this study. Understanding how teachers use classroom instruction to engage students, how they adapt their teaching and interaction strategies, how confident they are in communicating expectations to students, whether or not they use classroom discussion as a learning tool, and how well their formative assessment and feedback strategies are enhancing the learning environment are all crucial to improving the mathematical achievement culture of teachers. Alber (2015) further identified certain variables that are encapsulated in classroom teaching practices which may likely be enhanced via teacher tech-creativity, the variables selected are: 1) teacher clarity; 2) classroom discussion; 3) feedback; 4) formative assessment; 5) teacher-teacher collaboration. Though these practices are recurring teacher events in the classroom, their viability will necessarily depend on how the teacher can reinforce novel ideas to keep them enlightening. The novelty in idea formation here brings about creativity. These variables are discussed based on their possible relatedness to teacher creativity. The idea of "clear teaching behavior," which, according to Hattie (2012), outlines the abilities, information, attitudes, and values that students must acquire, is crucial to the idea of teacher clarity. When a teacher introduces a new subject to the class, it is best to explain the purpose and learning objectives. Then, the teacher can use creative and explicit visual aids to help students understand difficult concepts, and online resources like interactive whiteboards, educational apps, and video tutorials can help students learn more engagingly. Connecting and sharing resources via social media platforms facilitates learning about the most effective teaching methods for pupils. For pupils to see what the finished result looks like, it is best to additionally show them models or examples. Copyright © International Online Journal of Primary Education 5 5 IOJPE ISSN: 1300 – 915X www.iojpe.org 2024, volume 13, issue 1 International Online Journal of Primary Education 2024, volume 13, issue 1 educators learn to work together electronically in real-time or offline to discuss issues, advancements, and new advances, they can collaborate creatively. Teachers can also form collaboration teams and build productive relationships that can rid them of archaic and mundane approaches, skills, and practices. Another creative way to handle teacher-teacher collaboration is by encouraging peer observations; teachers can learn from each other when they observe each other teach and also provide feedback on what they have observed. Yet another creative way of handling teacher-teacher collaboration is by introducing professional learning communities; these are groups of educationists who meet regularly to discuss teaching practices, students’ learning, and other topics related to education. Some research works focused on creativity and technology used to enhance teacher’s performance or students’ learning. For instance, the findings of Fitriah (2018) revealed that teachers are aware of the importance of technology in creativity. Technology appears to help them explore their creativity and encourages learners’ creativity as well such that it helps transfer their creativity into reality, making the activities more authentic, and provides teaching materials on various topics. Similarly, Mgboro, Otuba, and Uda (2019) looked into how to use digital technology to increase teacher creativity and discovered that the social environment in which teachers work, external institutional forces, and internal creative personality traits all contribute to increased teacher creativity. In other words, using digital technology in secondary school instruction enables instructors to participate in the learning process. However, the sustainability of this type of teacher involvement depends on the presence of creative personality qualities and an environment that fosters creativity. Additionally, research by Li, Kim, and Palker (2022) showed that new technologies efficiently foster students' creativity, especially in interactive learning environments. Mathematics teachers can construct everyday creativity through the implementation of technology to improve their teaching practices. Technology does not provide ideas; technology can complement skills by providing a means of experimentation and exploration (Carlile & Jordan, 2012). Teachers who have not equipped and improved their tech-creativity skills may not be apt with the required and essential teaching practices, this can further hamper students’ performance in mathematics. Technology and creativity complement each other because technology applies practical ideas for the emergence of something new. IOJPE For a new thing, idea, concept, or method to be initiated and actualized, creative thinking must be involved. Hence, teachers of mathematics must consider harmonizing technology and creativity in the teaching and learning process. Teacher classroom practices are teaching culture operations in which teachers need to steer teaching and learning procedures in the classroom. Teachers of mathematics may necessarily need to upgrade, alternate, and sometimes vary the approaches they employ while delivering classroom practices. It is possible to go about this action fervently if teachers are abreast with recent relevant technological applications blended with creative thinking to enhance the smooth sailing of the teaching-learning process. Technological facilities provide the opening for teachers to select from numerous options what technological innovations are suitable for use to advance their teacher classroom practices and keep teachers well-informed about innovations in classroom practices. The essence of being tech-savvy for knowledge growth and the development of professional skills cannot be over-emphasized. This knowledge becomes eminent and necessary to sustain the teachers’ quest and zeal to enhance professional development and as well make their classroom practices prosper. IOJPE Formative assessment is described as a process in which students participate in the process through self-assessment, teachers adapt their instruction based on assessment evidence, and students receive feedback on their learning and suggestions for improvement (Black & Wiliam, 2009). Teachers must regularly and often evaluate their student's progress toward the topic's learning objectives or final result to give them insightful and correct feedback (summative evaluation). Students will find formative assessment enjoyable and less threatening if teachers use creative approaches such as: requesting students to mention subjects or topics they find hard to understand and then providing a specific worksheet or journal to students to express their thoughts. Asking class representatives to evaluate the performances of their classmates. Asking students to self-evaluate their learning growth and performances. Teachers can try to be creative in giving formative assessment by asking students to write a letter to a family member or friend on a sheet of paper or index card, explaining to them a new concept they have learned. A strong collaborative culture among teachers is one element that is widely accepted to support improvements in classroom and school environments. According to Richter and Pant (2016) and Kolleck, Schuster, Hartmann, and Grasel (2021), teachers are expected to work in teacher teams, collaborate closely with colleagues, and co-construct classroom methods to build trust connections within the team. Although it does not appear frequent, teacher collaboration has a lot to offer those who participate. In addition to providing instructors with the chance to exchange sound ideas and dispel misconceptions based on newly acquired knowledge, teacher collaboration with colleagues is crucial for the development of a professional community. Teaching can be exhausting and emotionally draining. Teachers face stress in their profession, whether it is from managing a particularly difficult student or juggling work and home life (or both). Fortunately, their colleagues may offer assistance during stressful times. Teachers who rely on one another for help, can build relationships based on empathy and trust. Establishing enduring professional and mentoring ties requires these frequent encounters. Supported teachers are more likely to give their students the same support (Arkansas State University, 2020). When 6 Copyright © International Online Journal of Primary Education 6 Research questions The following research questions (a derivative of Alber’s criteria) were answered in this study: The following research questions (a derivative of Alber’s criteria) were answered in t 1. What types of technological tools are available for use to enhance teacher classroom practice? p 2. To what extent does teacher tech-creativity foster behaviour affect teacher clarity in clas as a teacher classroom practice? 3. To what extent does teacher tech-creativity foster behaviour affect teacher clas discussions as a teacher classroom practice? 4. To what extent does teacher tech-creativity foster behaviour affect teacher feedback as a teacher classroom practice? 5. To what extent does teacher tech-creativity foster behaviour affect teacher formative assessment as a teacher classroom practice? 6. To what extent does teacher tech-creativity foster behaviour affect teacher-teacher collaboration as a teacher classroom practice? Hypotheses following hypotheses were tested at .05 level of significance: Ho1: Teacher tech-creativity fostering behaviour has no significant impact on teacher clarity as a teacher classroom practice. Ho2: Teacher tech-creativity fostering behaviour has no significant impact on teacher classroom discussion as a teacher classroom practice. Ho3: Teacher tech-creativity fostering behaviour has no significant impact on teacher feedback as a teacher classroom practice. Ho4: Teacher tech-creativity fostering behaviour has no significant impact on teacher formative assessment as a teacher classroom practice. Ho5: Teacher tech-creativity fostering behaviour has no significant impact on teacher-teacher collaboration as a teacher classroom practice. IOJPE ISSN: 1300 – 915X www.iojpe.org 2024, volume 13, issue 1 ISSN: 1300 – 915X www.iojpe.org International Online Journal of Primary Education 2024, volume 13, issue 1 mathematics teachers due to thoughts of their underperformance and students’ poor performance has caused teachers to lose their courage and relegate their classroom teaching practices. mathematics teachers due to thoughts of their underperformance and students’ poor performance has caused teachers to lose their courage and relegate their classroom teaching practices. Mathematics teachers may gloss over classroom teaching practices, considering them superficial. The truth is that these practices make or break the teaching and learning scenario in the classroom. Without acquiring the ethics of classroom teaching practices, teachers are half-baked and cannot be recognized as fit for the responsibility of teaching. Some teachers may have adopted the right practices, but without infusing technology and creativity into their teaching, which may have crippled the ingenuity, ferventness, and effectiveness of their teaching. With technological advancement which has exploded and gained ground in our school and classroom systems, it is unfair and illogical for teachers to imbibe teaching practices without blending them with tech-creativity. Teachers may need to try tech-creativity to see if it can foster their classroom teaching practices to redeem their lost courage and teaching expertise. This justifies the worry for this study. Statement of the problem Mathematics teachers may need to re-evaluate how they present their teaching instructions to erase doubts or fears of futile or unworkable classroom practices. Teachers may need to see if infusing creativity into lesson delivery can foster teaching practices. Teachers may also be worried if the way knowledge is transferred from teacher to student is adequate or appreciable. Teachers worry if students comprehend what they teach and how they go about the teaching, which is especially true for beginning teachers of mathematics. Mathematics teachers worry about these issues and more because of the existing poor students’ performance in mathematics, which may likely be attributed to how teachers impart knowledge during the teaching-learning process. The growing ambivalence in the minds of Copyright © International Online Journal of Primary Education 7 IOJPE ISSN: 1300 – 915X www.iojpe.org 2024, volume 13, issue 1 ISSN: 1300 – 915X www.iojpe.org 2024, volume 13, issue 1 ISSN: 1300 – 915X www.iojpe.org 2024, volume 13, issue 1 International Online Journal of Primary Education schools were used because they are mostly equipped with technologically driven facilities to aid and facilitate teaching and learning. schools were used because they are mostly equipped with technologically driven facilities to aid and facilitate teaching and learning. Two structured instruments developed by the researchers were used for data collection namely: The Mathematics Teacher Tech-Creativity Inventory (MTTI) was an adopted and modified version of the instrument by Rambe, Ndofirepi, and Dzansi (2016), and the Mathematics Teacher Classroom Practice Inventory (MTCPI) was developed by the researchers. The MTTI has three components; firstly, the biodata section; secondly, a selected list of technological tools used by mathematics teachers, such as desktops, calculators, internet surfing, and laptops, and the freedom for teachers to include any other tools they have used; lastly, twelve item statements on teachers’ level of creativity. These statements come with four options ranging strongly agree (SA-4), agree (A-3), disagree (D-2), strongly disagree (SD-1). A few of the draft items for MTTI are: “I enjoy trying out new mathematical ideas using technologically driven tools in class,” “I am willing to try any new technology-supported method even if there is a chance it could fail,” and lastly, “I continuously look at old problems with a fresh mindset guided by latest technology developments during mathematics lessons.” The MTCPI is an instrument with 25 items, which cover the five components of teacher clarity, teacher discussion, teacher feedback, formative assessment, and teacher-teacher collaboration with four options ranging from always (A-4), sometimes (S-3), rarely (R-2), never (N-1). It has two sections, the first is the biodata section, and the second section covers components of teacher classroom practices such as teacher clarity, teacher discussion, teacher feedback, formative assessment, and teacher-teacher collaboration. A few selected items from components of the MTCPI are: “give vivid explanations to students who lack the requisite knowledge,” “relate the lesson to students to have their opinions,” “call students privately to discuss areas of weakness and strengths,” “give assignments after each lesson to assess knowledge yet uncovered,” “take a cue from other teacher’s lesson plans for deeper understanding.” Both MTTI and MTCPI were trial-tested to establish their reliability coefficients which were obtained as .82 and .75 respectively. IOJPE The instruments were given out to three experts to ascertain face validity. Schools selected for the study were visited, permission was sought from various school Head- teachers then, the instruments were administered to the teachers. Correlation and histograms were used to answer the research questions while analysis of variance was used as inferential statistics to test the hypotheses at .05 level of significance. METHOD The research design used in this study was correlational, which evaluates the relationship between the predictor and the criterion variable. With little to no attempt to control unrelated variables, correlational research is a sort of nonexperimental study in which two variables are measured and the statistical relationship—that is, the correlation—between them is evaluated (Jhangiani, Chiang, Cuttler, & Leighton, 2020). The criterion variable was teacher classroom practices, and the predictor was teacher tech-creativity. The Makurdi Local Government Area in Benue State, Nigeria, is the research area. All of the private primary school instructors in the Makurdi local government area make up the study's population. In Makurdi Local Government Area, fifty private elementary schools provided a sample of seventy mathematics teachers. Schools were drawn using a systematic sampling procedure. Private Copyright © International Online Journal of Primary Education 8 Copyright © International Online Journal of Primary Education RESULTS Research Question 1 What types of technological tools are available for use to enhance teacher classroom practice? Research Question 1 What types of technological tools are available for use to enhance teacher classroom practice? Research Question 1 What types of technological tools are available for use to enhance teacher classroom practice? Figure 1. Technological tools that foster teacher classroom practices. Figure 1 shows technological tools listed by the researchers and others included by mathematics teachers. From Figure 1, the top three technological tools among others that mathematics teachers at the primary school level use most often to facilitate their teaching practices are, first interactive Figure 1. Technological tools that foster teacher classroom practices. Figure 1. Technological tools that foster teacher classroom practices. Figure 1. Technological tools that foster teacher classroom practices. Figure 1 shows technological tools listed by the researchers and others included by mathematics teachers. From Figure 1, the top three technological tools among others that mathematics teachers at the primary school level use most often to facilitate their teaching practices are, first interactive Copyright © International Online Journal of Primary Education 9 9 IOJPE ISSN: 1300 – 915X www.iojpe.org 2024, volume 13, issue 1 ISSN: 1300 – 915X www.iojpe.org 2024, volume 13, issue 1 International Online Journal of Primary Education whiteboard, second, the calculator, and third, internet surfing. Projectors and wall clocks are hardly used. esearch Question 2 IOJPE ISSN: 1300 – 915X www.iojpe.org 2024, volume 13, issue 1 ISSN: 1300 – 915X www.iojpe.org 2024, volume 13, issue 1 2024, volume 13, issue 1 International Online Journal of Primary Education From Table 1, the results have shown that there is a positive moderate relationship (R= .394) between teacher tech-creativity and teacher clarity as a classroom teaching practice. This means that a single increase in the value of teacher tech-creativity causes a relative increase in the value of teacher clarity in the same direction. Table 1 further shows that 15.6% of the variation in teacher clarity is accounted for by teacher tech-creativity. The histogram on Figure 3, further shows that the distribution is negatively skewed to the left, meaning that the mean value is less than the median value. Research Question 2 Research Question 2 To what extent does teacher tech-creativity foster behaviour affect teacher clarity as a teacher classroom practice? Q To what extent does teacher tech-creativity foster behaviour affect teacher clarity as a teacher classroom practice? Figure 2. Scatterplot for teacher-tech creativity and teacher clarity. Figure 2. Scatterplot for teacher-tech creativity and teacher clarity. Results from the scatterplot on Figure 2 show a low positive correlation in the responses between teacher-tech creativity and teacher clarity as a teacher classroom practice. Table 1. Correlation analysis of teacher tech-creativity on teacher clarity. Table 1. Correlation analysis of teacher tech-creativity on teacher clarity. e 1. Correlation analysis of teacher tech-creativity on teacher clarity. Copyright © International Online Journal of Primary Education 10 Table 1. Correlation analysis of teacher tech creativity on teacher clarity. Model Summary Model R R Square Adjusted R Square Std. Error of the Estimate 1 .394a .156 .143 .29210 a. Predictors : (Constant), MTTI b. Dependent Variable : Teacher Clarity c. R-Squared : .156 Histogram Dependent Variable: Teacher Clarity Frequency Regression Standardized Residual Mean = -3.89E-15; Std. Dev. = .993; N = 70 Figure 3. Histogram illustrating regression of teacher tech-creativity against teacher clarity. Model Summary Model R R Square Adjusted R Square Std. Error of the Estimate 1 .394a .156 .143 .29210 a. Predictors : (Constant), MTTI b. Dependent Variable : Teacher Clarity c. R-Squared : .156 Histogram Dependent Variable: Teacher Clarity Histogram g Dependent Variable: Teacher Clarity Frequency Regression Standardized Residual Mean = -3.89E-15; Std. Dev. = .993; N = 70 Figure 3. Histogram illustrating regression of teacher tech-creativity against teacher clarity. Dependent Variable: Teacher Clarity Frequency Regression Standardized Residual Regression Standardized Residual Mean = -3.89E-15; Std. Dev. = .993; N = 70 ; ; Figure 3. Histogram illustrating regression of teacher tech-creativity against teacher clarity. Copyright © International Online Journal of Primary Education 10 10 Research Question 3 To what extent does teacher tech-creativity foster behaviour affect teacher class discussions as a teacher classroom practice? Fi 4 S tt l t f t h t h ti it d t h l di i Figure 4. Scatterplot for teacher-tech creativity and teacher class discussions. Results from the scatterplot on Figure 4 show a low positive correlation in the responses between teacher-tech creativity and teacher class discussions as a teacher classroom practice. Table 2. Correlation analysis of teacher tech-creativity on teacher class discussions Table 2. Correlation analysis of teacher tech-creativity on teacher class discussions Model Summary Model R R Square Adjusted R Square Std. Error of the Estimate 1 .370a .137 .125 .32545 a. Predictors : (Constant), MTTI b. Dependent Variable : Classroom Discussion c. R-Squared : .137 Copyright © International Online Journal of Primary Education 11 IOJPE ISSN: 1300 – 915X www.iojpe.org International Online Journal of Primary Education 2024, volume 13, issue 1 Histogram Dependent Variable: Classroom Discussion Frequency Regression Standardized Residual Mean = -9.84E-15; Std. Dev. = .993; N = 70 Figure 5. Histogram illustrating regression of teacher tech-creativity against teacher classroom discussions. ISSN: 1300 – 915X www.iojpe.org 2024, volume 13, issue 1 Frequency g Mean = -9.84E-15; Std. Dev. = .993; N = 70 ; ; Figure 5. Histogram illustrating regression of teacher tech-creativity against teacher classroom discussions. From Table 2, the results have shown that there is a positive moderate relationship (R= .370) between teacher tech-creativity and teacher classroom discussions as a classroom teaching practice. This indicates that an increase in the value of teacher tech-creativity causes a relative increase in the value of teacher class discussions in the same direction. Table 2 further shows that 13.7% of the variation in teacher class discussions is accounted for, by teacher tech-creativity. The histogram in Figure 5, further shows that the distribution is random, meaning the data patterns were not clear and distinct. Research Question 4 Q To what extent does teacher tech-creativity foster behaviour affect teacher feedback as a teacher classroom practice? oom practice? Figure 6. Scatterplot for teacher-tech creativity and teacher feedback. Figure 6 Scatterplot for teacher tech creativity and teacher feedback Figure 6. Scatterplot for teacher-tech creativity and teacher feedback. Figure 6. Scatterplot for teacher-tech creativity and teacher feedback. Copyright © International Online Journal of Primary Education 12 International Online Journal of Primary Education Results from the scatterplot in Figure 6 show a low positive correlation in the responses between teacher-tech creativity and teacher feedback as a teacher classroom practice. Table 3. Correlation analysis of teacher tech-creativity and teacher feedback. Table 3. Correlation analysis of teacher tech creativity and teacher feedback. Model Summary Model R R Square Adjusted R Square Std. Error of the Estimate 1 .275a .076 .062 .37292 a. Predictors : (Constant), MTTI b. Dependent Variable : Feedback c. R-Squared : .076 Histogram Dependent Variable: Feedback Frequency Regression Standardized Residual Mean = -2.19E-16; Std. Dev. = .993; N = 70 Figure 7. Histogram illustrating regression of teacher tech-creativity versus teacher feedback. Model Summary Model R R Square Adjusted R Square Std. Error of the Estimate 1 .275a .076 .062 .37292 a. Predictors : (Constant), MTTI b. Dependent Variable : Feedback c. R-Squared : .076 Histogram Dependent Variable: Feedback g Dependent Variable: Feedback g Dependent Variable: Feedback Frequency Regression Standardized Residual Mean = -2.19E-16; Std. Dev. = .993; N = 70 Figure 7. Histogram illustrating regression of teacher tech-creativity versus teacher feedback. Dependent Variable: Feedback Frequency Regression Standardized Residual Mean = -2.19E-16; Std. Dev. = .993; N = 70 Figure 7. Histogram illustrating regression of teacher tech-creativity versus teacher feedback. Frequency Regression Standardized Residual Regression Standardized Residual g Mean = -2.19E-16; Std. Dev. = .993; N = 70 ; ; Figure 7. Histogram illustrating regression of teacher tech-creativity versus teacher feedback. From Table 3, the results have shown that there is a weak positive correlation (R= .275) between teacher tech-creativity and teacher feedback as a classroom teaching practice. This indicates that although both teacher tech-creativity and teacher feedback rise in response to one another, the relationship is not very strong. Table 3 shows that 7.6% of the variation in teacher feedback is accounted for by teacher tech- creativity. The histogram in Figure 7 further shows that the distribution is negatively skewed to the left, meaning the mean value is less than the median value. International Online Journal of Primary Education 2024, volume 13, issue 1 International Online Journal of Primary Education 2024, volume 13, issue 1 IOJPE IOJPE ISSN: 1300 – 915X www.iojpe.org International Online Journal of Primary Education 2024, volume 13, issue 1 ISSN: 1300 – 915X www.iojpe.org IOJPE www.iojpe.org International Online Journal of Primary Education 2024, volume 13, issue 1 Research Question 5 To what extent does teacher tech-creativity foster behaviour affect teacher formative assessment as a teacher classroom practice? 13 Copyright © International Online Journal of Primary Education IOJPE ISSN: 1300 – 915X www.iojpe.org 2024, volume 13, issue 1 International Online Journal of Primary Education 2024, volume 13, issue 1 ernational Online Journal of Primary Education 2024, volume 13, issu Figure 8. Scatterplot for teacher-tech creativity and teacher formative assessment. Figure 8 Scatterplot for teacher tech creativity and teacher formative assessment Figure 8. Scatterplot for teacher-tech creativity and teacher formative assessment. Figure 8. Scatterplot for teacher-tech creativity and teacher formative assessment. Results from scatterplot in Figure 8 show a low positive relationship in the responses between teacher tech-creativity and teacher formative assessment as a teacher classroom practice. Table 4. Correlation analysis of teacher tech-creativity and teacher formative assessment. able 4. Correlation analysis of teacher tech-creativity and teacher formative assessment. Table 4. Correlation analysis of teacher tech-creativity and teacher formative assessment. Table 4. Correlation analysis of teacher tech-creativity and teacher formative assessment. Model Summary Model R R Square Adjusted R Square Std. Error of the Estimate 1 .296a .087 .074 .42971 a. Predictors : (Constant), MTTI b. Dependent Variable : Formative Assessment c. R-Squared : .087 Histogram Dependent Variable: Formative Assessment Frequency Regression Standardized Residual Mean = -2.49E-15; Std. Dev. = .993; N = 70 Figure 9. Histogram illustrating regression of teacher tech-creativity versus teacher formative assessment. Table 4. Correlation analysis of teacher tech-creativity and teacher formative assessment. Model Summary Model R R Square Adjusted R Square Std. Error of the Estimate 1 .296a .087 .074 .42971 a. Predictors : (Constant), MTTI b. Dependent Variable : Formative Assessment c. R-Squared : .087 Histogram Dependent Variable: Formative Assessment Histogram g Dependent Variable: Formative Assessment Frequency ; ; igure 9. Histogram illustrating regression of teacher tech-creativity versus teacher formative assessment. From Table 4, the results have shown that there is a moderate positive correlation (R= .296) between teacher tech-creativity and teacher formative assessment as a classroom teaching practice. This indicates that although both variables of teacher tech-creativity and teacher formative assessment go up in response to one another, the relationship is not very strong. Table 4 further depicts that an 8.7% proportion in the variation of teacher formative assessment is predicted by teacher tech-creativity. The histogram in Figure 9, shows that the distribution is right-skewed and unimodal, meaning the mean value is greater than the median value, and both the mean and the median are greater than the mode. Copyright © International Online Journal of Primary Education 14 IOJPE ISSN: 1300 – 915X www.iojpe.org 2024, volume 13, issue 1 ISSN: 1300 – 915X www.iojpe.org 2024, volume 13, issue 1 Hypotheses 1 Teacher tech-creativity fostering behaviour has no significant impact on teacher clarity as a teacher classroom practice. Table 6. ANOVA results of teacher tech-creativity on teacher clarity. Model Sum of Squares df Mean Square F Sig. 1 Regression Residual 1.069 5.802 1 68 1.069 .085 12.527 .001 Total 6.871 69 a. Dependent Variable: Teacher Clarity b. Predictors: (Constant), MTTI Table 6. ANOVA results of teacher tech-creativity on teacher clarity. Results from Table 6 show that, the p value (.001) is less than alpha (.05); (F1, 68=12.53; p=.001<.05), this indicates statistical significance. Hence, we reject null hypothesis 1, then conclude that teacher tech- creativity fostering behaviour has a significant impact on teacher clarity as a teacher classroom practice. Results from Table 6 show that, the p value (.001) is less than alpha (.05); (F1, 68=12.53; p=.0 hi i di i i l i ifi H j ll h h i 1 h l d h h Results from Table 6 show that, the p value (.001) is less than alpha (.05); (F1, 68=12.53; p=.0 this indicates statistical significance. Hence, we reject null hypothesis 1, then conclude that teach creativity fostering behaviour has a significant impact on teacher clarity as a teacher classroom p Hypothesis 2 Teacher tech-creativity fostering behaviour has no significant impact on teacher classroom discussion as a teacher classroom practice. Table 7. ANOVA results of teacher tech-creativity on teacher classroom discussions. Table 7. ANOVA results of teacher tech-creativity on teacher classroom discussions. Model Sum of Squares df Mean Square F Sig. 1 Regression Residual 1.146 7.202 1 68 1.146 .106 10.815 .002 Total 8.348 69 a. Dependent Variable: Classroom Discussion b. Predictors: (Constant), MTTI Results from Table 7 show that, the p value (.002) is less than alpha (.05); (F1, 68=10.82, p=.002<.05), this indicates statistical significance. Hence, we reject null hypothesis 2, and then conclude that teacher tech-creativity fostering behaviour has a significant impact on teacher classroom discussions as a teacher classroom practice. IOJPE ISSN: 1300 – 915X www.iojpe.org 2024, volume 13, issue 1 ISSN: 1300 – 915X www.iojpe.org 2024, volume 13, issue 1 ISSN: 1300 – 915X www.iojpe.org International Online Journal of Primary Education 2024, volume 13, issue 1 From Table 5, the results have shown that there is a weak positive association (R= .285) between teacher tech-creativity and teacher-teacher collaboration as a classroom teaching practice. This indicates that although both variables of teacher tech-creativity and teacher-teacher collaboration go up in response to one another, the relationship is not very strong. Table 5 further depicts that an 8.1% proportion in the variation of teacher-teacher collaboration is predicted by teacher tech-creativity. The histogram in Figure 11, shows that the distribution is symmetric, meaning the data points are clustered around the mean, with fewer values away from the mean. Research Question 6 Q tent does teacher tech-creativity foster behaviour affect teacher-teacher collaboration as a i ? To what extent does teacher tech-creativity foster behaviour affect teacher-teacher collaboration as a teacher classroom practice? Figure 10 Scatterplot for teacher tech creativity and teacher teacher collaboration Figure 10. Scatterplot for teacher-tech creativity and teacher-teacher collaboration. Figure 10. Scatterplot for teacher-tech creativity and teacher-teacher collaboration. Results from the scatterplot in Figure 10 show a low positive relationship in the responses between teacher tech-creativity and teacher-teacher collaboration as a teacher classroom practice. Table 5. Correlation analysis of teacher tech-creativity and teacher-teacher collaboration. Table 5. Correlation analysis of teacher tech-creativity and teacher-teacher collaboration. Copyright © International Online Journal of Primary Education 15 Table 5. Correlation analysis of teacher tech-creativity and teacher-teacher collaboration. Model Summary Model R R Square Adjusted R Square Std. Error of the Estimate 1 .285a .081 .068 .47548 a. Predictors : (Constant), MTTI b. Dependent Variable : Teacher-teacher Collaboration c. R-Squared : .081 Histogram Dependent Variable: Teacher-teacher Collaboration Frequency Regression Standardized Residual Mean = -7.77E-16; Std. Dev. = .993; N = 70 Figure 11. Histogram regression of teacher tech-creativity versus teacher-teacher collaboration. Table 5. Correlation analysis of teacher tech-creativity and teacher-teacher collaboration. Model Summary Model R R Square Adjusted R Square Std. Error of the Estimate 1 .285a .081 .068 .47548 a. Predictors : (Constant), MTTI b. Dependent Variable : Teacher-teacher Collaboration c. R-Squared : .081 Histogram Dependent Variable: Teacher-teacher Collaboration Histogram Histogram Dependent Variable: Teacher-teacher Collaboration epe de t Va ab e: eac e teac e Co abo at o Frequency Regression Standardized Residual Mean = -7.77E-16; Std. Dev. = .993; N = 70 Figure 11. Histogram regression of teacher tech-creativity versus teacher-teacher collaboration. Frequency Frequency g Mean = -7.77E-16; Std. Dev. = .993; N = 70 ; ; Figure 11. Histogram regression of teacher tech-creativity versus teacher-teacher collaboration. igure 11. Histogram regression of teacher tech-creativity versus teacher-teacher collaboration. Copyright © International Online Journal of Primary Education 15 Hypothesis 3 yp Teacher tech-creativity fostering behaviour has no significant impact on teacher feedback as a teacher classroom practice. Table 8. ANOVA results of teacher tech-creativity on teacher feedback. Table 8. ANOVA results of teacher tech-creativity on teacher feedback. Model Sum of Squares df Mean Square F Sig. 1 Regression Residual .075 9.457 1 68 .775 .139 5.570 .021 Total 10.231 69 a. Dependent Variable: Feedback b. Predictors: (Constant), MTTI p b. Predictors: (Constant), MTTI Results from Table 8 show that, the p value (.021) is less than alpha (.05), (F1, 68= 5.57, p=.021<.05), this indicates statistical significance. Hence, we reject null hypothesis 3, then conclude that teacher tech-creativity fostering behaviour has a significant impact on teacher feedback as a teacher classroom practice. Copyright © International Online Journal of Primary Education 16 16 IOJPE ISSN: 1300 – 915X www.iojpe.org International Online Journal of Primary Education 2024, volume 13, issue 1 Hypothesis 5 Teacher tech-creativity fostering behaviour has no significant impact on teacher-teacher collaboration as a teacher classroom practice. Table 10. ANOVA results of teacher tech-creativity on teacher-teacher collaboration. Model Sum of Squares df Mean Square F Sig. 1 Regression Residual 1.359 15.373 1 68 1.359 .226 6.010 .017 Total 16.732 69 a. Dependent Variable: Teacher-teacher Collaboration b. Predictors: (Constant), MTTI Table 10. ANOVA results of teacher tech-creativity on teacher-teacher collaboration. Results from Table 10 indicate that, the p value (.017) is less than alpha (.05); (F1, 68=6.01; p=.017<.05), this shows statistical significance. Hence, we reject null hypothesis 5, then conclude that teacher tech-creativity fostering behaviour has a significant impact on teacher-teacher collaboration as a teacher classroom practice. Hypothesis 4 Teacher tech-creativity fostering behaviour has no significant impact on teacher formative assessment as a teacher classroom practice. Table 9. ANOVA results of teacher tech-creativity on teacher formative assessment. Model Sum of Squares df Mean Square F Sig. 1 Regression Residual 1.204 12.556 1 68 1.204 .185 6.519 .013 Total 13.760 69 a. Dependent Variable: Formative Assessment b. Predictors: (Constant), MTTI Results from Table 9 indicate that, the p value (.013) is less than alpha (.05); (F1, 68 =6.52; p= .013<.05), this shows statistical significance. Hence, we reject null hypothesis 4, then conclude that teacher tech-creativity fostering behaviour has a significant impact on teacher formative assessment as a teacher classroom practice. Ethics and Conflict of Interest All ethical rules were observed at all stages of the research. Authors declare that they acted in accordance with ethical rules in all processes of the research. Authors declare that there is no conflict of interest between the authors of this work. Recommendations Teachers of mathematics should consider utilizing technological tools blended with a creative mindset to help foster innovative approaches to instructions during lessons since this can advance classroom teaching practices. Promoting this practice can further impact teachers’ skills and professional development creatively to enhance teacher clarity, teacher classroom discussion, teacher feedback, teacher formative assessment, and teacher-teacher collaboration. IOJPE ISSN: 1300 – 915X www.iojpe.org 2024, volume 13, issue 1 ISSN: 1300 – 915X www.iojpe.org 2024, volume 13, issue 1 ISSN: 1300 – 915X www.iojpe.org International Online Journal of Primary Education 2024, volume 13, issue 1 and assessment; it further helps the teacher to gauge students’ comprehension and evaluate their teaching practices. Hence, using tech tools creatively to facilitate the process, enhances students’ learning growth and helps teachers better their future instructions and assessments. It is found in this study that teacher tech-creativity fostering behaviour has a significant impact on teacher formative assessment as a teacher classroom practice. It is along this line that Jewitt, Clark, and Hadjithoma-Garstka (2011) found that using digital learning and teaching resources provides a safer space for formative assessment and feedback. Elmahdi, Al-Hattami, and Fawzi (2018) findings also corroborate the findings of this research. It was discovered that using technology-based tools such as Plickers, enhanced formative assessment, and immediate feedback; using technology-based tools leads to creating an effective teaching and learning environment. Nicol (2008) found that technology supports assessment practices and can help teachers construct and present assessment tasks, make valid judgments of students’ progress, and support the production and delivery of marks. It is worthy of note that mathematics teachers necessarily need to be creatively selective with technological tools that can enhance formative assessment of mathematics instructions. Another finding from this research discloses that there is a significant impact of teacher-tech creativity on teacher-teacher collaboration. To buttress this finding, the work of Nwoke, Nwoga, and Emenyonu (2018) found that technology supports creativity in the classroom because it is a medium that requires interaction. Interaction comes as a result of collaboration, when people interact, they find a level ground and basis for which they can collaborate or work together to achieve meaningful results. It can be concluded that integrating technology and creativity into lesson instructions could advertently close the gap created by conventional teaching approaches, since is it rather ideal in its sense to promote transformative classroom practices that embrace creativity, technology, and education to help empower teachers to successfully implement instructions and educational goals. Technology alone will not enhance teaching and learning, but using it creatively has enhanced teacher clarity, teacher classroom discussion, teacher feedback, teacher formative assessment, and teacher-teacher collaboration as part of good teaching practice that can open new doors for learners and teachers. Limitations This work is limited to only private schools in Makurdi Local Government Area of Benue State, since they mandatorily make provision for necessary infrastructures and resources to handle technological tools; hence generalizations cannot be inferred on the entire Benue State. In Benue State, there are urban and rural districts where schools are located, rural schools will not have enough facilities and mechanisms to effectively man technological tools due to environmental conditions. Additionally, tech-creativity and teaching practices have not been observed but have been conducted by self-report data, which can be distorted because of different understandings of the items or biased due to social desirability (Safrudiannur, 2020). DISCUSSION, CONCLUSION, and RECOMMENDATIONS Findings from the research show that tech-creativity fostering behaviour significantly impacts teacher clarity in class. To support this finding, Costley (2014) found that technology provides meaningful experiences for both teacher and students; by using the computer, students believed that they understood the teacher and lesson better and as well were able to recall what was taught to them previous days. The synergy of tech-creativity and teacher verbal explanations brings about clarity of lessons because mathematics teachers have to be creative when bringing about relevant instructions via technology to enhance lesson clarity. The findings from this work show that tech-creativity fostering behaviour significantly impacts teacher classroom discussions. To support this finding, the work of Coffey (2012) found that integrating technology and peer-led discussions into teaching can produce class engagement and motivation. Herron (2012) discovered that using the internet to surf materials and get the information needed to foster learning and gainful knowledge, exposed students to mathematics activities at different levels as such students were able to engage in class and discuss their activities. When mathematics teachers find creative ways of internalizing and building technology into lesson delivery, this can foster and encourage class discussions. Another finding from this study discovered that teacher tech-creativity fostering behaviour had a significant impact on teacher feedback as a teacher classroom practice. Male, Burden, Martin, Hopkins, and Trala (2012) found that teachers reported that iPads as digital tools enabled them to provide better feedback to learners about learning. Feedback is an essential tool teachers use to inform their instruction 17 Copyright © International Online Journal of Primary Education REFERENCES Achor, E. E. (2022). Integrating technologies in curriculum development. Journal of Curriculum and Instruction, 13(2022), 4-33. Achor, E. E. (2020). Cognitive dimension in demystifying abstract concepts in physics. Benue State University Inaugural Lecture Series, No. 15. 20th February, 2020 Agogo, P. O. (2018). Promoting the culture of creativity and innovation in Nigeria education system. A paper presented at GRAMS School speech and prize giving day, held at the school premises in Makurdi on the 28th July. Alber, R. (2015). Five highly effective teaching practices. 12 September 2023 retrieved from https://www.edutopia.org/blog/5highly-effective Anderson, L. W., Krathwohl, D. R., & Bloom, B. S. (2001). (Benjamin S. A taxonomy for learning, teaching, and assessing: A revision of Bloom’s taxonomy of educational objectives / Editors, Lorin W. Anderson, David Krathwohl; Contributors, Peter W. Airasian ... [et Al.]. Complete ed. Longman. quincycollege.edu/wpcontent/uploads/Anderson- and-Krathwohl_Revised-Blooms-Taxonomy.pdf Arkansas State University (2020). The importance of teacher collaboration. Retrieved from The Importance of Teacher Collaboration \| A-State Online (astate.edu). https://degree.astate.edu/online-programs/education/master-of science/ed- leadership... Bay Atlantic University (2022). How does technology impact students’ learning? Retrieved from How Does Technology Impact Student Learning? (bau.edu) https://bau.edu>blog>technology... Benton, S., & Li, D. (2021). Teacher clarity: Cornerstone of effective teaching. IDEA paper, 83. Retrieved from Idea-Paper- 83_5-21v4.pdf (windows.net) Black, P., & Wiliam, D. (1998). Assessment and classroom learning. Assessment in Education, 5, 7-74. http://dx.doi.org/10.1080/0969595980050102 Black, P., & Wiliam, D. (2009). Developing the theory of formative assessment. Educational Assessment, Evaluation and Accountability, 21(1), 5–31. https://doi.org/10.1007/s11092-008-9068-5 Blazar, D., & Kraft, M. (2017). Teacher and teaching effects on students’ attitudes and behaviors. Educ. Eval. Polic 39, 146–170. BrckaLorenz, A., Ribera, T., Kinzie, J., & Cole, E. R. (2012). 10: Examining effective faculty practice: teaching clarity and student engagement. To Improve the Academy A Journal of Educational Development, 31(1), 148-159. https://doi.org/10.1002/j.2334-4822.2012.tb00679.x Cashin, W. E. (2011). Effective classroom discussions. IDEA Paper #49. Manhattan, KS: The IDEA Center.12 September 2023 retrieved from http://www.ideaedu.org/Portals/0/Uploads/Documents/IDEA%20Papers/IDEA%20Papers/IDEA_Paper_49.pdf. Effective Classroom Discussions \| IDEA (ideaedu.org) Carlile, O., & Jordan, A. (2012). Approaches to creativity: A guide for teachers. Maidenhead: Open University Pres Cicconi, M. (2013). Vygotsky meets technology: A reinvention of collaboration in the early childhood mathematics classroom. Early Childhood Education Journal, 42(1), 57–65. doi:10.1007/s10643-013-0582-9 Coffey, G. (2012). Literacy and technology: Integrating technology with small groups, peer-led discussions of literature. International Electronic Journal of Elementary Education, 4(2), 395-405. Cornelius-Ukpepi, B. U., & Aglazor, G. (2019). Corresponding Author Correspondence to Martha Mimi CHIANSON-AKAA, searchhbk@yahoo.com Correspondence to Martha Mimi CHIANSON-AKAA, searchhbk@yahoo.com IOJPE ISSN: 1300 – 915X www.iojpe.org 2024, volume 13, issue 1 International Online Journal of Primary Education Author Contributions All authors’ contributions to the article are equal in every aspect. All authors have read and agreed to the published version of this work. Copyright © International Online Journal of Primary Education 18 Fitriah, A. (2018). The role of technology in teachers’ creativity development in English teaching practices. Teflin 29(2), 177-193. http://dx.doi.org/10.15639/teflinjournal.v29i2/177-193 Gonscherowski, P., & Rott, B. (2022). Digital competencies of pre-/in-service teachers – An interview study. Twelfth Congress of the European Society for Research in Mathematics Education (CERME12), Feb. 2022, Bozen-Bolzano, Italy. HALid: hal-03747501. Gyuse, E. Y., Achor, E. E., & Chianson, M. M. (2014). How creative are secondary school students in Nigeria? In Z.C., Njoku (Ed.), STEM education and creativity. Proceedings of 55th Annual Conference of Science Teachers’ Association of Nigeria, Aug 18-23, 146-159. Available at https://ssrn.com/abstract=2487968 Haleem, A., Javaid, M., Qadri, M. A., & Suman, R (2022). Understanding the role of digital technologies in education: A review. Sustainable Operations and Computers, 3, 275-285. https://doi.org/10.1016/j.susoc.2022.05.004 Hattie, J. A (2012). Visible learning for teachers: Maximizing impact on learning. London, UK: Routledge. https://doi.org/10.4324/9780203181522 Hattie, J., & Timperly, H. (2007). The power of feedback. Review of Educational Research,77(1), Sage Journals. https://doi.org/10.3102/003465430298487 Henriksen, D., Mishra, P., & Fisser, P. (2016). Infusing creativity and technology in 21st century education: A syste for change. Educational Technology & Society, 19(3), 27–37. Herron, J. (2010). Implementation of technology in an elementary mathematics lesson: The experiences of pre-servic at one university. SRATE Journal, 19(1), 22-29. Jewitt, C., Clark, W., & Hadjithoma-Garstka, C. (2011). The use of learning platforms to organise learning in English primary and secondary schools. Learning, Media, and Technology, 36(4), 335-348. DOI:10.1080/17439884.2011.621955. Jhangiani, R. S., Chiang, C. A., Cuttler, C., & Leighton, D. C. (2020). Research methods in psychology (4th ed.). Kwantlen Polytechnic University Surrey, B.C. 15 November 2023. Retrieved from https://kpu.pressbooks.pub/psychmethods4e/ Jimin, N., Chianson-Akaa, M. M., & Amua, E. D. (2023). Teacher feedback techniques: An analyses of students’ self-efficacy in mathematics. International Journal of Research and Innovation in Social Science (IJRISS), Feb 21, 2023, 1316- 1326. Kolleck, N., Schuster, J., Hartmann, U., & Grasel, C. (2021). Teachers’ professional collaboration and trust relationships: An inferential social network analysis of teacher teams. Research in Education, 111(1), 89-107. DOI: 10.1177/00345237211031585. Kosko, K. W. (2012). Student enrollment in classes with frequent mathematical discussion and its longitudinal effect on mathematics achievement. The Mathematics Enthusiast, 9(1),111–148. DOI: https://doi.org/10.54870/15513440.1237 Lapeniene, D., & Dumciene, A. (2014). Teachers’ creativity: Different approaches and similar results. Procedia – Social and Behavioral Sciences, 116, 279–284. http://dx.doi.org/10.1016/j.sbspro.2014.01.208. Li, Y., Kim, M., & Palker, J. (2022). Emerging technologies to promote creativity in education: A systematic review. International Journal of Educational Research Open 3. https://doi.org/10.1016/j.ijedro.2022.100177 Lopez-Fernandez, O. (2021). Emerging health and education issues related to internet technologies and addictive problems. Int. REFERENCES Teacher classroom practice: A case study of teachers’ awareness of students’ learning styles in Calabar, Cross River State, Nigeria. International Journal of Quantitative and Qualitative Research Methods, 7(3), 1-7. https://www.researchgate.net/publication/333787198 Costley, K. C. (2014). The positive effects of technology on teaching and student learning. Department of Education (gov.). 20 October 2023 retrieved from https://eric.ed.gov/?id=ED554557 Darma, Q., Notosudjono, D., & Herfina, D. (2021). Strengthening teamwork, visionary leadership and self-efficacy in efforts to improve teacher’s creativity. Psychology and Education, 58(4), 3825–3837. Deeley, S. J. (2018) Using technology to facilitate effective assessment for learning and feedback in higher education. Assessment & Evaluation in Higher Education, 43(3), 439-448. DOI: 10.1080/02602938.2017.1356906 Copyright © International Online Journal of Primary Education 19 IOJPE ISSN: 1300 – 915X www.iojpe.org 2024, volume 13, issue 1 ISSN: 1300 – 915X www.iojpe.org 2024, volume 13, issue 1 International Online Journal of Primary Education Eiland, L. S., & Todd, T. J. (2019). Considerations when incorporating technology into classroom and experiential teaching. The Journal of Pediatric Pharmacology and Therapeutics, 24(4), 270-275. https://doi.org/10.5863/1551-6776- 24.4.270 Eiland, L. S., & Todd, T. J. (2019). Considerations when incorporating technology into classroom and experiential teaching. The Journal of Pediatric Pharmacology and Therapeutics, 24(4), 270-275. https://doi.org/10.5863/1551-6776- 24.4.270 Elmahdi, I., Al-Hattami, A., & Fawzi, H. (2018). Using technology for formative assessment to improve students’ learning. The Turkish Online Journal of Educational Technology, 17, 182-188. Elmahdi, I., Al-Hattami, A., & Fawzi, H. (2018). Using technology for formative assessment to improve students’ learning. The Turkish Online Journal of Educational Technology, 17, 182-188. European Space Agency (2023). What is technology. Retrieved from https://www.esa.int>what_is_techn.... ESA - What is technology? Fitriah, A. (2018). The role of technology in teachers’ creativity development in English teaching practices. Teflin Journal, 29(2), 177-193. http://dx.doi.org/10.15639/teflinjournal.v29i2/177-193 International Online Journal of Primary Education Mishra, P., Koehler, M. J., & Henriksen, D. (2011). The seven trans-disciplinary habits of mind: Extending the TPACK framework towards 21st century learning. Educational Technology, 51(2), 22–28. http://www.jstor.org/stable/44429913 Mishra, P., Koehler, M. J., & Henriksen, D. (2011). The seven trans-disciplinary habits of mind: Extending the TPACK framework towards 21st century learning. Educational Technology, 51(2), 22–28. http://www.jstor.org/stable/44429913 Nicol, D. (2008). Technology-supported assessment: A review of research. Unpublished manuscript available at http://www.reap.ac.uk/resources.html Technology-supported Assessment: A Review of Research (researchgate.net) Nicol, D. (2008). Technology-supported assessment: A review of research. Unpublished manuscript available at http://www.reap.ac.uk/resources.html Technology-supported Assessment: A Review of Research (researchgate.net) Nwoke, B. I., Nwoga, A. N., & Emenyonu, A. O. (2018). Impact of technology on secondary school students’ creativity in mathematics: A case for internationalization of education in Nigeria. Multidisciplinary Journal of Research Development, 27(1), 13-20. Nwoke-et-al-pp.-163-1741.pdf (unn.edu.ng) Oroujlou, N., & Vahedi, M. (2011). Motivation, attitude, and language learning. Proc. Soc. Behav. Sci. 29, 994-100 doi:10.1016/j.sbspro.2011.11.333. Motivation, attitude, and language learning (researchgate.net) Ortese, P. T, Yawe, A., & Akume, G. T. (2014). Psychology of learning. Makurdi: Eagle Prints. Puentedura, R. R. (2006). Transformation, technology, and education in the state of Maine. Retrieved from http://www.hippasus.com/rrpweblog/archives/2006_11.html Rankin, J., & Brown, V. (2016). Creative teaching method as a learning strategy for student midwives: A qualitative study. Nurse Educ. Today, 38, 93-100. https://doi.org/10.1016/j.nedt.2015.12.009. Creative teaching method as a learning strategy for student midwives: A qualitative study - PubMed (nih.gov) Rambe, P., Ndofirepi, T. M., & Dzansi, D. Y. (2016). Technological creativity and its influence on entrepreneurship intentions of vocational education students. Central University of Technology, South Africa. 13 August 2023 retrieved from https://www.researchgate.net/publication/308467442 Richter, D., & Pant, H. A. (2016). Teacher cooperation in Germany: A study on cooperative industrial relations among secondary school teachers. Gutersloh: Bertelsmann Foundation, Robert Bosch Stiftung, Stiftung Mercator and Deutsche Telekom Stiftung. 13 August 2023 retrieved from https://www.researchgate.net/publication/296677616_Lehrerkooperation_in_Deutschland_EineStudie_zu_koopera tiven_Arbeitsbeziehungen_bei_Lehrkraften_der_Sekundarstufe_I. https://doi.org/10.1177/1741143220945689 Rott, B., & Liljedahl, P. (2018, July). Creativity or imagination: Challenges with measuring creativity. Paper presented at 42nd Conference of the International Group for the Psychology of Mathematics Education, Umea, Sweden Safrudiannur, S. (2020). Measuring teachers’ beliefs: A comparison of three different approaches. In: Measuring Teachers’ Beliefs Quantitatively. Kölner Beiträge zur Didaktik der Mathematik. Springer Spektrum, Wiesbaden. https://doi.org/10.1007/978-3-658-30023-4_4 Sharma, S. (2023). Supporting student engagement with technology. Retrieved from https://www.edutopia.org/article/using- technology-support-student-engagement. Sierotowicz, T. (2015). What is technological creativity? Contemporary Global Perspectives on Gender Economy, 29. DOI:10.4018/978-1-4666-8611-3.ch014. Fitriah, A. (2018). The role of technology in teachers’ creativity development in English teaching practices. Teflin 29(2), 177-193. http://dx.doi.org/10.15639/teflinjournal.v29i2/177-193 J. Environ. Res. Public Health, 18(1), 321. https://doi.org/10.3390/ijerph18010321 Male, T., Burden, K., Martin, S., Hopkins, P., & Trala, C. (2012). iPad Scotland Evaluation. Retrieved from https://api.semanticscholar.org/CorpusID:203103531 Mgboro, C. U., & HU, O. F. U. (2019). Enhancing teacher creativity using digital technology. Journal of Education and Practice, 202111, 3(1), 2. https://doi.org/10.6897/IETITEM Mishra, P., & Koehler, M. J. (2006). Technological pedagogical content knowledge: A framework for teacher knowledge. Teachers College Record, 108(6), 1017-1054. https://doi/10.1111/j.1467-9620.2006.00684.x Copyright © International Online Journal of Primary Education 20 IOJPE ISSN: 1300 – 915X www.iojpe.org 2024, volume 13, issue 1 ISSN: 1300 – 915X www.iojpe.org 2024, volume 13, issue 1 International Online Journal of Primary Education Emmanuel Edoja ACHOR Prof. Emmanuel Edoja Achor is a Professor of Physics Education at the Benue State University, Makurdi, Nigeria. He is the former Head of Department, of Science and Mathematics, Education. He is a consultant data analyst. He is a former Consultant Researcher and Data Analyst, United Nation Development Project, and former NUC/World Bank Consultant- Lead, Higher Education Programme Specialist (LHEPS). His research areas are metacognitive awareness, curriculum development and teacher professional development. International Online Journal of Primary Education What is Technological Creativity \| IGI Global (igi-global.com) Sweller, J. (1998). Cognitive load during problem solving: Effects on learning. Cognitive Science, 12(2), 257-285. https://doi.org/10.1207/s15516709cog1202_4 Ugwuogo, C. C. (2011). Status of e-teaching and e-learning in Nigeria Colleges of Education (Unpublished PhD Thesis). Ebonyi State University, Abakaliki. UNESCO (2023). Technology in education. GEM Report. Retrieved from https://gem-report-2023.unesco.org/technology-in- education/ Vygotsky, L. (1978). Mind in society. London: Harvard University Press. Retrieved from https://gsi.berkeley.edu>sociol- con... Yalcinalp, S., & Avci, Ü. (2019). Creativity and emerging digital educational technologies: A systematic review. Turkish Online Journal of Educational Technology - TOJET, 18(3), 25-45. Zhang, L., & Zheng, Y. (2018). Feedback as an assessment for learning tool: How useful can it be? Assessment & Evaluation in Higher Education, 43(7), 1120-1132. DOI: 10.1080/02602938.2018.1434481 Copyright © International Online Journal of Primary Education 21 Martha Mimi CHIANSON-AKAA Martha Mimi Chianson-Akaa is a Senior Lecturer of Mathematics Education at Benue State University, Makurdi Nigeria. She is a para-counselor inducted into the University Counseling Unit and currently the Postgraduate Coordinator (department). Her research interest areas are classroom practices, teacher professional development and learning strategies. Her teaching emphasizes collaboration and equity to improve the quality of teaching-learning in the classroom. IOJPE ISSN: 1300 – 915X www.iojpe.org 2024, volume 13, issue 1 ISSN: 1300 – 915X www.iojpe.org 4 l 13 i 1 Copyright © International Online Journal of Primary Education Benjamin ROTT Dr. Benjamin Rott is Professor of Mathematics Education at the University of Cologne. His research focuses on mathematical problem solving, beliefs, and giftedness (including creativity). His recent works also focus on teachers' competencies and especially digital competencies. 22 Copyright © International Online Journal of Primary Education
https://openalex.org/W2963955318	https://portal.findresearcher.sdu.dk/files/152578439/Movement_behavior_profiles_and_obesity.pdf	English	null	Movement behavior profiles and obesity: a latent profile analysis of 24-h time-use composition among Danish workers	International journal of obesity	2,019	cc-by	8,026	Download date: 24. Oct. 2024 University of Southern Denmark Citation for pulished version (APA): Gupta, N., Hallman, D. M., Dumuid, D., Vij, A., Rasmussen, C. L., Jørgensen, M. B., & Holtermann, A. (2020). Movement behavior profiles and obesity: a latent profile analysis of 24-h time-use composition among Danish workers. International Journal of Obesity, 44(2), 409-417. https://doi.org/10.1038/s41366-019-0419-8 Citation for pulished version (APA): Gupta, N., Hallman, D. M., Dumuid, D., Vij, A., Rasmussen, C. L., Jørgensen, M. B., & Holtermann, A. (2020). Movement behavior profiles and obesity: a latent profile analysis of 24-h time-use composition among Danish workers. International Journal of Obesity, 44(2), 409-417. https://doi.org/10.1038/s41366-019-0419-8 Go to publication entry in University of Southern Denmark's Research Portal Terms of use This work is brought to you by the University of Southern Denmark. Unless otherwise specified it has been shared according to the terms for self-archiving. If no other license is stated, these terms apply: ARTICLE ARTICLE Movement behavior proﬁles and obesity: a latent proﬁle analysis of 24-h time-use composition among Danish workers Nidhi Gupta1 ●David M. Hallman2 ●Dorothea Dumuid3 ●Akshay Vij4 ●Charlotte Lund Ras Marie Birk Jørgensen6 ●Andreas Holtermann1,7 Received: 23 November 2018 / Revised: 7 May 2019 / Accepted: 26 May 2019 © The Author(s) 2019. This article is published with open access 1 National Research Centre for the Working Environment, Copenhagen, Denmark Terms of use This work is brought to you by the University of Southern Denmark. Unless otherwise specified it has been shared according to the terms for self-archiving. If no other license is stated these terms apply: • You may download this work for personal use only. y p y • You may not further distribute the material or use it for any profit-making activity or commercial Y f l di t ib t th URL id tif i thi i y p y • You may not further distribute the material or use it for any profit-makin at this document breaches copyright please contact us providing details and we will investigate your claim. enquiries to puresupport@bib.sdu.dk If you believe that this document breaches copyright please contact us providing details and we will investigate your claim. Please direct all enquiries to puresupport@bib.sdu.dk If you believe that this document breaches copyright please contact us providing details and we will investigate your claim. Please direct all enquiries to puresupport@bib.sdu.dk Download date: 24. Oct. 2024 International Journal of Obesity https://doi.org/10.1038/s41366-019-0419-8 Abstract Background/objectives An element of obesity prevention is increasing total physical activity energy expenditure. However, this approach does not incorporate the balance of various movement behaviors—physical activity, sedentary behaviors and sleep—across domains of the day. We aimed to identify time-use proﬁles over work and leisure, termed ‘movement behavior proﬁles’ and to investigate their association with obesity. Subjects/methods Eight-hundred-and-seven workers completed (a) thigh accelerometry and diaries to determine their 24-h composition of behaviors (sedentary and standing, light physical activity and moderate-to-vigorous physical activity at work and leisure, and time in bed) and (b) obesity measurements. Movement behavior proﬁles were determined using latent proﬁle analyses of isometric log-ratios of the 24-h composition, and labeled according to animal movement behavior traits. Linear d l li d t d t i th i ti b t ﬁl d b it models were applied to determine the association between proﬁles and obesity. Results Four proﬁles were identiﬁed, labeled as “Chimpanzees” (n = 226), “Lions” (n = 179), “Ants” (n = 244), and “Koalas” (n = 158). “Chimpanzees” work time was evenly distributed between behaviors while their leisure time was predominantly active. Compared to Chimpanzees, “Lions” were more active at work and sedentary during leisure and spent more time in bed; “Ants” were more active at work and during leisure; “Koalas” were more sedentary at work and leisure and spent similar time in bed. With “Chimpanzees” as reference, “Lions” had least favorable obesity indicators: +2.0 (95% conﬁdence interval [CI] 0.6, 3.4) %body fat, +4.3 cm (1.4, 7.3) waist circumference and +1.0 (2.0, 0.0) Body Mass Index (BMI), followed by “Koalas” +2.0 (0.4, 3.7) %body fat, +3.1 cm (0.1, 6.0) waist circumference, and +0.8 (−0.30, 1.94) BMI. No signiﬁcant differences were found between “Chimpanzees” and “Ants”. Conclusions Movement behavior proﬁles across work and leisure time-use compositions are associated with obesity. Achieving adequate balance between work and leisure movement behaviors should be further investigated as a potential obesity prevention strategy. Conclusions Movement behavior proﬁles across work and leisure time-use compositions are associated with obesity. Achieving adequate balance between work and leisure movement behaviors should be further investigated as a potential obesity prevention strategy. 2 Centre for Musculoskeletal Research, Department of Occupational Health Sciences and Psychology, University of Gävle, Gävle, Sweden 3 Alliance for Research in Exercise, Nutrition and Activity (ARENA), School of Health Sciences, University of South Australia, Adelaide, South Australia, Australia Introduction However, if proper recovery is not attained, a chronic disturbed state of homeostasis and excessive cumulative allostatic load [13] can induce adverse effects such as preferential deposition of adipose tissues, promotion of energy storage as fat, and insulin resistance [14]. Similarly, too little physical activity and excessive sedentary behavior can disturb homeostasis over time, causing an energy imbalance [6]. Both of these examples of imbalanced time use over the day may con- tribute to obesity. An existing behavioral approach to prevent obesity is to promote higher daily total energy expenditure by increasing the time spent in physical activity and reducing time spent sedentary [6]. However, this approach does not incorporate the recovery process from daily activities, shown to be of relevance for obesity [7–9]. Further, the approach of increasing total energy expenditure does not consider domains (i.e., work and leisure) of movement behaviors over the day. This could be important for obesity prevention as the pattern of movement behaviors, possibilities of var- iation and recovery and their health effects may depend on the domain in which they occur [10]. Therefore, an obesity prevention approach that accounts for recovery and domains of movement behaviors in a day could be useful. Abstract * Nidhi Gupta ngu@nfa.dk 4 Institute for Choice, University of South Australia, Adelaide, Australia 5 Section of Social Medicine, Department of Public Health, University of Copenhagen, Copenhagen, Denmark 2 Centre for Musculoskeletal Research, Department of Occupational Health Sciences and Psychology, University of Gävle, Gävle, Sweden 6 Department of Forensic Science, University of Copenhagen, Copenhagen, Denmark 7 Department of Sports Science and Clinical Biomechanics, University of Southern Denmark, Odense, Denmark 3 Alliance for Research in Exercise, Nutrition and Activity (ARENA), School of Health Sciences, University of South Australia, Adelaide, South Australia, Australia N. Gupta et al. Materials/subjects and methods This study used cross-sectional data from the Danish PHysical ACTivity cohort with Objective measurements (DPHACTO) cohort [19]. Workers from 15 companies engaged in three different sectors - cleaning, transport, and manufacturing - were recruited between December 2011 and March 2013. In total, 2107 workers were invited to participate, of which 1119 consented to participate. To be included, workers had to be able to participate during working hours. The pre-established exclusion criteria were: being in a management position, intern, pregnant and hav- ing fever on the day of testing or bandage allergy. An appropriate behavioral approach for obesity preven- tion could be to facilitate balanced distribution of time spent in movement behaviors across domains in a day. According to the principle of the homeostasis state of the body [11, 12], a balance can be obtained if the ‘stimulus’ on the body (e.g., physical activity) is balanced with sufﬁcient recovery (e.g., sedentary time and sleep). However, if proper recovery is not attained, a chronic disturbed state of homeostasis and excessive cumulative allostatic load [13] can induce adverse effects such as preferential deposition of adipose tissues, promotion of energy storage as fat, and insulin resistance [14]. Similarly, too little physical activity and excessive sedentary behavior can disturb homeostasis over time, causing an energy imbalance [6]. Both of these examples of imbalanced time use over the day may con- tribute to obesity. Data collection was conducted from spring 2012 to spring 2013. The consenting and eligible workers were invited to ﬁll in a web-based questionnaire and to perform physical examination tests and accelerometry measurements. All workers provided their written consent prior to par- ticipation. The present study was conducted according to the Helsinki declaration and approved by the Danish data protection agency and the local Ethics Committee (The Capital Region of Denmark, H-2-2012-011). Introduction behavioral patterns via an exploratory person-driven approach. One such approach—latent proﬁle analysis— detects groups with common patterns of time-use behaviors [17]. However, to date, no latent proﬁle analysis has dis- tinguished between work and leisure domains or accounted for the constrained nature of time use (i.e., every day can only have 24 h). It is now widely accepted that analysis of 24-hour time use should respect the compositional proper- ties of the data [18]. The worldwide rate of obesity has tripled over the last three decades [1]. In 2016, more than 1.9 billion adults (≥18 years) were overweight, out of which around 650 million were obese [1]. Obesity is a known risk factor for diabetes mellitus, hypertension, cardiovascular diseases and mortal- ity [2–5]. The increasing prevalence and adverse health effects of obesity necessitate more effective preventive interventions. Our study aimed to use compositional latent proﬁle analysis to explore if distinct time-use proﬁles based on movement behaviors during work and leisure time could be identiﬁed among Danish workers, and to determine whether these proﬁles were associated with obesity. interventions. An existing behavioral approach to prevent obesity is to promote higher daily total energy expenditure by increasing the time spent in physical activity and reducing time spent sedentary [6]. However, this approach does not incorporate the recovery process from daily activities, shown to be of relevance for obesity [7–9]. Further, the approach of increasing total energy expenditure does not consider domains (i.e., work and leisure) of movement behaviors over the day. This could be important for obesity prevention as the pattern of movement behaviors, possibilities of var- iation and recovery and their health effects may depend on the domain in which they occur [10]. Therefore, an obesity prevention approach that accounts for recovery and domains of movement behaviors in a day could be useful. An appropriate behavioral approach for obesity preven- tion could be to facilitate balanced distribution of time spent in movement behaviors across domains in a day. According to the principle of the homeostasis state of the body [11, 12], a balance can be obtained if the ‘stimulus’ on the body (e.g., physical activity) is balanced with sufﬁcient recovery (e.g., sedentary time and sleep). Accelerometry Balancing time use over a day is dependent on the inherent imposed constraints of the main domains of the day (i.e., work and leisure). For example, an ofﬁce worker is likely constrained to spend most of the work time being sedentary or standing. On the contrary, a cleaner is likely constrained to do prolonged walking/standing for several hours at work [15, 16]. The ofﬁce worker and the cleaner may therefore require different amounts of leisure time sedentary behavior, physical activity and sleep to achieve balanced 24-hour time use to prevent obesity. Workers attached a triaxial accelerometer (Actigraph GT3X+, Actigraph LLC, Florida, USA) on the right thigh for four consecutive days (4 × 24 h) including at least two working days [20]. Workers were also asked to complete a short paper- based diary noting the start and end of work periods, time in bed (going to bed and getting out of bed), non-wear time, and time of reference measurement (i.e., standing in an upright position for 15 s) during the measurement period. Workers were instructed to remove the device if it caused any kind of discomfort. To gain an understanding of how to best balance time spent in movement behaviors over various daily domains to prevent obesity, it may be useful to ﬁrst explore time-use The raw data from the accelerometer were downloaded using the Actilife software (v.5, ActiGraph LLC, Pensacola, Movement behavior proﬁles and obesity: a latent proﬁle analysis of 24-h time-use composition among. . . FL, USA) and later processed using a customized MATLAB program, Acti4 (The National Research Centre for the Working Environment, Copenhagen, Denmark and BAuA, Berlin, Germany). This program has shown to determine duration spent in various postures (lie, sit, and stand) and physical activity (walk, run, stair climb, and cycle) with high sensitivity (95–99%) and speciﬁcity (>99%) during stan- dardized conditions [21]. Time spent sedentary, standing still, and in light physical activity (LIPA; including moving [standing with movements], and slow walking), MVPA (including fast walking, running, stair climbing and cycling) at work and leisure and time in bed was determined based on procedures explained elsewhere [21]. sedentary behavior and sleep and obesity [7, 24, 25]. Age was determined using the workers’ unique Danish civil registration numbers. Statistical analyses All analyses were conducted in RStudio software (Version 0.99.893 – © 2009–2016) using the “Compositions” package [26] and in Mplus software [version 7.4, Muthen & Muthen [27]]. The data of time spent in all movement behaviors (sedentary, standing still, LIPA and MVPA at work and leisure, and time in bed) in a day are ‘compositional’ in nature. That is, these data exist in a constrained data space where the sum of all parts (i.e., movement behaviors in this case) always sum up to 100% (i.e., 24 h). Thus, increasing time spent in one behavior will inevitably substitute time spent in at least one other behavior within a day. This special property of the data needs to be addressed using compositional data analysis (CoDA; [28, 29]). The mean of the time spent sedentary, standing still, in LIPA, and MVPA, and a median of time-in-bed periods, across all valid days, was calculated for each worker. These summary statistics were chosen based on the distribution of the data. Accelerometry Smoking status was obtained from a single item “Do you smoke?” with four responses sum- marized into ‘smokers (smoking regularly, smoking occa- sionally)’ and ‘non-smokers (used to smoke not anymore, never smoked)’. Alcohol intake was determined using a single item “How much alcohol did you drink during the last week?” with responses in units per week. Poor dietary intake was obtained from two items; “How often do you usually eat/drink”—“Fast food, pizza, burger, shawarma, etc.?” and “Candy, ice cream, chocolate, soft drinks” with four responses (daily, 3–4/week, 1–2/week, and rarely). The responses for the two items were reversed and averaged where higher scores indicated worse dietary intake. A proxy measure of socioeconomic status was determined through a single question on predominant type of work (responses were “administration” named as “white-collar” and “pro- duction” named as “blue-collar” workers). Non-wear periods were recognized according to an automatic procedure [22]. All non-wear periods and non- working days were excluded from the analyses. A day consisted of 24 h starting from midnight. A work period was deﬁned as the self-reported work hours spent on primary occupation while the remaining hours, except time in bed, were considered as the leisure period. Time in bed, as a proxy of sleep time, was measured using information from self-reported diary that was conﬁrmed via visual checks of accelerometry data. A day was considered valid if it con- tained valid work, leisure, and time in bed periods. Work and leisure periods were considered valid if they comprised at least 4 h or 75% of the worker’s average work and leisure time. Time in bed was considered valid if it was at least 4 h in duration [23]. Workers who had measurements on at least one valid day were included in further analyses. Determination of movement behavior proﬁles and their association with obesity Waist circumference (WC) was measured two times hor- izontally midway between the top edge of the hip and lower ribs using a measurement tape (Seca, model 201) to the nearest 0.1 mm. The average of the two measurements was calculated. Weight and fat percentage (Fat%) were mea- sured using the Tanita (model BC418 MA) bio-impedance segmental body composition analyzer [47], to the nearest 0.1 kg/0.1%. Height without shoes was measured using a stadiometer (Seca, model 213) to the nearest 0.1 cm. Body mass index (BMI) was calculated as weight (kg) divided by height (m) squared. First, the compositional time-use data (work [sedentary, standing still, LIPA, and MVPA] and leisure [sedentary, standing still, LIPA, MVPA], and time in bed) were expressed as a set of eight isometrical log ratios (ilrs) using the default ilr transformation in the “Compositions” R package [26]. The ilrs contain all the relative information regarding the time-use composition, and can be used as real vectors in standard statistical models instead of the com- positional vectors of the raw minutes/day. Results Of the 1119 workers who consented to participate, 807 workers provided valid accelerometry data for at least one working day, including valid periods of time in bed, work and leisure. On average, workers wore the accelerometer for 7.6 h (SD 1.2) at work and 15.8 h (SD 1.5) at leisure— including time in bed (Mean 7.0, SD 1.0)—summing to 23.4 h (SD 1.2) per day. Table 1 shows the results of consecutive latent proﬁle models with 1 to 5-proﬁle solutions for 807 workers. The AIC values and entropy continued to decline from 1 to 5- proﬁle solutions but the BIC value was the lowest at the 4- proﬁle solution. Additionally, at the 5-proﬁle solution, the p-value of LMRT became non-signiﬁcant and one of the resulting proﬁles was very small (n = 8). Thus, for the further analyses, we chose the 4-proﬁle solution based on the goodness of ﬁt statistics and an evaluation of the bio- logical relevance of the proﬁles. Potential outliers in the multivariate compositional time- use data were identiﬁed using a principal components bi- plot [30]. Twelve outliers were identiﬁed based on a 0.999% probability ellipse. The sensitivity of the best latent proﬁle model to these outliers was tested by determining if the decision of selecting the best latent model remained the same before and after removal of these outliers. Results indicated high agreement between the two models (Cohen’s Kappa 0.97). Thus, the outliers were kept in the dataset and subsequent analyses. Figure 1 and Table 2 show the compositional mean of the daily time-use composition for the four movement behavior proﬁles. We named the four movement behavior proﬁles as “Chimpanzees”, “Lions”, “Koalas” and “Ants”. We con- sidered the “Chimpanzees” proﬁle to have the most balanced movement behavior proﬁle across the different domains of the day and used this proﬁle as a reference for comparisons. After choosing the best model, each worker was assigned to one of four proﬁles, based on their maximum posterior probability of being in that proﬁle [31]. Thereafter, each proﬁle was named to reﬂect similar behavioral proﬁles in the animal kingdom. For example, one proﬁle was named “Lions” as the workers in this proﬁle spent much of their work time being active (i.e., “hunting”) and most of their leisure time being sedentary or in bed. Confounders Second, the ilrs were used as inputs for compositional latent proﬁle models. Latent proﬁle analysis uses a ﬁnite mixture latent modeling approach to statistically derive subgroups that are homogenous with respect to their time-use Potential confounders were chosen a priori based on pre- vious research on the association between physical activity, N. Gupta et al. behaviors within a proﬁle and heterogeneous between pro- ﬁles. To identify the best ﬁtting latent model indicating the respective movement behavior proﬁles, we conducted consecutive latent models with one to ﬁve proﬁle solutions. Then, we chose the best model based on ﬁt statistics fol- lowed by an evaluation of the distinguished proﬁles according to their clinical relevance. The model ﬁt was judged based on the following ﬁt statistics and criteria: [1] Lo-Mendell-Rubin Test (LMRT): an inferential statistical test which compares a targeted proﬁle solution (e.g., 5- proﬁles) with a 1-less proﬁle solution (e.g., 4-proﬁles). We chose a 1-more proﬁle solution if the corresponding p-value was less than 0.05, otherwise a 1-less proﬁle solution was retained; [2] Akaike Information Criterion (AIC) and the Bayesian Information Criterion (BIC): these are indicators of the best balance between the simplicity of the model and the goodness of ﬁt. Lower values indicate a more parsi- monious model; [3] Entropy: ranges from 0 to 1, and describes the degree of certainty of classiﬁcation of each proﬁle solution; higher values indicate higher classiﬁcation certainty; and [4] Number of workers in each group not less than 50. As we did not have prior knowledge of the number of movement behaviors proﬁles or sufﬁcient information about expected effect sizes of the movement behavior proﬁles on the obesity measures from previous studies, we could not perform an a priori sample size calculation of sufﬁcient quality. Results This means that on average, Koalas are 2.3 times more (exp(0.81)) seden- tary at work compared to the whole population. LIPA light physical activity MVPA moderate to vigorous physical activity TIB time in bed Fig. 1 Compositional Proﬁle Plots indicating the relative difference between time-use compositional means of each proﬁle compared to the whole population. The proﬁles for Lions, Koalas and Ants are com- pared with Chimpanzees (black). Values on the Y axis represent the log of the ratio between a particular proﬁle mean and the population mean. A score of 0 for any behavior means that, on average, equal time is spent in that behavior by the whole population as by the proﬁle of interest. Positive and negative values represent higher and lower compositional means than the whole population. On the basis of the log-ratio value, the actual ratio between the composition mean of a particular proﬁle and whole population can be calculated. For example the log-ratio value for sedentary time at work for Koalas is 0.81. This means that on average, Koalas are 2.3 times more (exp(0.81)) seden- tary at work compared to the whole population. LIPA light physical activity, MVPA moderate to vigorous physical activity, TIB time in bed Fig. 1 Compositional Proﬁle Plots indicating the relative difference between time-use compositional means of each proﬁle compared to the whole population. The proﬁles for Lions, Koalas and Ants are com- pared with Chimpanzees (black). Values on the Y axis represent the log of the ratio between a particular proﬁle mean and the population mean. A score of 0 for any behavior means that, on average, equal time is spent in that behavior by the whole population as by the proﬁle of interest. Positive and negative values represent higher and lower reported to consume a poor diet ‘everyday’ and 23% reported themselves to be smokers. Results The “Chimpanzees” proﬁle (n = 226, 28% of the sample) was characterized by (i) a rather evenly distributed com- position of work behaviors (sedentary = 197 min; standing = 145 min; physical activity = 117 min), (ii) an active leisure time (physical activity = 114 min; standing = 121 min), and (iii) 440 min in bed. The time-use compositions of the four movement beha- vior proﬁles were described in terms of center (composi- tional mean of time spent in bed and in work and leisure sedentary, LIPA and MVPA) and multivariate dispersion (variation matrix) [32]. Table 1 Fit indices of the latent 1 to 5-proﬁle solutions (N = 807) Proﬁles AIC BIC Entropy LMRT (p-value) Min size 5 6522 6766 0.80 0.35 8 4 6541 6743 0.77 0.02 158 3 6732 6891 0.78 <0.01 210 2 7090 7208 0.82 <0.01 269 1 7949 8024 – – – AIC akaike information criterion, BIC Bayesian Information Criterion, LMRT Lo-Mendell-Rubin Test; min size reﬂects the minimum size of the proﬁle in each solution Table 1 Fit indices of the latent 1 to 5-proﬁle solutions (N = 807) Proﬁles AIC BIC Entropy LMRT (p-value) Min size Thereafter, we explored the linear association between membership of the movement behavior proﬁles (predictor) and obesity indicators (outcomes) included in separate models, adjusted for selected confounders—age, sex, smoking, alcohol, poor dietary habits and socioeconomic status. Sex, smoking, and socioeconomic status were treated as categorical variables, whilst age, alcohol intake, and poor dietary habits were treated as continuous variables. Movement behavior proﬁles and obesity: a latent proﬁle analysis of 24-h time-use composition among. . . Fig. 1 Compositional Proﬁle Plots indicating the relative difference between time-use compositional means of each proﬁle compared to the whole population. The proﬁles for Lions, Koalas and Ants are com- pared with Chimpanzees (black). Values on the Y axis represent the log of the ratio between a particular proﬁle mean and the population mean. A score of 0 for any behavior means that, on average, equal time is spent in that behavior by the whole population as by the proﬁle of compositional means than the whole population. On the basis of the log-ratio value, the actual ratio between the composition mean of a particular proﬁle and whole population can be calculated. For example the log-ratio value for sedentary time at work for Koalas is 0.81. Results Compared to the “Chimpanzees” proﬁle, “Lions” (n = 179, 22% of the sample) were characterized by (i) more physical activity and standing at work, (ii) more sedentary behavior and less physical activity at leisure, and (iii) slightly more time in bed; Compared to “Chimpanzees”; “Lions” were younger, included more men, blue-collar workers and smokers, had a lower prevalence of poor dietary intake and alcohol intake; “Ants” were older, included more women, fewer smokers; “Koalas” were older, included more women and white collar workers, fewer smokers, had a higher prevalence of poor dietary intake and alcohol intake. “Ants” (n = 244, 30% of the sample) were characterized by (i) more physical activity and standing at work, (ii) almost similar physical activity and standing but higher sedentary behavior at leisure, and (iii) similar time in bed. “Koalas” (n = 158, 20% of the sample) were character- ized by (i) more sedentary behavior and less physical activity and standing at work, (ii) higher sedentary behavior and less physical activity and standing at leisure (iii) slightly higher time in bed. Table 3 shows the linear associations between the four movement behavior proﬁles and obesity indicators, adjusted for potential confounders. Compared to “Chim- panzees”; “Lions” had signiﬁcantly higher BMI (kg/m2; +1.0; 95% CI 0.1, 2.0), WC (cm; +4.3; 1.4, 7.3), and fat% (%; +2.0; 0.6, 3.4); “Koalas” also had higher obesity indicators (WC, +3.1; 0.1, 6.2, fat%, +2.0; 0.4, 3.7), however the results were non-signiﬁcant for BMI. In our sample, “Ants” had slightly higher obesity indicators than “Chimpanzees”, but the differences were not statistically signiﬁcant. Table 2 shows the distribution of demographical, life- style, health and work-related indicators across the four movement behavior proﬁles. On average, workers in the “Chimpanzees” proﬁle were 44 years old, 64% of them were men and 87% of them were blue-collar workers. They consumed 4 units of alcohol per week, 40% of them N. Gupta et al. Results Table 2 Demographical, lifestyle, obesity and work-related descriptive statistics for the total population and the four movement behavior proﬁles Variables Total (n = 807) Chimpanzees (n = 226) Lions (n = 179) Koalas (n = 158) Ants (n = 244) n x̅ SD % n x̅ SD % n x̅ SD % n x̅ SD % n x̅ SD % Demographic variables Age (years) 807 45.1 9.7 226 44.0 9.0 179 43.4 11.3 158 45.7 8.7 244 47.0 9.5 Male 439 54.4 144 63.7 130 72.6 87 55.1 78 32.0 Lifestyle-related variables Alcohol intake (units/week) 761 4.5 6.0 216 4.3 5.1 171 5.1 6.7 152 4.8 6.0 222 3.9 6.3 Poor diet (0–3)* 792 1.8 1.0 222 1.9 1.1 177 1.7 1.0 157 2.1 1.0 236 1.8 1.1 Smokers 216 27.4 50 22.7 62 35.2 33 21.2 71 30.2 Obesity Health-related variables BMI (kg/m2) 790 27.2 4.8 221 26.9 4.6 172 27.8 5.1 157 27.0 4.4 240 27.3 4.8 WC (cm) 570 93.3 12.6 171 91.7 12.6 110 96.6 12.2 134 92.9 13.0 155 93.1 12.3 Fat (%) 790 29.1 9.3 222 27.0 9.2 175 27.8 9.5 157 29.0 7.9 236 32.2 9.5 Work-related variables Blue-collar workers 671 83.1 196 86.7 173 96.6 61 38.6 241 98.8 Compositional means of sedentary behaviors, standing still, LIPA, and MVPA at work and leisure and time in bed (total = 1440 min) Sedentary behavior at work 149 197 137 333 64 Standing still at work 137 145 167 68 156 LIPA at work 70 60 79 26 123 MVPA at work 60 57 76 27 79 Sedentary behavior at leisure 353 306 380 346 342 Standing still at leisure 103 121 62 95 122 LIPA at leisure 57 62 43 43 70 MVPA at leisure 45 52 28 39 52 Time in bed 466 440 468 462 432 LIPA light physical activity, MVPA moderate-to-vigorous physical activity, WC waist circumference, BMI body mass index; *0 = never, 3 = everyday; x̅ = mean e, obesity and work-related descriptive statistics for the total population and the four movement behavior proﬁles Table 2 Demographical, lifestyle, obesity and work-related descriptive statistics for the total population and the fo Discussion fatigue, thereby requiring less recovery time to obtain homeostasis. Additionally, they spent more time in physical activity during leisure, which has shown numerous health beneﬁts, including weight loss [40]. In agreement, we found that “Chimpanzees” had the most favorable obesity indi- cators compared to the three other proﬁles. On average, “Chimpanzees” had 27 kg/m2 (SD = 5) BMI, 92 cm (SD = 13) WC, and 27% (SD = 9) fat%. These results were robust even after adjusting for several potential confounders including age, gender, smoking, alcohol intake, poor dietary habits, and socioeconomic status. However, other factors such as psychosocial factors and total energy expenditure may have played a role that needs to be explored in future studies. This is the ﬁrst study to explore latent movement behavior proﬁles based on 24-h time-use compositions among a working population. Most similar previous studies (a) have been performed on children and adolescents [33–36] or (b) did not use 24-h data [37–39] or (c) did not use device- based measures of movement behaviors [37, 38]. We found that workers in this study could be divided into four distinct movement behavior proﬁles, that we named “Chimpan- zees”, “Lions”, “Koalas”, and “Ants”. Compared to the remaining movement behavior proﬁles, “Chimpanzees” were characterized by (a) relatively mod- erate occupational physical activity, (b) high leisure-time physical activity, and (c) slightly less time in bed. Although “Chimpanzees” had total time in physical activity similar to “Lions” and less than “Ants”, we theoretically considered this proﬁle to have the most balanced time-use composition. For instance, moderate (≈50% of the time) physical activity at work would have given “Chimpanzees” recovery opportunities and lowered the likelihood of work-related As expected, “Koalas” had clinically relevant higher obesity levels [41] compared with “Chimpanzees”. These results may be explained by their time-use proﬁle. Com- pared to “Chimpanzees”, “Koalas” were more sedentary and less physically active both at work and in leisure, and spent slightly more time in bed. This proﬁle could be considered to be the least favorable for obesity prevention because of (a) higher sedentary time and less physical Movement behavior proﬁles and obesity: a latent proﬁle analysis of 24-h time-use composition among. . . Table. Discussion Each model is adjusted for age, sex, smoking status, alcohol, poor dietary intake, and socioeconomic status; BMI body mass index, WC waist circumference, CI conﬁdence interval; “Chimpanzees” was set as a reference; results in bold are signiﬁcant at p < 0.05; β is the beta estimate that indicated estimated difference in obesity indicators between each animal proﬁle and “Chimpanzees” activity resulting in lower energy expenditure, and (b) the balance-driven approach (i.e., because of an imbalance in time spent in physical activity, sedentary behavior and sleep time [recovery] throughout a day). y g y Although not statistically signiﬁcant in our sample, “Ants” had 2.6 cm higher WC, 1.0% higher fat% and 0.4 kg/m2 higher BMI than “Chimpanzees”. We advocate similar future studies to conﬁrm these results. Theoretically, if this difference was statistically signiﬁcant, these results contradict the traditional obesity prevention approach advocating increasing total physical activity levels. Since “Ants” had overall higher total physical activity levels and consequently higher energy expenditure, this proﬁle would be considered to be the most favorable proﬁle for obesity prevention. However, this was not the case in our study. Our results rather would suggest that a predominance of physical activity and limited time to recover (by being sedentary and by spending more time asleep) might hamper homeostasis, thus not being optimal for preventing obesity. “Ants” were mostly females and thus genetically predis- posed to higher fat%. However, adjusting for gender did not change our results. This result thus supports the premise that the proﬁle of movement behaviors throughout the day is of relevance for obesity. However, as the difference between “Chimpanzees” and “Ants” was not statistically signiﬁcant, and the results are based on cross-sectional analyses, more similar research is needed to conﬁrm these ﬁndings in larger prospective cohorts. The implication of the ﬁndings The results suggest that obtaining a balance between time spent in physical activity over the main domains of the day whilst allowing for recovery (sedentary and bedtime), ought to be considered as a potential obesity prevention approach. Discussion 3 Multiple linear regression analysis of the association between the four movement behavior proﬁles and obesity indicators among 807 workers Variables Proﬁle β 95% CI p-value BMI (kg/m2) Chimpanzees Ref Lions 1.0 0.1, 2.0 0.04 Koalas 0.8 −0.3, 1.9 0.15 Ants 0.4 −0.5, 1.3 0.39 Chimpanzees Ref WC (cm) Lions 4.3 1.4, 7.2 <0.001 Koalas 3.1 0.1, 6.2 0.05 Ants 2.6 −0.2, 5.3 0.07 Chimpanzees Ref Fat (%) Lions 2.0 0.6, 3.4 0.01 Koalas 2.0 0.4, 3.7 0.02 Ants 1.0 −0.4, 2.4 0.16 Each model is adjusted for age, sex, smoking status, alcohol, poor dietary intake, and socioeconomic status; BMI body mass index, WC waist circumference, CI conﬁdence interval; “Chimpanzees” was set as a reference; results in bold are signiﬁcant at p < 0.05; β is the beta estimate that indicated estimated difference in obesity indicators between each animal proﬁle and “Chimpanzees” Table. 3 Multiple linear regression analysis of the association between the four movement behavior proﬁles and obesity indicators among 807 workers 4.3 cm higher WC, and 2.0% higher fat%. Overall, “Lions” total duration spent in LIPA and MVPA during a whole day —226 min— was in fact similar to “Chimpanzees”— 231 min. Therefore, according to the traditional obesity prevention approach, both proﬁles would be expected to have similar obesity levels. However, this was not the case. These results may rather favor the proposed balance-driven approach to prevent obesity. Compared to “Chimpanzees”, “Lions” performed more physical activity at work (~70% of the worktime), perhaps giving them less opportunity to recover at work. This may explain why they spent more time being sedentary during leisure (≈74% of waking time at leisure), and slightly more time in bed. “Lions” accrued most of their physical activity at work and recent evidence shows that work-time physical activity does not produce the same health beneﬁts as leisure-time physical activity [10, 42]. Leisure time physical activity is characterized by voluntary dynamic movements at conditioning intensity levels sufﬁcient to improve cardiorespiratory ﬁtness and metabolism, promoting obesity reduction [10]. In contrary, occupational physical activity is characterized by static load, heavy lifting, and monotonous postures, generally not con- ducted at sufﬁcient conditioning intensity and performed for longer periods with fewer recovery breaks [10], being less efﬁcient for preventing obesity. More research of detailed proﬁles based on intensity, bout durations and frequency of occupational and leisure-time movement behavior in relation to obesity are needed to conﬁrm these results. Strengths and limitations This study has several important strengths. First, time-use behaviors were derived from posture-identiﬁcation instead of using arbitrary thresholds based on counts per minute that are criticized for not accurately differentiating between sedentary and standing postures [43]. Time spent in various movement behaviors was determined using Acti4 software [21]. Acti4 has shown to identify postures and physical activities with high sensitivity (80%) and speciﬁcity (>90%) during semi-standardized and free-living conditions [21, 44]. Second, accelerometer wear-time was high in this study. The average wear time was 23.4 h (SD 1.2), unlike previous studies where participants had to remove accel- erometers during showering or swimming leading to reduced wear time [45, 46]. Third, the use of CoDA enabled the compositional nature of 24-hour time-use data to be Compared to “Chimpanzees”, “Lions” had clinically relevant higher obesity levels —1.0 kg/m2 higher BMI, N. Gupta et al. Conclusion 1. World Health Organisation. Obesity and overweight. http://www. who.int/news-room/fact-sheets/detail/obesity-and-overweight (2018). Accessed 15 Oct 2018. In conclusion, participants in this study could be grouped into four movement behavior proﬁles that we named “Chimpanzees”, “Lions”, “Koalas”, and “Ants”. “Chim- panzees” had the most balanced time-use proﬁle. Compared to “Chimpanzees”, “Lions” were highly physically active at work, more sedentary at leisure and had slightly more time in bed; “Koalas” were more sedentary at both domains and had slightly higher time in bed; “Ants” were physically active in both domains while spending similar time in bed. The movement behavior proﬁles were associated with obesity. Compared to “Chimpanzees”, “Lions” had the least favorable obesity indicators followed by “Koalas” and “Ants”. An approach of obtaining a balance between phy- sical activity stimulus and recovery at work and leisure may be promising for obesity prevention instead of only focus- ing on increasing physical activity or reducing sedentary behavior. 2. Singh GM, Danaei G, Farzadfar F, Stevens GA, Woodward M, Wormser D, et al. The age-speciﬁc quantitative effects of meta- bolic risk factors on cardiovascular diseases and diabetes: a pooled analysis. PLoS ONE. 2013;8:e65174. 3. Emerging Risk Factors Collaboration, Wormser D, Kaptoge S, Di Angelantonio E, Wood AM, Pennells L, et al. Separate and combined associations of body-mass index and abdominal adip- osity with cardiovascular disease: collaborative analysis of 58 prospective studies. Lancet. 2011;377:1085–95. 4. Must A, McKeown NM. The disease burden associated with overweight and obesity. In: De Groot LJ, Beck-Peccoz P, Chrousos G, Dungan K, Grossman A, Hershman JM, et al., edi- tors. South Dartmouth, Endotext. 2000. 5. The GBD 2015 Obesity Collaborators. Health effects of over- weight and obesity in 195 Countries over 25 Years. New Engl J Med. 2017;377:13–27. 6. Hill JO, Wyatt HR, Peters JC. Energy balance and obesity. Cir- culation. 2012;126:126–32. 7. Beccuti G, Pannain S. Sleep and obesity. Curr Opin Clin Nutr Metab Care. 2011;14:402–12. 8. Pannain S, Miller A, Van Cauter E. Sleep loss, obesity and dia- betes: prevalence, association and emerging evidence for causa- tion. Obesity and Metabolism. 2008;4:28–41. Conﬂict of interest The authors declare that they have no conﬂict of interest. Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. One limitation of the study is the cross-sectional design which cannot reveal the causality between proﬁles and obesity and thus the results of our study need to be veriﬁed using a prospective study design. Another limitation is the inability to adjust the results for total energy expenditure. In addition, we did not have an external dataset to allow us to determine the external validity of these proﬁles. Therefore, similar future studies should be conducted on other datasets to determine if these four proﬁles exist in other working populations, and if they are similarly associated with obe- sity. As this study included workers from cleaning, manu- facturing and transport sector only, our ﬁndings may be generalized to these sectors only. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons. org/licenses/by/4.0/. Code availability 9. Chaput J-P, Dutil C. Lack of sleep as a contributor to obesity in adolescents: impacts on eating and activity behaviors. Int J Behav Nutr Phys Activity. 2016;13:103. The R codes for our analyses are available upon request. 10. Holtermann A, Krause N, van der Beek AJ, Straker L. The physical activity paradox: six reasons why occupational physical activity (OPA) does not confer the cardiovascular health beneﬁts that leisure time physical activity does. Br J Sports Med. 2017;52:149–50. Acknowledgements We would like to acknowledge the Danish work environment research fund (journal number 01–2015–03) for ﬁnan- cially supporting this study and the Danish government (satspulje) for providing funds to collect DPHACTO cohort data. We also would like to thank the participants and the entire DPHACTO cohort research group at the National Research Centre for the Working Environment, Copenhagen, Denmark. We would like to thank Darren Dahly from HRB Clinical Research Facility, Cork School of Public Health, Uni- versity College Cork, Cork, Ireland for his valuable help in the sta- tistical analysis in this study. 11. Kellmann M. Underrecovery and overtraining: different concepts- similar impacts? In: Kellmann M, editor. Enhancing recovery: preventing underperformance in athletes. Human kinetics. 2002;3–24. 11. Kellmann M. Underrecovery and overtraining: different concepts- similar impacts? In: Kellmann M, editor. Enhancing recovery: preventing underperformance in athletes. Human kinetics. 2002;3–24. Compliance with ethical standards respected in the analysis. Fourth, we obtained domain- speciﬁc behavior information, as movement behaviors may contribute differently to obesity depending on the domain they occur in [20]. Finally, the study used latent proﬁling analysis which resulted in meaningfully interpretable clus- ters based on multivariate data. Conﬂict of interest The authors declare that they have no conﬂict of interest. Movement behavior proﬁles and obesity: a latent proﬁle analysis of 24-h time-use composition among. . . 12. Powers SK, Howley ET. Exercise physiology: theory and appli- cation to ﬁtness and performance. 4th ed. USA: McGraw Hill; 2001. 29. Pedisic Z. Measurement issues and poor adjustments for physical activity and sleep undermine sedentary behaviour research—the focus should shift to the balance between sleep, sedentary beha- viour, standing and activity. Kinesiology. 2014;46:135–46. 13. McEwen BS. Stress, adaptation, and disease: allostasis and allo- static load. Ann N Y Acad Sci. 2006;840:33–44. 30. Filzmoser P, Hron K, Reimann C. Interpretation of multivariate outliers for compositional data. Computers Geosci. 2012;39:77–85. 14. Virtue S, Vidal-Puig A. Adipose tissue expandability, lipotoxicity and the metabolic syndrome—an allostatic perspective. Biochim Biophys Acta. 2010;1801:338–49. 31. Berlin KS, Williams NA, Parra GR. An introduction to latent vari- able mixture modeling (part 1): overview and cross-sectional latent class and latent proﬁle analyses. J Pediatr Psychol. 2014;39:174–87. 15. Korshoj M, Krustrup P, Jespersen T, Sogaard K, Skotte JH, Holtermann A. A 24-h assessment of physical activity and cardio- respiratory ﬁtness among female hospital cleaners: a pilot study. Ergonomics. 2013;56:935–43. 32. Pawlowsky-Glahn V, Egozcue JJ, Tolosana-Delgado R. Modeling and analysis of compositional data. Chichester, UK: John Wiley & Sons; 2015. 16. Korshøj M, Lidegaard M, Krustrup P, Jørgensen MB, Søgaard K, Holtermann A. Long term effects on risk factors for cardiovas- cular disease after 12-months of aerobic exercise intervention—a worksite RCT among cleaners. PLoS ONE. 2016;11:e0158547–e. 33. Patnode CD, Lytle LA, Erickson DJ, Sirard JR, Barr-Anderson DJ, Story M. Physical activity and sedentary activity patterns among children and adolescents: a latent class analysis approach. J Phys Activity Health. 2011;8:457–67. 17. Hunt E, McKay EA, Dahly DL, Fitzgerald AP, Perry IJ. A person- centred analysis of the time-use, daily activities and health-related quality of life of Irish school-going late adolescents. Qual Life Res. 2015;24:1303–15. 34. Ferrar K, Golley R. Adolescent diet and time use clusters and associations with overweight and obesity and socioeconomic position. Health Educ Behav. 2015;42:361–9. p 35. Trilk JL, Pate RR, Pfeiffer KA, Dowda M, Addy CL, Ribisl KM, et al. A cluster analysis of physical activity and sedentary behavior patterns in middle school girls. J Adol Health. 2012;51:292–8. 18. Pedišić Ž, Dumuid D, Olds T. Integrating sleep, sedentary beha- viour, and physical activity research in the emerging ﬁeld of time- use epidemiology: deﬁnitions, concepts, statistical methods, the- oretical framework, and future directions. Kinesiology. 2017;49:252–69. 36. Ferrar K, Olds T, Maher C. More than just physical activity: time use clusters and proﬁles of Australian youth. J Sci Med Sport. 2013;16:427–32. 37. Wennman H, Kronholm E, Partonen T, Tolvanen A, Peltonen M, Vasankari T, et al. Physical activity and sleep proﬁles in Finnish men and women. BMC Public Health. 2014;14:82. 19. Jørgensen MB, Korshøj M, Lagersted-Olsen J, Villumsen M, Mortensen OS, Skotte J, et al. Physical activities at work and risk of musculoskeletal pain and its consequences: protocol for a study with objective ﬁeld measures among blue-collar workers. BMC Musculoskel Disord. 2013;14:213-2474-14-213. 38. Charreire H, Casey R, Salze P, Kesse-Guyot E, Simon C, Chaix B, et al. Leisure-time physical activity and sedentary behavior clusters and their associations with overweight in middle-aged French adults. Int J Obesity. 2010;34:1293. 20. Gupta N, Heiden M, Aadahl M, Korshoj M, Jorgensen MB, Holtermann A. What is the effect on obesity indicators from replacing prolonged sedentary time with brief sedentary bouts, standing and different types of physical activity during working days? A cross-sectional accelerometer-based study among blue- collar workers. PLoS ONE. 2016;11:e0154935. 39. Wolvers MDJ, Bussmann JBJ, Bruggeman-Everts FZ, Boerema ST, van de Schoot R, Vollenbroek-Hutten MMR. Physical beha- vior proﬁles in chronic cancer-related fatigue. Int J Behav Med. 2018;25:30–7. 21. Skotte J, Korshoj M, Kristiansen J, Hanisch C, Holtermann A. Detection of physical activity types using triaxial accelerometers. J Phys Act Health. 2014;11:76–84. 40. Swift DL, Johannsen NM, Lavie CJ, Earnest CP, Church TS. The role of exercise and physical activity in weight loss and main- tenance. Prog Cardiovasc Dis. 2014;56:441–7. 41. Eakin EG, Reeves MM, Marshall AL, Dunstan DW, Graves N, Healy GN, et al. Living Well with Diabetes: a randomized con- trolled trial of a telephone-delivered intervention for maintenance of weight loss, physical activity and glycaemic control in adults with type 2 diabetes. BMC Public Health. 2010;10:1–15. 22. Gupta N, Stordal CC, Hallman D, Korshøj M, Gomes CI, Hol- termann A. Is objectively measured sitting time associated with low back pain? A cross-sectional investigation in the NOMAD study. PLoS ONE. 2015;10:e0121159. 23. Rasmussen CL, Palarea-Albaladejo J, Bauman A, Gupta N, Nabe- Nielsen K, Jorgensen MB, et al. Does physically demanding work hinder a physically active lifestyle in low socioeconomic workers? A compositional data analysis based on accelerometer data. Int J Environ Res Public Health. 2018;15:E1306. 42. Holtermann A, Hansen JV, Burr H. S›gaard K, Sj›gaard G. The health paradox of occupational and leisure-time physical activity. Br J Sports Med. 2012;46:291–5. 43. Freedson P, Bowles HR, Troiano R, Haskell W. Assessment of physical activity using wearable monitors: recommendations for monitor calibration and use in the ﬁeld. Med Sci Sports Exerc. 2012;44(1 Suppl 1):S1–4. 24. Heinonen I, Helajärvi H, Pahkala K, Heinonen OJ, Hirvensalo M, Pälve K, et al. Sedentary behaviours and obesity in adults: the cardiovascular risk in young Finns study. BMJ Open. 2013;3: e002901. 44. Stemland I, Ingebrigtsen J, Christiansen CS, Jensen BR, Hanisch C, Skotte J, et al. Validity of the Acti4 method for detection of physical activity types in free-living settings: comparison with video analysis. Ergonomics. 2015;58:953–65. 25. Colley RC, Michaud I, Garriguet D. Reallocating time between sleep, sedentary and active behaviours: Associations with obesity and health in Canadian adults. Health Rep. 2018;29:3–13. 26. van den Boogaart GK, Tolosana-Delgado R, Bren M. Composi- tions: compositional data analysis (R package). R (version 1.40- 2). 2018. 45. Vanhelst J, Béghin L, Drumez E, Coopman S, Gottrand F. Awareness of wearing an accelerometer does not affect physical activity in youth. BMC Med Res Methodol. 2017;17:99. 27. Muthén LK, Muthén BO. Mplus user’s guide. 8th ed. Los Angeles, CA: Muthén & Muthén; 1998–2017. 46. Vanhelst J, Mikulovic J, Bui-Xuan G, Dieu O, Blondeau T, Fardy P, et al. Comparison of two ActiGraph accelerometer generations in the assessment of physical activity in free living conditions. BMC Res Notes. 2012;5:187. 28. Aitchison J. The statistical analysis of compositional data. UK: Chapman & Hall Ltd; 1986. p. 416.
https://openalex.org/W2888682661	https://europepmc.org/articles/pmc6117419?pdf=render	English	null	The Double Burdens of Mental Health Among AIDS Patients With Fully Successful Immune Restoration: A Cross-Sectional Study of Anxiety and Depression in China	Frontiers in psychiatry	2,018	cc-by	6,103	ORIGINAL RESEARCH published: 24 August 2018 doi: 10.3389/fpsyt.2018.00384 Edited by: Specialty section: This article was submitted to Public Mental Health, a section of the journal Frontiers in Psychiatry Specialty section: This article was submitted to Public Mental Health, a section of the journal Frontiers in Psychiatry Received: 02 March 2018 Accepted: 30 July 2018 Published: 24 August 2018 Methods: In this cross-sectional study, we described clinical and psychosocial variables related to depression and anxiety in 4103 HIV-infected persons. Doctors assessed anxiety and depression by asking patients whether they had experienced anxiety or depression in the prior month. Patients also self-administered the Hospital Anxiety and Depression (HAD) scale; those with score ≥8 on HAD-A/D were considered to be at high risk of anxiety or depression. Received: 02 March 2018 Accepted: 30 July 2018 Published: 24 August 2018 Received: 02 March 2018 Accepted: 30 July 2018 Published: 24 August 2018 Citation: Xiaojie Huang 1†, Kathrine Meyers 2†, Xinchao Liu 3†, Xia Li 4, Tong Zhang 1, Wei Xia 1, Jiahua Hou 1, Aixin Song 1, Haolan He 5, Chongxi Li 6, Shenghua He 5, Weiping Cai 5, Huolin Zhong 5, Chengyu Huang 7, Shuiqing Liu 8, Hui Wang 9, Xuemei Ling 10, Ping Ma 11, Rongxia Ye 12, Gang Xiao 13, Taisheng Li 3, Ding Ding 14, Kristine Yaffe 15, Hui Chen 16, Yaokai Chen 7 and Hao Wu 1* Edited by: Edited by: Jutta Lindert, University of Applied Sciences Emden Leer, Germany Edited by: Jutta Lindert, University of Applied Sciences Emden Leer, Germany Edited by: Jutta Lindert, University of Applied Sciences Emden Leer, Germany Reviewed by: Victor Lasebikan, University of Ibadan, Nigeria Costin Roventa, Spitalul Clinic Colentina, Romania Correspondence: Hao Wu whdoc@sina.com Yaokai Chen yaokaichen@hotmail.com Hui Chen chenhui@ccmu.edu.cn Reviewed by: Victor Lasebikan, University of Ibadan, Nigeria Costin Roventa, Spitalul Clinic Colentina, Romania 1 Center for Infectious Diseases, Beijing You’an Hospital, Capital Medical University, Beijing, China, 2 The Aaron Diamond AIDS Research Center, The Rockefeller University, New York, NY, United States, 3 Infectious Diseases Department, Peking Union Medical College Hospital, Beijing, China, 4 Infectious Diseases Department, Yunnan AIDS Care Center, Kunming, China, 5 Institute of Infectious Diseases, The Eighth People’s Hospital of Guangzhou, Guangzhou, China, 6 Infectious Diseases Department, The Third People’s Hospital of Kunming, Kunming, China, 7 Department of Infectious Diseases, Chongqing Infectious Disease Medical Center, Chongqing, China, 8 Department of Infectious Diseases, Guiyang Public Health Clinical Center, Guiyang, China, 9 Department of Clinical AIDS Research, The Third People’s Hospital of Shenzhen, Shenzhen, China, 10 Department of Hematology, The Third People’s Hospital of Hengyang, Hengyang, China, 11 Department of Infectious Disease, The Second Afﬁliated Hospital of Medical School of the Southeast University, Tianjin, China, 12 Department of Infectious Diseases, The Sixth People’s Hospital of Hangzhou, Hangzhou, China, 13 Department of Infectious Diseases, The First Hospital of Changsha, Changsha, China, 14 Shanghai Public Health Clinical Center, Shanghai, China, 15 Department of Psychiatric and Neurology and Department of Epidemiology and Statistics, University of California, San Francisco, San Francisco, CA, United States, 16 School of Biomedical Engineering, Capital Medical University, Beijing, China Correspondence: Hao Wu whdoc@sina.com Yaokai Chen yaokaichen@hotmail.com Hui Chen chenhui@ccmu.edu.cn †These authors have contributed equally to this work †These authors have contributed equally to this work Background: Anxiety and depression continue to be signiﬁcant comorbidities for people with HIV infection. We investigated the prevalence of and factors associated with anxiety and depression among adult HIV-infected patients across China. Background: Anxiety and depression continue to be signiﬁcant comorbidities for people with HIV infection. We investigated the prevalence of and factors associated with anxiety and depression among adult HIV-infected patients across China. INTRODUCTION treatment clinics across China. All participants provided written informed consent to complete a survey and have their medical data abstracted from their medical records. The study was approved by the Beijing You’an Hospital institutional review board. Over half of all HIV-1-infected individuals suﬀer from mental health disorders (1–3) and anxiety and depression are common comorbidities in HIV-infected populations (4–6). The impact of mental health issues on HIV patients in resource-limited settings is often underestimated due to a lack of education and awareness both among physicians and patients, resulting in insuﬃcient and ad-hoc screening and diagnosis of mental health conditions in this population (4, 7). The pathophysiology of anxiety/depression among HIV-infected patients is unclear, but may be related to clinical factors (e.g., the ability of HIV to infect the central nervous system (CNS) or impact of antiretroviral medications) or psychosocial in nature (e.g., availability of social support, issues related to substance abuse) (8–10). Recent studies have largely focused on the impact of speciﬁc antiretroviral agents (e.g., efavirenz) on mental health (11, 12). Regardless of its etiology, anxiety and depression is clinically important in this population and has the potential to impact quality of life, adherence to anti-retroviral medications, cognition, cause sleep disturbance and may weaken patients’ immune system (13–16). Each participant completed the Hospital Anxiety and Depression (HAD) scale questionnaire, which consists of seven items each relating to depression (HAD-D) and anxiety (HAD-A) respectively. High risk of anxiety and depression was deﬁned as a HAD score ≥8 on each subscale (21). Before self-administration of the HAD, clinicians asked patients if they had experienced any depressed mood or anxiety in the previous month and results were coded yes vs. no. y Demographic, behavioral, and psychosocial data were collected through self-administered survey. Clinical data was abstracted from medical records. This included HIV- speciﬁc data, including date of HIV diagnosis, history of medical conditions, current CD4+ cell counts and HIV RNA levels, and antiretroviral therapy (ART) regimen. The Pittsburgh Sleep Quality Index (PSQI) questionnaire, a 19- item questionnaire that assesses seven sleep components, was completed by each participant to evaluate sleep disturbance during the prior month. We used the PSQI cut oﬀof a global score >5 on the PSQI instrument (15) (sensitivity of 90% and speciﬁcity of 87%) to deﬁne sleep disturbance in this cohort (22). INTRODUCTION There are currently no guidelines to manage psychiatric disorders in the HIV clinic setting in China, despite the reality that these disorders adversely aﬀect the course of HIV infection (17). Major anxiety and depression frequently go unrecognized and untreated and may foster severe consequences, such as rapid disease progression, suicide, high-risk behaviors and immune system impairment (18–20). In this study, we characterized the prevalence of symptoms of anxiety and depression in Chinese HIV-infected patients, identiﬁed risk factors, and sought to assess clinical impacts of anxiety and/or depression on treatment outcomes. We also investigated the utility of the Hospital Anxiety and Depression (HAD) instrument to identify missed-diagnoses when compared to a clinician-initiated one-item question asking patients to self-report anxiety and depression in the previous month. STATISTICAL ANALYSIS Descriptive statistics are presented as means with standard deviations (SDs), or counts with proportions, as appropriate. Two sample t-tests were used to compare means, and χ2-tests were used to compare proportions. Logistic regression analysis was used to investigate associations between demographic, behavioral, psychosocial, and clinical factors and anxiety or depression. Odds ratios (ORs) for depression or anxiety were estimated with 95% conﬁdence intervals (CIs). Factors with a P-value < 0.10 in univariate Logistic models were initially included in the multivariate Logistic model and were then eliminated using backward selection. Crude ORs are reported from univariate analysis; adjusted ORs are reported from multivariable logistic regression. All P-values were 2-sided, and Citation: Huang X, Meyers K, Liu X, Li X, Zhang T, Xia W, Hou J, Song A, He H, Li C, He S, Cai W, Zhong H, Huang C, Liu S, Wang H, Ling X, Ma P, Ye R, Xiao G, Li T, Ding D, Yaffe K, Chen H, Chen Y and Wu H (2018) The Double Burdens of Mental Health Among AIDS Patients With Fully Successful Immune Restoration: A Cross-Sectional Study of Anxiety and Depression in China. Front. Psychiatry 9:384. doi: 10.3389/fpsyt.2018.00384 Results: Associations between socio-demographic, psychosocial, and ART-related clinical factors and risk of depression or anxiety were investigated using multivariable logistic regression. Among patients assessed between 9/2014 and 11/2015, 27.4% had symptoms of anxiety, 32.9% had symptoms of depression, and 19.0% had both. Recentness of HIV diagnoses (P = 0.046) was associated with elevated odds of anxiety. Older age (P = 0.004), higher educational attainment (P < 0.001), employment (P = 0.001), support from family / friends (P < 0.001), and sleep disturbance (P < 0.001), and number of ART regimen switches (P = 0.046) were associated with risk of August 2018 \| Volume 9 \| Article 384 Frontiers in Psychiatry \| www.frontiersin.org Huang et al. Mental Health Among HIV Patients depression, while neither sex nor transmission route showed any associations. There were no signiﬁcant associations with HIV-speciﬁc clinical factors including current CD4+ T cell count and current viral load. Conclusions: Prevalence of symptoms of anxiety and depression is high in this cohort of treatment-experienced HIV patients. Psychological and social-demographic factors, rather than HIV disease status, were associated with risk of depression and anxiety. This ﬁnding highlights the need to deliver interventions to address the mental health issues affecting HIV-infected persons with fully successful immune restoration across China. Keywords: HIV-1, anxiety, depression, Hospital Anxiety and Depression (HAD) scale, mental health, prevalence, risk factors Frontiers in Psychiatry \| www.frontiersin.org Keywords: HIV-1, anxiety, depression, Hospital Anxiety and Depression (HAD) scale, mental health, prevalence, risk factors Characteristics of Study Population Characteristics of Study Population 4103 HIV-infected persons were eligible for data anlaysis with the mean age being 37.6 ± 11.7 years (Table 1). Over three- quarters of the cohort were male patients. The cohort represented a diverse sample of educational achievement and employment status. Over 40% were infected through anal sex. The median time from HIV diagnosis to study enrollment was 27 months (interquartile range [IQR], 11–58 months); median duration of ART was 18 months (IQR, 6–43 months); 15.5% had a current CD4+ T cell count below 200 cells/mm3, and 40.8% were virally- suppressed. Three-quarters of patients were on ﬁrst-line ART regimen (Table 1). Over 60% had disclosed their HIV status to their family members and close to 70% expressed feeling supported by their family members. In contrast, just over one quarter had disclosed their status to a friend. METHODS We conducted a cross-sectional study among 4724 HIV-infected adults on treatment, collecting data from patients at 20 HIV August 2018 \| Volume 9 \| Article 384 Frontiers in Psychiatry \| www.frontiersin.org 2 Mental Health Among HIV Patients Huang et al. P-values < 0.05 were considered signiﬁcant. Analyses were conducted using SPSS 21.0. Prevalence of Depression and Anxiety 1,349 (32.9%) and 1,125 (27.4%) HIV-infected persons had symptoms of depression and anxiety based on the HAD-D and HAD-A, respectively, among whom 779 persons (19.0%) had symptoms consistent with both depression and anxiety. Out of 1,344 patients whose HAD-D score indicated likely depression, 715 (53.2%) did not report feeling depressed in the previous month in response to the clinician-initiated question (Table 2). Meanwhile, 578 (21.0%) of those whose HAD-D score suggested that they were not depressed responded that they had in fact experienced depressed mood in the last month. Frontiers in Psychiatry \| www.frontiersin.org DISCUSSION This study represents one of the largest epidemiologic studies of the prevalence of symptoms of anxiety and depression among HIV-infected persons in the ART era as measured by the validated and frequently-used HAD scale (22–25). Our estimates of 32.9% prevalence of depressive symptoms and 27.4% prevalence of anxiety symptoms are lower than those reported in a systematic review of studies measuring mental health burden among people living with HIV in China that reported median prevalence of depression and anxiety of 60.4 and 43.1% respectively (2). However the authors of that review caution that their data may not be generalizable due to low quality of studies and high risk of bias in the samples (2) as many of these studies were conducted among former plasma donors and injecting drug users who were infected in the ﬁrst waves of the HIV epidemic in China and may not be representative of more recently diagnosed individuals who tend to be infected through sexual contact (2). In addition, our study was conducted among patients currently accessing ART whereas earlier studies included both treated and untreated patients. A recent study of current depression among recently-diagnosed men who have sex with men (MSM) found 36% prevalence of depression, (26) in line with our ﬁnding of 34% depression prevalence among recently- diagnosed patients. People who inject drugs had the highest prevalence of depression and anxiety, in line with a rich literature documenting the comorbidities of mental health and substance use (4, 27, 28). Despite a high prevalence of anxiety and depression among HIV-infected persons, less than 5% of patients with borderline or likely depression were regularly using anti-depressants. Prior studies have also noted that most patients in China with anxiety or depression remain untreated and often have a poor understanding of available treatment options (20, 35). This is perhaps not surprising given the chronic and severe undersupply of mental health services in China (36, 37). We did not ﬁnd a relationship between high likelihood of depression and anxiety and clinical factors like immune function. However frequent switching of ART regimens was associated with higher proportion of depression, which makes sense: someone who has had trouble achieving or maintaining viral suppression is likely to experience anxiety and depression related to anticipated downward progression of their disease. Unlike in other studies (11, 19), patients on EFV-based regimens did not have elevated odds of anxiety or depression. Factors Associated With Depression and Anxiety Patient report of depression Depression diagnosis based on HAD-D≥8 Total Presence Absence Presence 629 (46.8) 578 (21.0) 1,207 Absence 715 (53.2) 2,169 (79.0) 2,884 Total 1,344 2,747 4,091 people in other contexts, including ones that could be group- based, web-based, dyadic or individual (30, 31). Age older than 30 and less education were also associated with increased odds of depression in this cohort, a trend that has been seen in other studies as well (32). In our study the length of time since HIV diagnosis was found to be associated with high anxiety symptoms, with a signiﬁcant association between shorter duration since diagnosis and anxiety. Anxiety can be considered a normal emotional response to the reality of living with HIV and this may explain the higher rate of anxiety in the ﬁrst several months after diagnosis (20). This ﬁnding is in line with that of another study that found high rates of depression in relation to duration of infection (6), suggesting that the provision of mental health services at the time of diagnosis could be critical to manage the risk of depression and anxiety among recently-diagnosed individuals. Positive-aﬀect skills interventions to support recently-diagnosed individuals have been shown to improve psychological health, decrease the use of anti-depressants, and support adjustment to HIV-positive status (33) and could be piloted in Chinese patient populations. number of ART regimens, and sleep disturbance. There were no signiﬁcant associations with HIV-speciﬁc clinical factors including current CD4+ T cell count and current viral load. In the ﬁnal multivariate model, factors associated with depression based on the HAD included age (P = 0.004), education (P < 0.001), and employment (P = 0.001), while factors associated with anxiety included time since HIV diagnosis (P = 0.046), number of ART regimen (P = 0.046), employment (P < 0.001), support from family/friends (P < 0.001), and sleep disturbance (P < 0.001) (Table 3). Consistent with other studies, we found the patients who reported high levels of support from family or friends had lower rates of symptoms of anxiety and depression (10, 34). This suggests that interventions designed to build the capacity of family and friends to support newly-diagnosed patients in the acceptance of their disease and in developing positive coping strategies could have mental health beneﬁts. Factors Associated With Depression and Anxiety In univariate analyses, factors associated with likely depression (HAD-D ≥8) or anxiety (HAD-A ≥8) included age, sex, education, transmission route, employment, support from family/friends, time of diagnosed with HIV infection, drug adherence, duration of EFV exposure, duration of ART, TABLE 1 \| Demographic, clinical, and psychosocial characteristics of 4103 HIV patients. Demographic factors Clinical factors Psychosocial factors Age, years Current CD4 cell count, cells/mm3 Family history of mental illness <30 1,175 (28.5) <50 128 (3.1) Yes 85 (2.1) 30–50 2,306 (56.2) 50–199 506 (12.3) No 4,004 (97.6) >50 569 (13.9) 200–499 2,160 (52.6) Missing 14 (0.3) Missing 53 (1.3) ≥500 1,272 (31.0) Personal history of mental illness Gender Missing 37 (0.9) Yes 65 (1.6) Male 3,204 (78.1) Current viral load No 4,026 (98.1) Female 831 (20.3) Undetectable 1,672 (40.8) Missing 12 (0.3) Missing 68 (1.7) Detectable 1,781(43.4) Current sleep medication use Education Missing 650 (15.8) Yes 236 (5.8) <High school 1,528 (37.2) Time since HIV diagnosis No 3,867 (94.2) High school 988 (24.1) <3 months 297 (7.2) Current antidepressant use >High school 1,527 (37.2) ≥3 months 374 (91.3) Yes 197 (4.8) Missing 60 (1.5) Missing 58 (1.4) No 3,884 (94.7) Transmission route Adherence to ART Missing 22 (0.5) Heterosexual 1,589 (38.9) ≥95 3,642 (88.8) HIV status disclosed to friends Homosexual/bisexual 1,730 (42.2) <95 461 (11.2) Yes 1,092 (26.6) Blood transfusion 39 (1.0) Current EFV-based regimen No 2,996(73.0) PWID 216 (5.3) Yes 2,944 (71.8) Missing 15 (0.4) Unknown 520 (12.7) No 1,159 (28.2) HIV status disclosed to family Marital status Duration of EFV exposure Yes 2,464 (60.1) Married 1,698 (41.4) 0 month 1,159 (28.2) No 1,628 (38.7) Divorced 456 (11.1) <3 months 681 (16.6) Missing 11 (0.3) Widowed 132 (3.2) ≥3 months 2,217 (54.0) Support from family/friends Single 1,718 (41.9) Missing 46 (1.1) Yes 2,803 (68.3) Missing 99 (2.4) Duration of ART No 1,239 (30.2) Employment <1 year 1,514 (36.9) Missing 61 (1.5) White collar 1,158 (28.2) ≥1 year 2,528 (61.6) Blue collar 1,835 (44.7) Missing 61 (1.5) Unemployed 280 (6.8) TABLE 1 \| Demographic, clinical, and psychosocial characteristics of 4103 HIV patients. August 2018 \| Volume 9 \| Article 384 3 Mental Health Among HIV Patients Huang et al. TABLE 2 \| Agreement of depression diagnosis based on HAD-D and self-report [n (%)]. Frontiers in Psychiatry \| www.frontiersin.org DISCUSSION However, this is likely due to a bias in the sample in that patients who had suﬀered CNS side eﬀects like anxiety/depression from EFV may have switched regimens prior to this cross-sectional study, leaving only patients who did not suﬀer EFV-related side eﬀects in the sample. Asking patients to report experience of anxiety or depression in the previous month resulted in signiﬁcant under-reporting of depression when compared to the HAD scales. Low recognition of mental health needs have been well studied in the US and Western Europe (8) and leads to under-utilization of mental health services. Fewer studies have been conducted in China, though two recent studies among rural residents Students had higher rates of both anxiety and depression, an eﬀect that remained even after controlling for age. As infections among young people and students continue to rise in China (29), it will become increasingly important to develop mental health interventions that have been shown to be eﬀective for young August 2018 \| Volume 9 \| Article 384 4 Mental Health Among HIV Patients Huang et al. Huang et al. Mental Health Among HIV Patients TABLE 3 \| Demographic and clinical factors associated with depression and anxiety. August 2018 \| Volume 9 \| Article 384 DISCUSSION Factors Depression (HAD-D≥8) Anxiety (HAD-A≥8) % of subjects Crude OR (95% CI) Adjusted OR (95% CI) % of subjects Crude OR (95% CI) Adjusted OR (95% CI) DEMOGRAPHIC AND PSYCHOSOCIAL FACTORS Age, years ‡& <30 26.3% 1 1 26.5% 1.17 (0.93–1.48) 30–50 35.6% 1.55 (1.33–1.81) 1.35 (1.13–1.62) 28.9% 1.32 (1.07–1.64) >50 34.6% 1.48 (1.20–1.84) 1.29 (1.00–1.68) 23.6% 1 Gender # Female 37.5% 1.31 (1.12–1.54) 29.6% 1.15 (0.97–1.36) Male 31.5% 1 26.7% 1 Education ‡ < High school 39.9% 1.96 (1.68–2.29) 1.89 (1.54–2.31) 27.4% 1.04 (0.88–1.22) High school 33.8% 1.51 (1.27–1.80) 1.45 (1.19–1.78) 29.0% 1.13 (0.94–1.35) > High school 25.3% 1 1 26.7% 1 Transmission route #& Heterosexual 34.7% 1 25.7% 1 Homosexual/bisexual 28.8% 0.76 (0.66–0.88) 27.7% 1.11 (0.95–1.30) Blood transfusion 35.9% 1.06 (0.54–2.05) 30.8% 1.29 (0.65–2.57) PWID 48.6% 1.78 (1.34–2.37) 35.6% 1.61 (1.19–2.17) Unknown 34.0% 0.97 (0.79–1.20) 28.1% 1.13(0.91–1.41) Marital status Married 35.3% 1 26.7% 1 Divorced 37.7% 1.11 (0.90–1.38) 31.6% 1.27 (1.01–1.59) Widowed 35.6% 1.01 (0.70–1.47) 31.1% 1.24 (0.84–1.82) Single 28.6% 0.74 (0.64–0.85) 26.8% 1.01 (0.87–1.17) Employment ‡$ White collar 25.9% 1 1 24.9% 1 1 Blue collar 33.5% 1.44 (1.22–1.69) 1.05 (0.86–1.28) 27.0% 1.12 (0.95–1.32) 1.17 (0.97–1.41) Unemployed 29.3% 1.18 (0.89–1.58) 1.02 (0.72–1.44) 23.2% 0.91 (0.67–1.24) 0.93 (0.67–1.31) Student 42.5% 2.12 (1.75–2.56) 1.49 (1.18–1.87) 33.3% 1.51(1.24–1.83) 1.57 (1.26–1.95) Support from family/friends ‡$ Yes 29.1% 1 1 24.4% 1 1 No 41.2% 1.71 (1.48–1.96) 1.80 (1.54–2.10) 33.7% 1.57 (1.36–1.82) 1.60 (1.36–1.87) CLINICAL FACTORS Current CD4 count, cells/mm3 <50 32.8% 1 22.7% 1 50–199 35.0% 1.10 (0.73–1.66) 29.4% 1.43 (0.90–2.25) 200–499 33.5% 1.03 (0.71–1.51) 28.2% 1.34 (0.88–2.05) ≥500 31.3% 0.93 (0.63–1.37) 25.9% 1.19 (0.77–1.84) Current viral load Undetectable 34.7% 1.11 (0.97–1.28) 27.8% 1.06 (0.91–1.23) Detectable 32.4% 1 26.7% 1 Time since HIV diagnosis $ <3 months 34.0% 1.05 (0.82–1.35) 34.3% 1.42 (1.10–1.82) 1.33 (1.00–1.76) ≥3 months 32.9% 1 26.9% 1 1 (Continued) DEMOGRAPHIC AND PSYCHOSOCIAL FACTORS AUTHOR CONTRIBUTIONS XH, HC, KM, and XinL led the analysis and writing of this manuscript. XH, KM, HaW, YC, KY, HC, and JH contributed to the ﬁnal version. HaW is the Principle Investigator of and designed the study. XiaL, TZ, WX, HH, CL, SH, WC, HZ, CH, SL, HuW, XuL, PM, RY, GX, TL, DD, HC, YC, AS were involved in managing the data collection. All authors reviewed and approved the ﬁnal version of the manuscript. Our study had several potential limitations. The cross- sectional study design did not allow for assessment of the temporal relationship between anxiety/depression and factors (e.g., depression improvement over time or pre- and post- ART), the direction of causality, or assessment of whether anxiety/depression were transient or chronic in nature. Among our study’s strengths is its large size and geographic diversity across 14 provinces in China and inclusion of many sub- populations. Also important was its use of validated measures to assess risk for anxiety and depression and that could easily be integrated into routine clinical practice (9, 22, 40). CLINICAL FACTORS While introducing an initial screening question could be one way to identify a proportion of people who self-identify as experiencing mental health issues and these could be referred to mental health services, the low recognition of depression and anxiety in the population suggests that relying on such a question alone would miss a large proportion of patients who are identiﬁed as at risk of depression and anxiety through the HAD screening instrument. The introduction of a standardized assessment of mental health into HIV services could be done at hospitals where patients access ART or through the Center for Disease Control system that manages follow up and could be an eﬀective way to identify patients who could beneﬁt from clinical evaluation given that these mental health issues are treatable. In summary, the high prevalence of anxiety and depression symptoms among treatment-experienced HIV-infected individuals suggests that routinely administering a standardized mental health assessment to newly-diagnosed individuals and perhaps students would be a useful ﬁrst step to identify those in need of further clinical evaluation. In order for the introduction of such an assessment to translate to better mental health outcomes, HIV clinics would need to identify optimal models for the provision of mental health interventions to their patients, whether through on-site service provision or a strong referral system to mental health professionals and other social support services. CLINICAL FACTORS August 2018 \| Volume 9 \| Article 384 Frontiers in Psychiatry \| www.frontiersin.org 5 Mental Health Among HIV Patients Huang et al. TABLE 3 \| Continued Factors Depression (HAD-D≥8) Anxiety (HAD-A≥8) % of subjects Crude OR (95% CI) Adjusted OR (95% CI) % of subjects Crude OR (95% CI) Adjusted OR (95% CI) Adherence to ART #& ≥95% 32.0% 1 26.7% 1 <95% 39.5% 1.38 (1.13–1.69) 33.4% 1.38 (1.12–1.70) Duration of EFV exposure # 0 month 37.0% 1 27.1% 1 <3 months 31.9% 0.79 (0.64–0.98) 30.5% 1.18 (0.95–1.47) ≥3months 31.2% 0.77 (0.67–0.90) 26.7% 0.98 (0.84–1.15) Duration of ART # <1 year 30.2% 1 29.0% 1 ≥1 year 34.8% 1.23 (1.08–1.41) 26.6% 1.13 (0.98–1.30) Number of ART Regimens #$ <3 32.6% 1 27.2% 1 1 ≥3 45.5% 1.72 (1.09–2.71) 37.7% 1.62 (1.01–2.57) 1.71 (1.01–2.90) Sleep disturbance ‡$ Yes 46.5% 3.01 (2.62–3.46) 3.21 (2.77–3.72) 43.6% 4.22 (3.63–4.91) 4.10 (3.51–4.78) No 22.4% 1 1 15.5% 1 1 ‡P < 0.05 in both univariate analysis and multivariate analysis for depression. #P < 0.05 in univariate analysis for depression. $P < 0.05 in both univariate analysis and multivariate analysis for anxiety. &P < 0.05 in univariate analysis for anxiet. HAD, hospital anxiety and depression; OR, odds ratio; CI, conﬁdence interval; ART, antiretroviral therapy. The bold values are adjusted OR and 95% CI after controlling several confounders. reported low recognition of depression and anxiety (38) and low awareness of where to access mental health services (39). While introducing an initial screening question could be one way to identify a proportion of people who self-identify as experiencing mental health issues and these could be referred to mental health services, the low recognition of depression and anxiety in the population suggests that relying on such a question alone would miss a large proportion of patients who are identiﬁed as at risk of depression and anxiety through the HAD screening instrument. The introduction of a standardized assessment of mental health into HIV services could be done at hospitals where patients access ART or through the Center for Disease Control system that manages follow up and could be an eﬀective way to identify patients who could beneﬁt from clinical evaluation given that these mental health issues are treatable. reported low recognition of depression and anxiety (38) and low awareness of where to access mental health services (39). Frontiers in Psychiatry \| www.frontiersin.org REFERENCES anti-retroviral therapy: a cross sectional study. PLoS ONE (2014) 9:e98962. doi: 10.1371/journal.pone.0098962 1. Whetten K, Reif S, Whetten R, and Murphy-McMillan LK. Trauma, mental health, distrust, and stigma among HIV-positive persons: implications for eﬀective care. Psychosom Med. (2008) 70:531–8. doi: 10.1097/PSY.0b013e31817749dc 1. Whetten K, Reif S, Whetten R, and Murphy-McMillan LK. Trauma, mental health, distrust, and stigma among HIV-positive persons: implications for eﬀective care. Psychosom Med. (2008) 70:531–8. doi: 10.1097/PSY.0b013e31817749dc 13. Miners A, Phillips A, Kreif N, Rodger A, Speakman A, Fisher M, et al. Health-related quality-of-life of people with HIV in the era of combination antiretroviral treatment: a cross-sectional comparison with the general population. Lancet HIV (2014) 1:e32–40. doi: 10.1016/S2352-3018(14)70018-9 2. Niu L, Luo D, Liu Y, Silenzio VM, Xiao S. The mental health of people living with HIV in China, 1998-2014: a systematic review. PLoS ONE (2016) 11:e0153489. doi: 10.1371/journal.pone.0153489 2. Niu L, Luo D, Liu Y, Silenzio VM, Xiao S. The mental health of people living with HIV in China, 1998-2014: a systematic review. PLoS ONE (2016) 11:e0153489. doi: 10.1371/journal.pone.0153489 14. Shacham E, Onen NF, Donovan MF, Rosenburg N, Overton ET. Psychiatric diagnoses among an HIV-infected outpatient clinic population. J Int Assoc Provid AIDS Care (2016) 15:126–30. doi: 10.1177/2325957414553846 3. Li J, Mo PK, Kahler CW, Lau JT, Du M, Dai Y, et al. Prevalence and associated factors of depressive and anxiety symptoms among HIV-infected men who have sex with men in China. AIDS Care (2016) 28:465–70. doi: 10.1080/09540121.2015.1118430 15. McGuire JL, Kempen JH, Localio R, Ellenberg JH, Douglas SD. Immune markers predictive of neuropsychiatric symptoms in HIV-infected youth. Clin Vaccine Immunol. (2015) 22:27–36. doi: 10.1128/CVI.004 63-14 4. Charlson FJ, Baxter AJ, Cheng HG, Shidhaye R, Whiteford HA. The burden of mental, neurological, and substance use disorders in China and India: a systematic analysis of community representative epidemiological studies. Lancet (2016) 388:376–89. doi: 10.1016/S0140-6736(16)30590-6 16. Huang X, Li H, Meyers K, Xia W, Meng Z, Li C, et al. Burden of sleep disturbances and associated risk factors: a cross-sectional survey among HIV- infected persons on antiretroviral therapy across China. Sci Rep (2017) 7:3657. doi: 10.1038/s41598-017-03968-3 5. Olagunju AT, Adeyemi JD, Ogbolu RE, Campbell EA. A study on epidemiological proﬁle of anxiety disorders among people living with HIV/AIDS in a sub-Saharan Africa HIV clinic. AIDS Behav. (2012) 16:2192–7. doi: 10.1007/s10461-012-0250-x 17. Sun W, Wu M, Qu P, Lu C, Wang L. REFERENCES Psychological well-being of people living with HIV/AIDS under the new epidemic characteristics in China and the risk factors: a population-based study. Int J Infect Dis. (2014) 28:147–52. doi: 10.1016/j.ijid.2014.07.010 6. Pappin M, Wouters E, Booysen FL. Anxiety and depression amongst patients enrolled in a public sector antiretroviral treatment programme in South Africa: a cross-sectional study. BMC Public Health (2012) 12:244. doi: 10.1186/1471-2458-12-244 18. Garey L, Bakhshaie J, Sharp C, Neighbors C, Zvolensky MJ, Gonzalez A. Anxiety, depression, and HIV symptoms among persons living with HIV/AIDS: the role of hazardous drinking. AIDS Care (2015) 27:80–5. doi: 10.1080/09540121.2014.956042 7. Ian E, Gwen CL, Soo CT, Melissa C, Chun-Kai H, Eosu K, et al. The burden of HIV-associated neurocognitive disorder (HAND) in the Asia-Paciﬁc region and recommendations for screening. Asian J Psychiatr. (2015) 22:182–9. doi: 10.1016/j.ajp.2015.10.009 19. Yehia BR, Cui W, Thompson WW, Zack MM, McKnight-Eily L, DiNenno E, et al. HIV testing among adults with mental illness in the United States. AIDS Patient Care STDS (2014) 28:628–34. doi: 10.1089/apc.2014.0196 8. Heaton RK, Franklin DR Jr, Deutsch R, Letendre S, Ellis RJ, Casaletto K, et al. Neurocognitive change in the era of HIV combination antiretroviral therapy: the longitudinal CHARTER study. Clin Infect Dis. (2015) 60:473–80. doi: 10.1093/cid/ciu862 20. Nacher M, Adriouch L, Godard Sebillotte C, Hanf M, Vantilcke V, El Guedj M, et al. Predictive factors and incidence of anxiety and depression in a cohort of HIV-positive patients in French Guiana. AIDS Care (2010) 22:1086–92. doi: 10.1080/09540121003599232 9. Heywood W, Lyons A. HIV and elevated mental health problems: diagnostic, treatment, and risk patterns for symptoms of depression, anxiety, and stress in a national community-based cohort of gay men living with HIV. AIDS Behav (2016) 20:1632–45. doi: 10.1007/s10461-016-1324-y 21. Christodoulou C, Michopoulos J, Tournikioti K, Douzenis A, Bouras G, Seretis D, et al. Hospital anxiety and depression scale. a quantitative analysis in medical outpatients, psychiatric outpatients and normal subjects. Psychiatriki (2010) 21:279–86. 10. Familiar I, Murray S, Ruisenor-Escudero H, Sikorskii A, Nakasujja N, Boivin MJ, et al. Socio-demographic correlates of depression and anxiety among female caregivers living with HIV in rural Uganda. AIDS Care (2016) 28:1–5. doi: 10.1080/09540121.2016.1191609 22. Buysse DJ, Reynolds CF III, Monk TH, Berman SR, Kupfer DJ. The pittsburgh sleep quality index: a new instrument for psychiatric practice and research. Psychiatry Res. (1989) 28:193–213. doi: 10.1016/0165-1781(89)90047-4 23. Zigmond AS, Snaith RP. The hospital anxiety and depression scale. Acta Psychiatr Scand. ACKNOWLEDGMENTS The authors thank the site leaders and other SAD study group members, Dr. Zhihao Meng (Longtan Hospital of Guangxi), FUNDING This work was supported by the Chinese Government 13th Five-Year Plan (2017ZX10201101, 2018ZX10302104-001, August 2018 \| Volume 9 \| Article 384 6 Mental Health Among HIV Patients Huang et al. 2018ZX10302104), Major Project of Beijing Municipal Science and Technology Committee (D161100000416003, D171100000517003), the National Natural Science Foundation of China (No. 81571973), the NSFC-NIH Biomedical collaborative research program (81761128001), The Capital Health Research and Development of Special Fund (2016-1-2182), and Beijing Key Laboratory (No. BZ0089). Jinsong Bai (the Third People’s Hospital of Kunming), XuL (the Third People’s Hospital of Hengyang), Daling Tan (the People’s Hospital of Luzhai), Fuxiang Wang (the First Aﬃliated Hospital of Harbin Medical University), Lianguo Ruan (Wuhan Medical Treatment Center), Hongxin Zhao (Beijing Ditan Hospital), Hongxia Wei (the Second Hospital of Nanjing Aﬃliated to Southeast University Medical College), Yanfen Liu (the Fourth People’s Hospital of Nanning), JianhuaYu (the Sixth People’s Hospital of Hangzhou), Hongzhou Lu (Shanghai Public Health Clinical Center Aﬃliated to Fudan University), Min Wang (the First People’s Hospital of Changsha), whom they acknowledge as critical in the development, implementation, and analysis of this study. REFERENCES (1983) 67:361–70. doi: 10.1111/j.1600-0447.1983.tb09716.x 11. Gaida R, Truter I, Grobler C, Kotze T, Godman B. A review of trials investigating efavirenz-induced neuropsychiatric side eﬀects and the implications. Expert Rev Anti Infect Ther. (2016) 14:377–88. doi: 10.1586/14787210.2016.1157469 24. Darko DF, McCutchan JA, Kripke DF, Gillin JC, Golshan S. Fatigue, sleep disturbance, disability, and indices of progression of HIV infection. Am J Psychiatry (1992) 149:514–20. doi: 10.1176/ajp.149.4.514 12. Kelly CM, van Oosterhout JJ, Ngwalo C, Stewart RC, Benjamin L, Robertson KR, et al. HIV associated neurocognitive disorders (HAND) in Malawian adults and eﬀect on adherence to combination 25. Di Riso D, Bobbio A, Chessa D, Lis A, Mazzeschi C. Analysis of the interplay between depression, anxiety, and psychological resources in adolescence using self-report measures. Int J Psychiatry August 2018 \| Volume 9 \| Article 384 Frontiers in Psychiatry \| www.frontiersin.org 7 Huang et al. Mental Health Among HIV Patients Clin Pract. (2014) 18:103–11. doi: 10.3109/13651501.2014.8 90227 symptoms. Women Health (2016) 57:1–15. doi: 10.1080/03630242.2016. 1157126 symptoms. Women Health (2016) 57:1–15. doi: 10.1080/03630242.2016. 1157126 26. Tao J, Vermund SH, Lu H, Ruan Y, Shepherd BE, Kipp AM, et al. Impact of depression and anxiety on initiation of antiretroviral therapy among men who have sex with men with newly diagnosed HIV infections in China. AIDS Patient Care STDs (2017) 31:96–104. doi: 10.1089/apc.2016.0214 35. Rodkjaer L, Laursen T, Balle N, Sodemann M. Depression in patients with HIV is under-diagnosed: a cross-sectional study in Denmark. HIV Med. (2010) 11:46–53. doi: 10.1111/j.1468-1293.2009.00741.x j 36. Qian J. Mental health care in China: providing services for under-treated patients. J Mental Health Policy Econom. (2012) 15:179–86. 27. Chibanda D, Benjamin L, Weiss HA, Abas M. Mental, neurological, and substance use disorders in people living with HIV/AIDS in low- and middle-income countries. J Acquir Immune Deﬁc Syndr. (2014) 67(Suppl 1):S54–67. doi: 10.1097/QAI.00000000000 00258 37. Wu P, Li LP, Jin J, Yuan XH, Liu X, Fan B, et al. Need for mental health services and service use among high school students in China. Psychiatric Serv. (2012) 63:1026–31. doi: 10.1176/appi.ps.201200090 38. Li J, Li X, Hu S, Yu Y, Yan XF, Jiang LX. ST-segment elevation myocardial infarction in the eastern urban China: from 2001 to 2011. Chin J Cardiol (2016) 44:303–8. doi: 10.3760/cma.j.issn.0253-3758. 28. Ali MM, Dean D Jr, Lipari R, Dowd WN, Aldridge AP, Novak SP. The mental health consequences of nonmedical prescription drug use among adolescents. J Ment Health Policy Econ. Frontiers in Psychiatry \| www.frontiersin.org August 2018 \| Volume 9 \| Article 384 REFERENCES (2015) 18:3–15. 39. Yu Y, Liu ZW, Hu M, Liu HM, Yang JP, Zhou L, et al. Mental health help- seeking intentions and preferences of rural Chinese adults. PLoS ONE (2015) 10:e0141889. doi: 10.1371/journal.pone.0141889 29. McLaughlin K. HIV infections are spiking among young gay Chinese. Science (2017) 355:1359. doi: 10.1126/science.355.6332.1359 30. Li HH, Holroyd E, Lau J, Li X. Stigma, subsistence, intimacy, face, ﬁlial piety, and mental health problems among newly HIV-diagnosed men who have sex with men in China. J Assoc Nurses AIDS Care (2015) 26:454–63. doi: 10.1016/j.jana.2015.02.004 40. Dejman M, Ardakani HM, Malekafzali B, Moradi G, Gouya MM, Shushtari ZJ, et al. Psychological, social, and familial problems of people living with HIV/AIDS in Iran: a qualitative study. Int J Prev Med. (2015) 6:126. doi: 10.4103/2008-7802.172540 31. Chen L, Wang L, Qiu XH, Yang XX, Qiao ZX, Yang YJ, et al. Depression among Chinese university students: prevalence and socio-demographic correlates. PLoS ONE (2013) 8:e58379. doi: 10.1371/journal.pone.0058379 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. 32. Thielke SM, Diehr P, Unutzer J. Prevalence, incidence, and persistence of major depressive symptoms in the Cardiovascular Health Study. Aging Ment Health (2010) 14:168–76. doi: 10.1080/136078609030 46537 Copyright © 2018 Huang, Meyers, Liu, Li, Zhang, Xia, Hou, Song, He, Li, He, Cai, Zhong, Huang, Liu, Wang, Ling, Ma, Ye, Xiao, Li, Ding, Yaﬀe, Chen, Chen and Wu. 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. 33. Moskowitz JT, Carrico AW, Duncan LG, Cohn MA, Cheung EO, Batchelder A, et al. Randomized controlled trial of a positive aﬀect intervention for people newly diagnosed with HIV. J Consul Clin Psychol. (2017) 85:409–23. doi: 10.1037/ccp0000188 34. Cederbaum JA, Rice E, Craddock J, Pimentel V, Beaver P. Social networks of HIV-positive women and their association with social support and depression August 2018 \| Volume 9 \| Article 384 Frontiers in Psychiatry \| www.frontiersin.org 8
https://openalex.org/W4392896535	https://www.cancersp.com/jour/article/download/233/265	Russian	null	Mitochondrial transplantation: new challenges for cancer	Ûžno-Rossijskij onkologičeskij žurnal	2,024	cc-by	8,632	4.0 Южно-Российский онкологический журнал South Russian Journal of Cancer Tом 5 № 1, 2024 Митохондриальная трансплантация – новые вызовы раку О. И. Кит, Е. М. Франциянц, А. И. Шихлярова, И. В. Нескубина༈ ФГБУ «Национальный медицинский исследовательский центр онкологии» Министерства здравоохранения Российской Федерации, г. Ростов-на-Дону, Российская Федерация ༈ neskubina.irina@mail.ru Южно-Российский онкологический журнал. 2024. Т. 5, № 1. С. 60-70 https://doi.org/10.37748/2686-9039-2024-5-1-7 https://elibrary.ru/ymkxii 3.1.6. Онкология, лучевая терапия ОБЗОР 4.0 Южно-Российский онкологический журнал South Russian Journal of Cancer Tом 5 № 1, 2024 Митохондриальная трансплантация – новые вызовы раку О. И. Кит, Е. М. Франциянц, А. И. Шихлярова, И. В. Нескубина༈ ФГБУ «Национальный медицинский исследовательский центр онкологии» Министерства здравоохранения Российской Федерации, г. Ростов-на-Дону, Российская Федерация ༈ neskubina.irina@mail.ru Южно-Российский онкологический журнал. 2024. Т. 5, № 1. С. 60-70 https://doi.org/10.37748/2686-9039-2024-5-1-7 https://elibrary.ru/ymkxii 3.1.6. Онкология, лучевая терапия ОБЗОР 4.0 Южно-Российский онкологический журнал South Russian Journal of Cancer Tом 5 № 1, 2024 Митохондриальная трансплантация – новые вызовы раку О. И. Кит, Е. М. Франциянц, А. И. Шихлярова, И. В. Нескубина༈ Южно-Российский онкологический журнал. 2024. Т. 5, № 1. С. 60-70 https://doi.org/10.37748/2686-9039-2024-5-1-7 https://elibrary.ru/ymkxii 3.1.6. Онкология, лучевая терапия ОБЗОР ациональный медицинский исследовательский центр онкологии» Министерства здравоохранения Российской Федерации, Д Р й Ф РЕЗЮМЕ В представленном обзоре обсуждаются вопросы, касающиеся уникальности митохондрий, обеспечивающих нор- мальные клеточные функции, в то же время их участие во многих патологических состояниях организма, а также анализируется существующая литература с целью разъяснения эффективности трансплантации митохондрий при лечении злокачественных заболеваний. Являясь важными и полуавтономными органеллами в клетках, они способны адаптировать свои функции к потребностям соответствующего органа. Возможность митохондрий перепрограмми- роваться важна для всех типов клеток, которые могут переключаться между состоянием покоя и пролиферацией. Вместе с тем митохондрии опухолей претерпевают адаптивные изменения для ускорения размножения опухолевых клеток в кислой и гипоксической микросреде. Согласно появляющимся данным стало известно, что митохондрии могут выходить за границы клеток, перемещаться между клетками организма. Межклеточный перенос митохондрий естественным образом происходит у людей как нормальный механизм восстановления поврежденных клеток. Выявленный физиологический митохондриальный перенос стал основой для создания современной формы транс- плантации митохондрий, включая аутологичную (изогенную), аллогенную и даже ксеногенную трансплантацию. В настоящее время экзогенные здоровые митохондрии используются для лечения некоторых карцином, включая рак молочной железы, рак поджелудочной железы и глиому. Исследование функциональной активности здоровых митохондрий привело к обнаружению и доказательству того, что женские митохондрии обладают более высокой эффективностью подавления пролиферации опухолевых клеток, чем мужские митохондрии. Вместе с тем были описаны тканеспецифические половые различия в морфологии митохондрий и окислительной способности, и лишь немногие исследования показали функциональные половые различия митохондрий при терапии. Рассмотренные в обзоре исследования показывают, что трансплантация митохондрий может быть специфически нацелена на опухоль, с предоставлением доказательств изменений в функции опухоли после введения митохондрий. Таким образом, появление интереснейших данных об уникальных функциях митохондрий свидетельствуют об очевидной необходимости митохондриальной трансплантации. Ключевые слова: митохондрии, митохондриальная терапия, митохондриальный перенос, злокачественные нов образования Для цитирования: Кит О. И., Франциянц Е. М., Шихлярова А. И., Нескубина И. В. Митохондриальная трансплантация – новые вызовы раку. Южно-Российский онкологический журнал. 2024; 5(1): 60-70. https://doi.org/10.37748/2686-9039-2024-5-1-7, https://elibrary.ru/ymkxii Для корреспонденции: Нескубина Ирина Валерьевна – к.б.н., старший научный сотрудник лаборатории изучения патогенеза злокачественных опухолей, ФГБУ «Национальный медицинский исследовательский центр онкологии» Министерства здравоохранения Российской Федерации, г. Ростов-на-Дону, Российская Федерация Адрес: 344037, Российская Федерация, г. Ростов-на-Дону, ул. 14 линия, д. 63 E-mail: neskubina.irina@mail.ru ORCID: https://orcid.org/0000-0002-7395-3086 SPIN: 3581-8531, AuthorID: 794688 ResearcherID: AAG-8731-2019 S A th ID 6507509066 Для корреспонденции: Нескубина Ирина Валерьевна – к.б.н., старший научный сотрудник лаборатории изучения патогенеза злокачественных опухолей, ФГБУ «Национальный медицинский исследовательский центр онкологии» Министерства здравоохранения Российской Федерации, г. Ростов-на-Дону, Российская Федерация Адрес: 344037, Российская Федерация, г. Ростов-на-Дону, ул. 14 линия, д. Mitochondrial transplantation: new challenges for cancer O. I. Kit, E. M. Frantsiyants, A. I. Shikhlyarova, I. V. Neskubina ༈ National Medical Research Centre for Oncology, Rostov-on-Don, Russian Federation National Medical Research Centre for Oncology, Rostov-on-Don, Russian Federation ༈ neskubina.irina@mail.ru РЕЗЮМЕ 63 E-mail: neskubina.irina@mail.ru ORCID: https://orcid.org/0000-0002-7395-3086 SPIN: 3581-8531, AuthorID: 794688 ResearcherID: AAG-8731-2019 Scopus Author ID: 6507509066 еспонденции: Нескубина Ирина Валерьевна – к.б.н., старший научный сотрудник лаборатории изучения патогенеза злокачественных , ФГБУ «Национальный медицинский исследовательский центр онкологии» Министерства здравоохранения Российской Федерации, -на-Дону, Российская Федерация Д у р ц Адрес: 344037, Российская Федерация, г. Ростов-на-Дону, ул. 14 линия, д. 63 E il k bi i i @ il Финансирование: финансирование данной работы не проводилось Конфликт интересов: Кит О. И. является членом редакционной коллегии журнала «Южно-Российский онкологический журнал» с 2019 г., но не имеет никакого отношения к решению опубликовать эту статью. Статья прошла принятую в журнале процедуру рецензирования. Об иных конфликтах интересов авторы не заявляли Статья поступила в редакцию 28.02.2023; одобрена после рецензирования 12.08.2023; принята к публикации 27.02.2024 © Кит О. И., Франциянц Е. М., Шихлярова А. И., Нескубина И. В., 2024 60 South Russian Journal of Cancer. 2024. Vol. 5, No. 1. P. 60-70 https://doi.org/10.37748/2686-9039-2024-5-1-7 https://elibrary.ru/ymkxii REVIEW ABSTRACT (Biol.), senior researcher at the laboratory for the study of the pathogenesis of malignant tumors, National Medical Research Centre for Oncology, Rostov-on-Don, Russian Federation Address: 63 14 line str., Rostov-on-Don 344037, Russian Federation E-mail: neskubina.irina@mail.ru ORCID: https://orcid.org/0000-0002-7395-3086 SPIN: 3581-8531, AuthorID: 794688 ResearcherID: AAG-8731-2019 Scopus Author ID: 6507509066 ABSTRACT This review discusses the uniqueness of mitochondria providing normal cellular functions and at the same time involved in many pathological conditions, and also analyzes the scientific literature to clarify the effectiveness of mitochondrial transplan- tation in cancer treatment. Being important and semi-autonomous organelles in cells, they are able to adapt their functions to the needs of the corresponding organ. The ability of mitochondria to reprogram is important for all cell types that can switch between resting and proliferation. At the same time, tumor mitochondria undergo adaptive changes to accelerate the reproduc- tion of tumor cells in an acidic and hypoxic microenvironment. According to emerging data, mitochondria can go beyond the boundaries of cells and move between the cells of the body. Intercellular transfer of mitochondria occurs naturally in humans as a normal mechanism for repairing damaged cells. The revealed physiological mitochondrial transfer has become the basis for a modern form of mitochondrial transplantation, including autologous (isogenic), allogeneic, and even xenogenic trans- plantation. Currently, exogenous healthy mitochondria are used in treatment of several carcinomas, including breast cancer, pancreatic cancer, and glioma. Investigation of the functional activity of healthy mitochondria demonstrated and confirmed the fact that female mitochondria are more efficient in suppressing tumor cell proliferation than male mitochondria. However, tissue-specific sex differences in mitochondrial morphology and oxidative capacity were described, and few studies showed functional sex differences in mitochondria during therapy. The reviewed studies report that mitochondrial transplantation can be specifically targeted to a tumor, providing evidence for changes in tumor function after mitochondrial administration. Thus, the appearance of the most interesting data on the unique functions of mitochondria indicates the obvious need for mitochondrial transplantation. Keywords: mitochondria, mitochondrial therapy, mitochondrial transfer, malignant tumors For citation: Kit O. I., Frantsiyants E. M., Shikhlyarova A. I., Neskubina I. V. Mitochondrial transplantation: New challenges for cancer. South Russian Journal of Cancer. 2024; 5(1): 60-70. (In Russ.). https://doi.org/10.37748/2686-9039-2024-5-1-7, https://elibrary.ru/ymkxii For correspondence: Irina V. Neskubina – Cand. Sci. (Biol.), senior researcher at the laboratory for the study of the pathogenesis of m National Medical Research Centre for Oncology, Rostov-on-Don, Russian Federation Address: 63 14 line str Rostov on Don 344037 Russian Federation For correspondence: Irina V. Neskubina – Cand. Sci. For citation: Kit O. I., Frantsiyants E. M., Shikhlyarova A. I., Neskubina I. V. Mitochondrial transplantation: New challenges for cancer. South Russian Journal of Cancer. 2024; 5(1): 60-70. (In Russ.). https://doi.org/10.37748/2686-9039-2024-5-1-7, https://elibrary.ru/ymkxii ВВЕДЕНИЕ для конкретных органов и тканей. Например, мито- хондрии в печени в основном участвуют в биосин- тетических функциях, а митохондрии в сердце или мышцах в основном осуществляют продукцию аде- нозинтрифосфорная кислота (АТФ). Митохондрии в адипоцитах играют решающую роль в регуляции дифференцировки адипоцитов, чувствительности к инсулину и адаптивного термогенеза [12]. Ана- лиз митохондриального протеома, выделенного из различных тканей, таких как мозг, печень, сердце и почки крыс, показал митохондриальную гетеро- генность, специализирующуюся на различных функ- циях между тканями. Аномалии в митохондриях на- рушают основные физиологические функции, такие как производство АТФ, окислительное фосфорили- рование, производство активных форм кислорода (АФК) и регуляция Ca2+, все это считается митохон- дриальной дисфункцией. Кроме того, эти уникаль- ные органеллы, имеющие важное значение для нормальной клеточной функции, могут участвовать во многих патологических состояниях. Митохон- дрии присутствуют в каждой клетке человеческого организма, за исключением красных кровяных те- лец – эритроцитов. Выработка АТФ митохондриями приводит к образованию небольшого количества потенциально разрушительных свободных радика- лов, известных как АФК. Эти радикалы являются вторичными мессенджерами в жизненно важных клеточных сигнальных каскадах для нормальных биологических процессов. Однако накопление по- бочных продуктов производства АТФ может нанести вред клетке и спровоцировать повреждение клеточ- ных органелл, а также нарушение метаболических процессов [13]. Митохондрии сыграли фундаментальную роль в эволюции сложных организмов. Являясь важ- ными и полуавтономными органеллами в клетках, они способны адаптировать свои функции к потреб- ностям соответствующего органа. Митохондрии могут перепрограммировать свое целевое назна- чение на требуемый результат: на исключитель- ное снабжение энергией для поддержания работы клеток сердечной мышцы в течение всей жизни или контролировать обменные процессы в секре- тирующих органах, например поддерживать работу гепатоцитов и печени. Возможность митохондрий перепрограммироваться важна для всех типов кле- ток, которые могут переключаться между состоя- нием покоя и пролиферацией, таких как стволовые клетки и иммунные клетки. Большинство хрониче- ских заболеваний характеризуются нарушением регуляции митохондрий, что выявлено при сер- дечно-сосудистых заболеваниях, метаболическом синдроме, нейродегенеративных заболеваниях, нарушениях иммунной системы и злокачественных новообразованиях [1–7]. Целью данного обзора явилась оценка новых возможностей в терапии злокачественных новооб- разований при митохондриальной трансплантации. Funding: this work was not funded Funding: this work was not funded Conflict of interest: Kit O. I. has been the member of the editorial board of the South Russian Journal of Cancer since 2019, however he has no relation to the decision made upon publishing this article. The article has passed the review procedure accepted in the journal. The authors did not declare any other conflicts of interest The article was submitted 28.02.2023; approved after reviewing 12.08.2023; accepted for publication 27.02.2024 61 South Russian Journal of Cancer 2024. Vol. 5, No. 1. P. 60-70 Kit O. I., Frantsiyants E. M., Shikhlyarova A. I., Neskubina I. V.༈ Mitochondrial transplantation: new challenges for cancer South Russian Journal of Cancer 2024. Vol. 5, No. 1. P. 60-70 South Russian Journal of Cancer 2024. Vol. 5, No. 1. P. 60-70 Kit O. I., Frantsiyants E. M., Shikhlyarova A. I., Neskubina I. V.༈ Mitochondrial transplantation: new challenges for cancer Функциональная и дисфункциональная множественность митохондрий Злокачественные опухоли неизменно перестраи- вают свой метаболизм, способствуя клеточной пла- стичности с адаптацией к постоянно меняющейся доступности питательных веществ и приобретению черт агрессивного заболевания, включая способ- ность к метастазированию. Метаболизм рака дол- гое время приравнивался к преимущественному использованию гликолиза опухолевыми клетками даже при наличии кислорода, так называемому эффекту Варбурга [8]. Однако теперь известно, что функции митохондрий в метаболизме опухоли более широкие: использование окислительной биоэнерге- тики, изменение окислительно-восстановительного баланса, включение множественных механизмов выживания клеток и ретроградной экспрессии ядер- ных генов, а также влияние на первичное и мета- статическое распространение злокачественной опухоли [9–11]. Интересно, что точно так же, как и дифференцированные клетки, митохондрии вы- полняют специализированные функции, уникальные Очевидно, что митохондрии – это важнейшие ор- ганеллы, отвечающие за выживание клеток и апо- птоз. Здоровые митохондрии необходимы для под- держания нормального функционирования клеток. Вместе с тем накопленные данные исследований указывает на то, что митохондрии опухолей пре- терпевают адаптивные изменения для ускорения размножение опухолевых клеток в кислой и гип- оксической микросреде [14]. Все больше данных свидетельствует о том, что метаболизм и функции митохондрий незаменимы при онкогенезе и про- грессировании рака, а это делает митохондрии и их функции вероятными мишенями для противоопу- холевой терапии [15]. Хотя механизмы митохондриального перепро- граммирования при раке в последнее время полу- 62 Южно-Российский онкологический журнал 2024. Т. 5, № 1. С. 60-70 Кит О. И., Франциянц Е. М., Шихлярова А. И., Нескубина И. В.༈ Митохондриальная трансплантация – новые вызовы раку Южно-Российский онкологический журнал 2024. Т. 5, № 1. С. 60-70 Кит О. И., Франциянц Е. М., Шихлярова А. И., Нескубина И. В.༈ Митохондриальная трансплантация – новые вызовы раку митохондрий и митохондриального генома» [22]. Митохондриальный межклеточный перенос спо- собствует интеграции митохондрий в эндоген- ную митохондриальную сеть клеток-реципиентов, способствуя изменению их биоэнергетического статуса и других функциональных свойств клеток- реципиентов не только in vitro, но и in vivo. Более того, трансклеточный перенос митохондриальных генов может иметь серьезные последствия в пато- физиологии митохондриальной дисфункции [23]. чили более пристальное внимание, роль приспо- собленности органелл в этом процессе широко не рассматривалась [16, 17]. Фактически, микросреда, в которой растет опухоль, крайне неблагоприятна для митохондрий, поскольку неустойчивые кон- центрации кислорода и окислительные радикалы могут нарушить целостность органелл, дезинтегри- ровать регулирование множества функций митохон- дрий и активировать гибель клеток [18]. Поэтому то, как митохондрии справляются с потерей своей «функциональной формы», остается еще не поня- тым, и влияние некачественных или поврежденных митохондрий на признаки опухоли не изучено [19]. Функциональная и дисфункциональная множественность митохондрий Сообщалось, что межклеточный перенос мито- хондрий естественным образом происходит у лю- дей как нормальный механизм восстановления поврежденных клеток [24, 25]. Это физиологическое явление вдохновило исследователей для создания современной формы трансплантации митохондрий, включая аутологичную (изогенную), аллогенную и даже ксеногенную трансплантацию [4, 26, 27]. Учи- тывая, что митохондриальная дисфункция может быть в центре разрушительных патологических состояний, перенос митохондрий, называемый трансплантацией митохондрий, обладает высо- ким терапевтическим потенциалом в современ- ной медицине. Движение митохондрий как основа для митохондриальной терапии В другом исследова- нии также было замечено, что ксеногенный перенос митохондрий, выделенных из ткани печени мыши, в клетки человека, лишенные функциональных митохондрий (клетки ρ 0), восстанавливает функ- цию дыхания [32]. Эти результаты доказывают воз- можность лечения митохондриальных заболеваний с помощью митохондриальной трансплантации. Движение митохондрий как основа для митохондриальной терапии P. 60-70 Kit O. I., Frantsiyants E. M., Shikhlyarova A. I., Neskubina I. V.༈ Mitochondrial transplantation: new challenges for cancer South Russian Journal of Cancer 2024. Vol. 5, No. 1. P. 60-70 Kit O. I., Frantsiyants E. M., Shikhlyarova A. I., Neskubina I. V.༈ Mitochondrial transplantation: new challenges for cancer стая доставка митохондрий в кровеносные сосуды выполнялись для лечения острого повреждения почек или повреждения легких [37]. Недавнее ис- следование показало существование в перифери- ческой крови человека неповрежденных и функцио- нальных митохондрий [26]. Более того, существует много доказательств того, что в крови имеется мно- жество митохондриальных компонентов, таких как бесклеточная циркулирующая мтДНК, везикулы митохондриального происхождения и пептиды митохондриального происхождения, и эти компо- ненты увеличиваются при заболевании [38–40]. Хотя значение их присутствия в крови и их связь с заболеванием неясны, присутствие этих компо- нентов демонстрирует, что митохондрии могут иг- рать регулирующую сигнал роль через циркуляцию в отдаленных клетках, даже если они фрагменти- рованы. Соответственно, внутрисосудистое введе- ние митохондрий может быть многообещающим, если заранее понять существование митохондрий в крови, биологическую роль митохондриальных компонентов. доказали, что митохондриальный перенос между клетками часто запускается множественными внутриклеточными и внеклеточными событиями клетки-реципиента. Эти события могут действовать как сигналы «найди меня» или «спаси меня», рекру- тируя соответствующие донорские митохондрии для предоставления их клеткам-реципиентам [13]. доказали, что митохондриальный перенос между клетками часто запускается множественными внутриклеточными и внеклеточными событиями клетки-реципиента. Эти события могут действовать как сигналы «найди меня» или «спаси меня», рекру- тируя соответствующие донорские митохондрии для предоставления их клеткам-реципиентам [13]. Несколько исследований in vitro показали, что межклеточный перенос митохондрий происходит естественным образом. Когда DsRed-меченные митохондрии, выделенные из мезенхимальных клеток (EMC), происходящих из эндометриальных желез матки человека, были совместно инкубиро- ваны с изогенными EMC в течение 24 часов, с помо- щью визуализации живых флуоресцентных клеток наблюдали накопление экзогенных митохондрий в цитоплазме реципиентов [31]. В другом исследова- нии также было замечено, что ксеногенный перенос митохондрий, выделенных из ткани печени мыши, в клетки человека, лишенные функциональных митохондрий (клетки ρ 0), восстанавливает функ- цию дыхания [32]. Эти результаты доказывают воз- можность лечения митохондриальных заболеваний с помощью митохондриальной трансплантации. Несколько исследований in vitro показали, что межклеточный перенос митохондрий происходит естественным образом. Когда DsRed-меченные митохондрии, выделенные из мезенхимальных клеток (EMC), происходящих из эндометриальных желез матки человека, были совместно инкубиро- ваны с изогенными EMC в течение 24 часов, с помо- щью визуализации живых флуоресцентных клеток наблюдали накопление экзогенных митохондрий в цитоплазме реципиентов [31]. Движение митохондрий как основа для митохондриальной терапии Эндосимбиотическая теория предполагает, что митохондрии когда‑то были первичными свободно- живущими одноклеточными организмами, которые, возможно, были поглощены более крупными, веро- ятно, анаэробными клеточными организмами, что- бы использовать их для более эффективного аэроб- ного производства энергии [20]. Это «усыновление» и миллиарды лет эволюции привели к усложнению эукариот. Доказательством этой теории является то, что митохондрии содержат свою собственную ДНК (мтДНК) в форме кольцевой ДНК, аналогичной той, что обнаружена в бактериях, а также содержит два липидных бислоя. Митохондрии, как и бакте- рии, оснащены внутриклеточным механизмом, необходимым для производства 13 собственных митохондриальных белков, но при этом используют ядерную ДНК для производства других ключевых белков. Именно благодаря этому эндосимбиотиче- скому происхождению возможна интернализация митохондрий клетками-реципиентами [21]. Митохондриальная трансплантация – это инно- вационная стратегия лечения митохондриальной дисфункции, позволяющая преодолеть ограниче- ния терапии с использованием агентов. Замена, трансплантация или перенос митохондрий – это новое вмешательство и лечение для пациентов с диагнозом митохондриальное заболевание [28]. Митохондриальный перенос основан на концеп- ции таргетной терапии тРНК. Стратегии лечения митохондриальной дисфункции обычно делятся на следующие категории: усиление митохондриаль- ного биогенеза; уменьшение дисфункциональных митохондрий и замена их активными; доставка или замена дисфункциональных компонентов; вмешательство в последствия митохондриальной дисфункции и перепрограммирование митохондри- ального генома [29, 30]. Считается, что митохон- дрии сохраняются в клетках на протяжении всей их жизни. Предпосылкой для митохондриального переноса является то, что клетка может воспри- нимать множество различных сигналов окружаю- щей среды и впоследствии осуществлять поглоще- ние, перенос, обработку и интеграцию чужеродного материала. Какие сигналы запускают митохон- дриальный перенос имеет большое значение для дальнейшей теории и лечения. Текущие данные Появляющиеся данные показывают, что мито- хондрии могут выходить за границы клеток, пе- ремещаться между клетками млекопитающих, радикально бросая вызов известным до сих пор концепциям внутриклеточной сегрегации митохон- дрий и наследования митохондриальной ДНК – мтДНК. Их сигнальная роль может распространять- ся на межклеточную коммуникацию, показывая, что митохондриальный геном и даже целые ми- тохондрии действительно мобильны и могут опо- средовать передачу информации между клетками. Этот недавно открытый процесс мобильного пере- носа митохондрий и мтДНК был назван «момио- мой», чтобы обозначить все «мобильные функции 63 South Russian Journal of Cancer 2024. Vol. 5, No. 1. P. 60-70 Kit O. I., Frantsiyants E. M., Shikhlyarova A. I., Neskubina I. V.༈ Mitochondrial transplantation: new challenges for cancer South Russian Journal of Cancer 2024. Vol. 5, No. 1. P. 60-70 Kit O. I., Frantsiyants E. M., Shikhlyarova A. I., Neskubina I. V.༈ Mitochondrial transplantation: new challenges for cancer South Russian Journal of Cancer 2024. Vol. 5, No. 1. Дисфункциональное доминирование злокачественных митохондрий и возможность противодействия Поэтому в этом плане трансплантация гетеро- логичных митохондрий неизбежна [53]. Основными возможными проблемами трансплантации гетеро- генных митохондрий являются реакции иммунной системы и молекулярный паттерн, связанный с по- вреждением (DAMP). Следует отметить, что во всех предыдущих исследованиях сообщалось только об одной инъекции митохондрий. А что произойдет по- сле серии инъекций митохондрий в поврежденные ткани? McCully J. D. и соавт. (2017) провели исследо- вание, чтобы узнать поведение иммунной системы после прямых или непрямых аутогенных и аллоген- ных инъекций, однократных и серийных инъекций, а также различного количества изолированных митохондрий (1 × 105, 1 × 106 или 1 × 107 митохон- дрий). Полученные данные показали, что уровень профилей иммунной системы, включая IL‑1, IL‑4, IL‑6, IL‑12, IL‑18, IP‑10, макрофагальный воспалительный белок MIP‑1 α и MIP‑1 β не изменился. Единичные или серийные инъекции митохондрий не показали наличия DAMP в тканях реципиента [54]. Ramirez- Barbieri G. и соавт. (2019) исследовали иммунный ответ и связанные с повреждением молекулярные паттерны (DAMPs) у мышей после однократных или многократных внутрибрюшинных инъекций алло- Накапливающиеся данные исследований пока- зывают, что митохондрии опухоли претерпевают адаптивные изменения для ускорения быстрой пролиферации опухолевых клеток в кислой и гип- оксической микросреде [14]. Таким образом, пред- полагается, что введение здоровых митохондрий в опухолевые клетки обладает высокой эффектив- ностью в предотвращении роста опухоли [44]. В на- стоящее время экзогенные здоровые митохондрии используются для лечения нескольких карцином, включая рак молочной железы, рак поджелудоч- ной железы и глиому, и была показана отличная противоопухолевая эффективность здоровых митохондрий [45–47]. При этом авторы на основе полученных биохимических данных отмечали факт того, что здоровые митохондрии после митохондри- альной трансплантации могут значительно снижать способность к окислительному фосфорилированию (OXPHOS) и индуцировать апоптоз в опухолевых клетках. Однако молекулярный сигнальный меха- низм этого процесса остается не ясен. Дисфункциональное доминирование злокачественных митохондрий и возможность противодействия Понимание их участия в эффективности МТ было бы ценным для снижения возможных рисков. При существующем митохондриальном заболевании возможна трансплантация митохондрий, получен- ных из аутологичных клеток, без воспаления и ауто- иммунных реакций [52]. Некоторые исследователи полагают, что аутологичная трансплантация мито- хондрий может иметь более эффективные резуль- таты. Однако в ряде случаев, включая заболевания, связанные с митохондриями, или у некоторых наи- более тяжелых пациентов выделение собственных митохондрий невозможно. С другой стороны, неко- торым пациентам требуется несколько серий инъек- ций. Поэтому в этом плане трансплантация гетеро- логичных митохондрий неизбежна [53]. Основными возможными проблемами трансплантации гетеро- генных митохондрий являются реакции иммунной системы и молекулярный паттерн, связанный с по- вреждением (DAMP). Следует отметить, что во всех предыдущих исследованиях сообщалось только об одной инъекции митохондрий. А что произойдет по- сле серии инъекций митохондрий в поврежденные ткани? McCully J. D. и соавт. (2017) провели исследо- вание, чтобы узнать поведение иммунной системы после прямых или непрямых аутогенных и аллоген- ных инъекций, однократных и серийных инъекций, а также различного количества изолированных митохондрий (1 × 105, 1 × 106 или 1 × 107 митохон- дрий). Полученные данные показали, что уровень профилей иммунной системы, включая IL‑1, IL‑4, IL‑6, IL‑12 IL‑18 IP‑10 макрофагальный воспалительный хондрии рассматриваются как системные посред- ники в межклеточных коммуникациях [49]. Известно также, что митохондрии могут быть поглощены различными типами клеток, как было показано в исследованиях in vitro и in vivo [50]. Кроме того, митохондрии в крови могут активировать иммунную систему за счет увеличения активности фагоцитов и Т-клеток, что может в определенной степени уси- лить противоопухолевый эффект митохондрии [51]. дрии из клеток MCF‑12A также могут быть пере- несены в клеточные линии MCF‑7 рака молочной железы человека, что сопровождается повышенной чувствительностью к химиотерапии доксорубици- ном, абраксаном или карбоплатином [43]. Это пер- вая публикация, касающаяся переноса митохон- дрий, способствующих апоптозу злокачественных клеток и повышению чувствительности к лекар- ственным средствам. р у фф др [ ] На сегодняшний день в некоторых исследованиях обсуждаются иммунные реакции, происходящие во время митохондриальной трансплантации – МТ. Понимание их участия в эффективности МТ было бы ценным для снижения возможных рисков. При существующем митохондриальном заболевании возможна трансплантация митохондрий, получен- ных из аутологичных клеток, без воспаления и ауто- иммунных реакций [52]. Некоторые исследователи полагают, что аутологичная трансплантация мито- хондрий может иметь более эффективные резуль- таты. Однако в ряде случаев, включая заболевания, связанные с митохондриями, или у некоторых наи- более тяжелых пациентов выделение собственных митохондрий невозможно. С другой стороны, неко- торым пациентам требуется несколько серий инъек- ций. Дисфункциональное доминирование злокачественных митохондрий и возможность противодействия В дополнение к наблюдаемому переносу мито- хондрий в экспериментах in vitro представляется актуальным возможность введения митохондрий непосредственно в живые организмы. Митохон- дрии, используемые для инъекций, могут быть аутологичными, аллогенными или даже ксеноген- ными. Doulamis I. P. и соавт. вводили аллогенные или аутологичные митохондрии мышечных клеток в поврежденные участки сердца крыс с диабетом, оба варианта митохондрий приводили к восстанов- лению функции левого желудочка и уменьшению размера инфаркта [33]. Митохондрии можно вво- дить непосредственно в поврежденную область или в другое место. Например, Lin H. S. и соавт. вво- дили митохондрии в селезенку для лечения ишеми- чески поврежденной печени [34]. Кроме того, в про- шлом исследователи чаще вводили митохондрии непосредственно в региональную ишемическую зону для восстановления повреждения миокарда, а недавно решили вводить митохондрии в левое коронарное устье или коронарную артерию [33, 35]. Локальная внутримозговая или системная внутри- артериальная инъекция митохондрий может зна- чительно восстановить область инфаркта мозга и гибель нейрональных клеток [36]. Кроме того, внутриартериальная инъекция или внутрисосуди- Митохондрии злокачественных клеток играют ключевую роль во взаимодействии опухолевых клеток с микроокружением опухоли [41]. Как по- казали недавние научные исследования, опухоли состоят не только из злокачественных клеток, они представляют собой сложную систему опухолевых и неопухолевых клеток, которые создают симбио- тические отношения в микроокружении опухоли, способствуя выживанию и устойчивости к химио- терапии. Злокачественные клетки способны вы- теснять целые митохондрии или некоторые из их компонентов, включая мтДНК, цитохром С и фор- милированные пептиды в микроокружение опу- холи [42]. Они, в свою очередь, функционируют как ассоциированные с повреждением молекулярные паттерны (DAMP), высвобождающиеся из повре- жденных или «умирающих» клеток и активируют врожденную иммунную систему. Elliott R. L. и соавт. (2012) установили, что мито- хондрии, очищенные от иммортализованных, не- трансформированных клеток эпителия молочной железы MCF‑12A, могут успешно проникать в кле- точные линии рака молочной железы человека и подавлять их в зависимости от дозы. Митохон- 64 Южно-Российский онкологический журнал 2024. Т. 5, № 1. С. 60-70 Кит О. И., Франциянц Е. М., Шихлярова А. И., Нескубина И. В.༈ Митохондриальная трансплантация – новые вызовы раку хондрии рассматриваются как системные посред- ники в межклеточных коммуникациях [49]. Известно также, что митохондрии могут быть поглощены различными типами клеток, как было показано в исследованиях in vitro и in vivo [50]. Кроме того, митохондрии в крови могут активировать иммунную систему за счет увеличения активности фагоцитов и Т-клеток, что может в определенной степени уси- лить противоопухолевый эффект митохондрии [51]. На сегодняшний день в некоторых исследованиях обсуждаются иммунные реакции, происходящие во время митохондриальной трансплантации – МТ. Особенности митохондрий, связанные с полом др , Митохондрии являются почти исключительным наследием матери в эволюции, и при транспланта- ционной терапии могут возникнуть половые раз- личия в функционировании митохондрий. Ранее сообщалось, что митохондрии самок животных (женские митохондрии) более чувствительны к стрессу и лучше приспособлены для борьбы с неблагоприятными состояниями, поэтому предпо- лагалось, что женские митохондрии обладают раз- личной активностью в противоопухолевом росте по сравнению с митохондриями самцов [56]. Механизм митохондриального проникновения, иммунные реакции Межклеточный перенос митохондрий проис- ходит посредством туннельных нанотрубок (ТНТ), внеклеточных везикул (ВВ) и слияния клеток. Недавно в крови и кондиционированной среде для культивирования клеток наблюдали свобод- ные от клеток и цитоплазматической мембраны функционально активные митохондрии [48]. Хотя роль внеклеточных митохондрий в межклеточной коммуникации еще предстоит полностью понять, практические подходы, направленные на перенос интактных митохондрий в клетки-мишени, были разработаны ранее. Механизм проникновения митохондрий в клет- ки может быть связан с эндоцитозом, опосредо- ванным макропиноцитозом, поскольку ингибитор макропиноцитоза может предотвращать интерна- лизацию митохондрий клетками. Более того, мито- 65 South Russian Journal of Cancer 2024. Vol. 5, No. 1. P. 60-70 Kit O. I., Frantsiyants E. M., Shikhlyarova A. I., Neskubina I. V.༈ Mitochondrial transplantation: new challenges for cancer South Russian Journal of Cancer 2024. Vol. 5, No. 1. P. 60-70 Kit O. I., Frantsiyants E. M., Shikhlyarova A. I., Neskubina I. V.༈ Mitochondrial transplantation: new challenges for cancer South Russian Journal of Cancer 2024. Vol. 5, No. 1. P. 60-70 Kit O. I., Frantsiyants E. M., Shikhlyarova A. I., Neskubina I. V.༈ Mitochondrial transplantation: new challenges for cancer South Russian Journal of Cancer 2024. Vol. 5, No. 1. P. 60-70 Kit O. I., Frantsiyants E. M., Shikhlyarova A. I., Neskubina I. V.༈ Mitochondrial transplantation: new challenges for cancer генных митохондрий и обнаружили, что уровни цито- кинов и мтДНК в сыворотке не увеличивались ни после аутологичной, ни после аллогенной митохон- дриальной инъекции [55]. дование продемонстрировало новое понимание функции митохондрий при развитии меланомы и предполагает, что здоровые митохондрии инги- бируют пролиферацию опухолевых клеток, пред- отвращая транскрипцию опухолевых генов. Общая понижающая регуляция генов приводит к остановке клеточного цикла и застою клеточной пролифера- ции, а также к активации аутофагии и апоптоза, что в конечном итоге приводит к очевидному ингибиро- ванию роста меланомы после митохондриальной трансплантационной терапии [59]. ЗАКЛЮЧЕНИЕ 5, № 1. С. 60-70 Кит О. И., Франциянц Е. М., Шихлярова А. И., Нескубина И. В.༈ Митохондриальная трансплантация – новые вызовы раку Поскольку митохондрии легко получить из культивируемых клеток, а технология выделения и сохранения митохондрий становится все более зрелой, ожидается, что в будущем будут созданы крупномасштабные центры донорства митохон- дрий. Таким образом, когда аутологичная транс- плантация не может быть выполнена, можно во- время найти подходящего донора митохондрий. того, в настоящее время не существует метода дли- тельного хранения митохондрий, поэтому их следует использовать сразу после выделения. Следова- тельно, протокол оптимального метода выделения и хранения митохондрий, который поддерживает их целостность и обеспечивает более длительную выживаемость, должен быть разработан для обес- печения возможности клинического использования. того, в настоящее время не существует метода дли- тельного хранения митохондрий, поэтому их следует использовать сразу после выделения. Следова- тельно, протокол оптимального метода выделения и хранения митохондрий, который поддерживает их целостность и обеспечивает более длительную выживаемость, должен быть разработан для обес- печения возможности клинического использования. Список источников Hayes JD, Dinkova-Kostova AT, Tew KD. Oxidative Stress in Cancer. Cancer Cell. 2020 Aug 10;38(2):167–197. https://doi.org/10.1016/j.ccell.2020.06.001 10. Hayes JD, Dinkova-Kostova AT, Tew KD. Oxidative Stress in Cancer. Cancer Cell. 2020 Aug 10;38(2):167–197. https://doi.org/10.1016/j.ccell.2020.06.001 11. Martinez-Reyes I, Cardona LR, Kong H, Vasan K, McElroy GS, Werner M, et al. Mitochondrial ubiquinol oxidation is neces- sary for tumour growth. Nature. 2020 Sep;585(7824):288–292. https://doi.org/10.1038/s41586-020-2475-6 11. Martinez-Reyes I, Cardona LR, Kong H, Vasan K, McElroy GS, Werner M, et al. Mitochondrial ubiquinol oxidation is neces- sary for tumour growth. Nature. 2020 Sep;585(7824):288–292. https://doi.org/10.1038/s41586-020-2475-6 12. Lee JH, Park A, Oh KJ, Lee SC, Kim WK, Bae KH. The role of adipose tissue mitochondria: regulation of mitochondrial func- tion for the treatment of metabolic diseases. Int J Mol Sci. 2019 Oct 4;20(19):4924. https://doi.org/10.3390/ijms20194924 12. Lee JH, Park A, Oh KJ, Lee SC, Kim WK, Bae KH. The role of adipose tissue mitochondria: regulation of mitochondrial func- tion for the treatment of metabolic diseases. Int J Mol Sci. 2019 Oct 4;20(19):4924. https://doi.org/10.3390/ijms20194924 12. Lee JH, Park A, Oh KJ, Lee SC, Kim WK, Bae KH. The role of adipose tissue mitochondria: regulation of mitochondrial func- tion for the treatment of metabolic diseases. Int J Mol Sci. 2019 Oct 4;20(19):4924. https://doi.org/10.3390/ijms20194924 13. Liu Z, Sun Y, Qi Z, Cao L, Ding S. Mitochondrial transfer/transplantation: an emerging therapeutic approach for multiple tion for the treatment of metabolic diseases. Int J Mol Sci. 2019 Oct 4;20(19):4924. https://doi.org/10.3390/ijms20194924 13. Liu Z, Sun Y, Qi Z, Cao L, Ding S. Mitochondrial transfer/transplantation: an emerging therapeutic approach for multiple diseases. Cell Biosci. 2022 May 19;12(1):66. https://doi.org/10.1186/s13578-022-00805-7 13. Liu Z, Sun Y, Qi Z, Cao L, Ding S. Mitochondrial transfer/transplantation: an emerging therapeutic approach for multiple diseases. Cell Biosci. 2022 May 19;12(1):66. https://doi.org/10.1186/s13578-022-00805-7 13. Liu Z, Sun Y, Qi Z, Cao L, Ding S. Mitochondrial transfer/transplantation: an emerging therapeutic approach for multiple diseases. Cell Biosci. 2022 May 19;12(1):66. https://doi.org/10.1186/s13578-022-00805-7 14. Jing X, Yang F, Shao C, Wei K, Xie M, Shen H, et al. Role of hypoxia in cancer therapy by regulating the tumor microenviron- ment. Mol Cancer. 2019 Nov 11;18(1):157. https://doi.org/10.1186/s12943-019-1089-9 14. Jing X, Yang F, Shao C, Wei K, Xie M, Shen H, et al. Role of hypoxia in cancer therapy by regulating the tumor microenviron- ment. Mol Cancer. 2019 Nov 11;18(1):157. https://doi.org/10.1186/s12943-019-1089-9 15. Dong L, Gopalan V, Holland O, Neuzil J. Mitocans Revisited: Mitochondrial Targeting as Efficient Anti-Cancer Therapy. ЗАКЛЮЧЕНИЕ Сегодня митохондриям придается гораздо боль- шее значение, чем «энергетическая установка» в клетках. Митохондриальная трансплантационная терапия была активной областью исследований для лечения заболеваний, связанных с дисфунк- цией митохондрий, от исследований на животных до клинических испытаний. Тем не менее, специ- фический механизм, обеспечивающий противоопу- холевую активность здоровых митохондрий, еще предстоит охарактеризовать. Механизм межкле- точного переноса митохондрий до сих пор остается частично понятым и требует дальнейшего изучения, при этом его таргетирование может обеспечить новые возможности в терапии злокачественных новообразований. Доказательства того, что мито- хондриальный перенос может происходить сход- ным образом в солидных и гематологических опу- холевых клетках, еще больше повышают важность этого процесса как основы для трансплантации митохондрий. Кроме того, участие митохондриаль- ного переноса при прогрессировании рака и раз- витии химиорезистентности может объяснить еще неясные механизмы действия некоторых противо- опухолевых препаратов. Доказано, что терапев- тический эффект трансплантации митохондрий является потенциальным методом лечения забо- леваний, связанных с митохондриями. Однако есть несколько проблем, которые необходимо преодо- леть, чтобы лечение болезни с помощью транс- плантации митохондрий могло быть эффективно применено людям. В ряде сообщений описывались тканеспецифи- ческие половые различия в морфологии митохон- дрий и окислительной способности, при этом лишь немногие исследования показали функциональные различия митохондрий при терапии. Вместе с тем было показано, что митохондрии женщин имеют более высокое содержание белка и способность к производству АТФ, чем у мужчин [57]. По имею- щимся ограниченным данным, женские митохон- дрии имеют более благоприятную митохондриаль- но-ядерную коммуникацию в ответ на стресс по сравнению с мужскими митохондриями [58]. Yu Z. и соавт. (2021) оценили активность митохон- дрий, выделенных от самок и самцов мышей, и ре- зультаты показали, что женские митохондрии про- являли более высокую активность и способность вырабатывать АТФ, чем мужские митохондрии. Впо- следствии противоопухолевые митохондриальные эффекты в ряде экспериментов как на моделях in vitro, так и in vivo, доказали, что женские митохон- дрии обладают более высокой эффективностью подавления пролиферации опухолевых клеток, чем мужские митохондрии. Исследование также пока- зало, что женские митохондрии могут вызывать более устойчивый стресс-ответ на транскрипцию гена, чем мужские митохондрии в опухолевых клет- ках, предполагая, что женские митохондрии более чувствительны к гипоксическому микроокружению опухоли, чем мужские митохондрии, и в конечном итоге приводят к более сильному противоопухо- левому эффекту. Авторы использовали интактные митохондрии для изучения их противоопухолевой активности при внутривенном введении. Это иссле- В большинстве исследований подчеркивается, что изоляция митохондрий должна быть завер- шена в короткие сроки при низкой температуре, поскольку они очень чувствительны, а их актив- ность и выживаемость быстро снижаются. Кроме 66 Южно-Российский онкологический журнал 2024. Т. Список источников 1. Schirrmacher V. Mitochondria at Work: New Insights into Regulation and Dysregulation of Cellular Energy Supply and Me- tabolism. Biomedicines. 2020 Nov 22;8(11):526. https://doi.org/10.3390/biomedicines8110526 1. Schirrmacher V. Mitochondria at Work: New Insights into Regulation and Dysregulation of Cellular Energy Supply and Me- tabolism. Biomedicines. 2020 Nov 22;8(11):526. https://doi.org/10.3390/biomedicines8110526 2. Simoes ICM, Morciano G, Lebiedzinska-Arciszewska M, Aguiari G, Pinton P, Potes Y, et al. The mystery of mitochon- dria-ER contact sites in physiology and pathology: A cancer perspective. Biochim Biophys Acta Mol Basis Dis. 2020 Oct 1;1866(10):165834. https://doi.org/10.1016/j.bbadis.2020.165834 2. Simoes ICM, Morciano G, Lebiedzinska-Arciszewska M, Aguiari G, Pinton P, Potes Y, et al. The mystery of mitochon- dria-ER contact sites in physiology and pathology: A cancer perspective. Biochim Biophys Acta Mol Basis Dis. 2020 Oct 1;1866(10):165834. https://doi.org/10.1016/j.bbadis.2020.165834 2. Simoes ICM, Morciano G, Lebiedzinska-Arciszewska M, Aguiari G, Pinton P, Potes Y, et al. The mystery of mitochon- dria-ER contact sites in physiology and pathology: A cancer perspective. Biochim Biophys Acta Mol Basis Dis. 2020 Oct 1;1866(10):165834. https://doi.org/10.1016/j.bbadis.2020.165834 3. Bruni F. Mitochondria: From Physiology to Pathology. Life (Basel). 2021 Sep 21;11(9):991. https://doi.org/10.3390/life11090991 4. Kit OI, Frantsiyants EM, Neskubina IV, Cheryarina ND, Shikhlyarova AI, Przhedetskiy YuV, et al. Influence of standard and stimulated growth of B16/F10 melanoma on AIF levels in mitochondria in cells of the heart and other somatic organs in female mice.Cardiometry. 2021;(18):113–120. https://doi.org/10.18137/cardiometry.2021.18.113120, EDN: UYZTGH 5. Popov LD. One step forward: extracellular mitochondria transplantation. Cell Tissue Res. 2021 Jun;384(3):607–612. 4. Kit OI, Frantsiyants EM, Neskubina IV, Cheryarina ND, Shikhlyarova AI, Przhedetskiy YuV, et al. Influence of standard and stimulated growth of B16/F10 melanoma on AIF levels in mitochondria in cells of the heart and other somatic organs in female mice.Cardiometry. 2021;(18):113–120. https://doi.org/10.18137/cardiometry.2021.18.113120, EDN: UYZTGH 5. Popov LD. One step forward: extracellular mitochondria transplantation. Cell Tissue Res. 2021 Jun;384(3):607–612. https://doi.org/10.1007/s00441-021-03428-5 6. Xu Y, Yu Y, Yang B, Hui J, Zhang C, Fang H, et al. Extracellular Mitochondrial Components and Effects on Cardiovascular Disease. DNA Cell Biol. 2021 Sep;40(9):1131–1143. https://doi.org/10.1089/dna.2021.0087 7. Zhou H, Ren J, Toan S, Mui D. Role of mitochondrial quality surveillance in myocardial infarction: From bench Ageing Res Rev. 2021 Mar;66:101250. https://doi.org/10.1016/j.arr.2020.101250 8. Kossenkov AV, Milcarek A, Notta F, Jang GH, Wilson JM, Gallinger S, et al. Mitochondrial fitness and cancer risk 2022;17(10):e0273520. https://doi.org/10.1371/journal.pone.0273520 9. Bock FJ, Tait SWG. Mitochondria as multifaceted regulators of cell death. Nat Rev Mol Cell Biol. 2020 Feb;21(2):85–100. https://doi.org/10.1038/s41580-019-0173-8 10. Список источников Doulamis IP, Guariento A, Duignan T, Kido T, Orfany A, Saeed MY, et al. Mitochondrial transplantation by intra-arterial injection for acute kidney injury. Am J Physiol Renal Physiol. 2020 Sep 1;319(3):F403–F413. https://doi.org/10.1152/ajprenal.00255.2020 28. Park A, Oh M, Lee SJ, Oh KJ, Lee EW, Lee SC, et al. Mitochondrial Transplantation as a Novel Therapeutic Strategy for Mi- tochondrial Diseases. Int J Mol Sci. 2021 Apr 30;22(9):4793. https://doi.org/10.3390/ijms22094793 28. Park A, Oh M, Lee SJ, Oh KJ, Lee EW, Lee SC, et al. Mitochondrial Transplantation as a Novel Therapeutic Strategy for Mi- tochondrial Diseases. Int J Mol Sci. 2021 Apr 30;22(9):4793. https://doi.org/10.3390/ijms22094793 29. Russell OM, Gorman GS, Lightowlers RN, Turnbull DM. Mitochondrial Diseases: Hope for the Future. Cell. 2020 Apr 2;181(1):168–188. https://doi.org/10.1016/j.cell.2020.02.051 29. Russell OM, Gorman GS, Lightowlers RN, Turnbull DM. Mitochondrial Diseases: Hope for the Future. Cell. 2020 Apr 2;181(1):168–188. https://doi.org/10.1016/j.cell.2020.02.051 30. Sidarala V, Pearson GL, Parekh VS, Thompson B, Christen L, Gingerich MA, et al. Mitophagy protects β ce matory damage in diabetes. JCI Insight. 2020 Dec 17;5(24):e141138. https://doi.org/10.1172/jci.insight.141 31. Kitani T, Kami D, Kawasaki T, Nakata M, Matoba S, Gojo S. Direct human mitochondrial transfer: a novel concept b endosymbiotic theory. Transplant Proc. 2014 May;46(4):1233–1236. https://doi.org/10.1016/j.transproceed.20 32. Katrangi E, D’Souza G, Boddapati SV, Kulawiec M, Singh KK, Bigger B, et al. Xenogenic transfer of isolated murine mitochon- dria into human rho0 cells can improve respiratory function. Rejuvenation Res. 2007 Dec;10(4):561–570. https://doi.org/10.1089/rej.2007.0575 33. Doulamis IP, Guariento A, Duignan T, Orfany A, Kido T, Zurakowski D, et al. Mitochondrial transplantation for myocardial protection in diabetic hearts. Eur J Cardiothorac Surg. 2020 May 1;57(5):836–845. https://doi.org/10.1093/ejcts/ezz326 33. Doulamis IP, Guariento A, Duignan T, Orfany A, Kido T, Zurakowski D, et al. Mitochondrial transplantation for myocardial protection in diabetic hearts. Eur J Cardiothorac Surg. 2020 May 1;57(5):836–845. https://doi.org/10.1093/ejcts/ezz326 34. Lin HC, Liu SY, Lai HS, Lai IR. Isolated mitochondria infusion mitigates ischemia-reperfusion injury of the liver in rats. Shock. ( ) h d b f 34. Lin HC, Liu SY, Lai HS, Lai IR. Isolated mitochondria infusion mitigates ischemia-reperfusion injury of the liver in rats. Shock. 2013 Mar;39(3):304–310. https://doi.org/10.1097/SHK.0b013e318283035f 35. Blitzer D, Guariento A, Doulamis IP, Shin B, Moskowitzova K, Barbieri GR, et al. Delayed Transplantation of Autologous Mi- tochondria for Cardioprotection in a Porcine Model. Ann Thorac Surg. 2020 Mar;109(3):711–719. 35. Blitzer D, Guariento A, Doulamis IP, Shin B, Moskowitzova K, Barbieri GR, et al. Список источников Hosseinian S, Ali Pour P, Kheradvar A. Prospects of mitochondrial transplantation in clinical medicine: Aspirations and challenges. Mitochondrion. 2022 Jul;65:33–44. https://doi.org/10.1016/j.mito.2022.04.006 21. Hosseinian S, Ali Pour P, Kheradvar A. Prospects of mitochondrial transplantation in clinical medicine: Aspirations and challenges. Mitochondrion. 2022 Jul;65:33–44. https://doi.org/10.1016/j.mito.2022.04.006 22. Singh B, Modica-Napolitano JS, Singh KK. Defining the momiome: Promiscuous information transfer by mobile mitochondria and the mitochondrial genome. Semin Cancer Biol. 2017 Dec;47:1–17. https://doi.org/10.1016/j.semcancer.2017.05.004 22. Singh B, Modica-Napolitano JS, Singh KK. Defining the momiome: Promiscuous information transfer by mobile mitochondria and the mitochondrial genome. Semin Cancer Biol. 2017 Dec;47:1–17. https://doi.org/10.1016/j.semcancer.2017.05.004 and the mitochondrial genome. Semin Cancer Biol. 2017 Dec;47:1–17. https://doi.org/10.1016/j.semcancer.2017.05.004 23. Shanmughapriya S, Langford D, Natarajaseenivasan K. Inter and Intracellular mitochondrial trafficking in health and dis- ease. Ageing Res Rev. 2020 Sep;62:101128. https://doi.org/10.1016/j.arr.2020.101128 23. Shanmughapriya S, Langford D, Natarajaseenivasan K. Inter and Intracellular mitochondrial trafficking in health and dis- ease. Ageing Res Rev. 2020 Sep;62:101128. https://doi.org/10.1016/j.arr.2020.101128 23. Shanmughapriya S, Langford D, Natarajaseenivasan K. Inter and Intracellular mitochondrial trafficking in health and dis- ease. Ageing Res Rev. 2020 Sep;62:101128. https://doi.org/10.1016/j.arr.2020.101128 24. Levoux J, Prola A, Lafuste P, Gervais M, Chevallier N, Koumaiha Z, et al. Platelets Facilitate the Wound-Healing Capability of Mesenchymal Stem Cells by Mitochondrial Transfer and Metabolic Reprogramming. Cell Metab. 2021 Feb 2;33(2):283– 299. https://doi.org/10.1016/j.cmet.2020.12.006 24. Levoux J, Prola A, Lafuste P, Gervais M, Chevallier N, Koumaiha Z, et al. Platelets Facilitate the Wound-Healing Capability of Mesenchymal Stem Cells by Mitochondrial Transfer and Metabolic Reprogramming. Cell Metab. 2021 Feb 2;33(2):283– 299. https://doi.org/10.1016/j.cmet.2020.12.006 25. Walters HE, Cox LS. Intercellular Transfer of Mitochondria between Senescent Cells through Cytoskeleton-Supported In- tercellular Bridges Requires mTOR and CDC42 Signalling. Oxid Med Cell Longev. 2021;2021:6697861. https://doi.org/10.1155/2021/6697861 25. Walters HE, Cox LS. Intercellular Transfer of Mitochondria between Senescent Cells through Cytoskeleton-Supported In- tercellular Bridges Requires mTOR and CDC42 Signalling. Oxid Med Cell Longev. 2021;2021:6697861. https://doi.org/10.1155/2021/6697861 26. Ali Pour P, Hosseinian S, Kheradvar A. Mitochondrial transplantation in cardiomyocytes: foundation, methods, and out- comes. Am J Physiol Cell Physiol. 2021 Sep 1;321(3):C489–C503. https://doi.org/10.1152/ajpcell.00152.2021 26. Ali Pour P, Hosseinian S, Kheradvar A. Mitochondrial transplantation in cardiomyocytes: foundation, methods, and out- comes. Am J Physiol Cell Physiol. 2021 Sep 1;321(3):C489–C503. https://doi.org/10.1152/ajpcell.00152.2021 27. Doulamis IP, Guariento A, Duignan T, Kido T, Orfany A, Saeed MY, et al. Mitochondrial transplantation by intra-arterial injection for acute kidney injury. Am J Physiol Renal Physiol. 2020 Sep 1;319(3):F403–F413. https://doi.org/10.1152/ajprenal.00255.2020 27. Список источников Int J Mol Sci. 2020 Oct 26;21(21):7941. https://doi.org/10.3390/ijms21217941 15. Dong L, Gopalan V, Holland O, Neuzil J. Mitocans Revisited: Mitochondrial Targeting as Efficient Anti-Cancer Therapy. Int J Mol Sci. 2020 Oct 26;21(21):7941. https://doi.org/10.3390/ijms21217941 16. Jia D, Park JH, Jung KH, Levine H, Kaipparettu BA. Elucidating the Metabolic Plasticity of Cancer: Mitochondrial Repro- gramming and Hybrid Metabolic States. Cells. 2018 Mar 13;7(3):21. https://doi.org/10.3390/cells7030021 16. Jia D, Park JH, Jung KH, Levine H, Kaipparettu BA. Elucidating the Metabolic Plasticity of Cancer: Mitochondrial Repro- gramming and Hybrid Metabolic States. Cells. 2018 Mar 13;7(3):21. https://doi.org/10.3390/cells7030021 17. Li J, Agarwal E, Bertolini I, Seo JH, Caino MC, Ghosh JC, et al. The mitophagy effector FUNDC1 controls mitochondrial re- programming and cellular plasticity in cancer cells. Sci Signal. 2020 Jul 28;13(642):eaaz8240. 17. Li J, Agarwal E, Bertolini I, Seo JH, Caino MC, Ghosh JC, et al. The mitophagy effector FUNDC1 controls mitochondrial re- programming and cellular plasticity in cancer cells. Sci Signal. 2020 Jul 28;13(642):eaaz8240. https://doi.org/10.1126/scisignal.aaz8240 17. Li J, Agarwal E, Bertolini I, Seo JH, Caino MC, Ghosh JC, et al. The mitophagy effector FUNDC1 controls mitochondrial re- programming and cellular plasticity in cancer cells. Sci Signal. 2020 Jul 28;13(642):eaaz8240. https://doi.org/10.1126/scisignal.aaz8240 18. Damaghi M, West J, Robertson-Tessi M, Xu L, Ferrall-Fairbanks MC, Stewart PA, et al. The harsh microenvironment in early breast cancer selects for a Warburg phenotype. Proc Natl Acad Sci U S A. 2021 Jan 19;118(3):e2011342118. https://doi.org/10.1073/pnas.2011342118 18. Damaghi M, West J, Robertson-Tessi M, Xu L, Ferrall-Fairbanks MC, Stewart PA, et al. The harsh microenvironment in early breast cancer selects for a Warburg phenotype. Proc Natl Acad Sci U S A. 2021 Jan 19;118(3):e2011342118. https://doi.org/10.1073/pnas.2011342118 67 South Russian Journal of Cancer 2024. Vol. 5, No. 1. P. 60-70 19. Humpton TJ, Alagesan B, DeNicola GM, Lu D, Yordanov GN, Leonhardt CS, et al. Oncogenic KRAS Induces NIX-Mediated Mitophagy to Promote Pancreatic Cancer. Cancer Discov. 2019 Sep;9(9):1268–1287. https://doi.org/10.1158/2159-8290.CD-18-1409 19. Humpton TJ, Alagesan B, DeNicola GM, Lu D, Yordanov GN, Leonhardt CS, et al. Oncogenic KRAS Induces NIX-Mediated Mitophagy to Promote Pancreatic Cancer. Cancer Discov. 2019 Sep;9(9):1268–1287. https://doi.org/10.1158/2159-8290.CD-18-1409 20. Margulis L. Symbiotic theory of the origin of eukaryotic organelles; criteria for proof. Symp Soc Exp Biol. 1975;(29):21–38. 21. Hosseinian S, Ali Pour P, Kheradvar A. Prospects of mitochondrial transplantation in clinical medicine: Aspirations and challenges. Mitochondrion. 2022 Jul;65:33–44. https://doi.org/10.1016/j.mito.2022.04.006 g y y g y g p y p p ( ) 21. Kit O. I., Frantsiyants E. M., Shikhlyarova A. I., Neskubina I. V.༈ Mitochondrial transplantation: new challenges for cancer South Russian Journal of Cancer 2024. Vol. 5, No. 1. P. 60-70 Kit O. I., Frantsiyants E. M., Shikhlyarova A. I., Neskubina I. V.༈ Mitochondrial transplantation: new challenges for cancer Список источников Delayed Transplantation of Autologous Mi- tochondria for Cardioprotection in a Porcine Model. Ann Thorac Surg. 2020 Mar;109(3):711–719. https://doi.org/10.1016/j.athoracsur.2019.06.075 https://doi.org/10.1016/j.athoracsur.2019.06.075 36. Huang PJ, Kuo CC, Lee HC, Shen CI, Cheng FC, Wu SF, et al. Transferring Xenogenic Mitochondria Provides Neural Protec- tion Against Ischemic Stress in Ischemic Rat Brains. Cell Transplant. 2016;25(5):913–927. https://doi org/10 3727/096368915X689785 36. Huang PJ, Kuo CC, Lee HC, Shen CI, Cheng FC, Wu SF, et al. Transferring Xenogenic Mitochondria Provides Neural Protec- tion Against Ischemic Stress in Ischemic Rat Brains. Cell Transplant. 2016;25(5):913–927. https://doi.org/10.3727/096368915X689785 37. Patananan AN, Sercel AJ, Wu TH, Ahsan FM, Torres A, Kennedy SAL, et al. Pressure-Driven Mitochondrial Transfer Pipeline Generates Mammalian Cells of Desired Genetic Combinations and Fates. Cell Rep. 2020 Dec 29;33(13):108562. https://doi.org/10.1016/j.celrep.2020.108562 38. Lindqvist D, Wolkowitz OM, Picard M, Ohlsson L, Bersani FS, Fernström J, et al. Circulating cell-free mitochondrial DNA, but not leukocyte mitochondrial DNA copy number, is elevated in major depressive disorder. Neuropsychopharmacology. 2018 Jun;43(7):1557–1564. https://doi.org/10.1038/s41386-017-0001-9 39. Miller B, Kim SJ, Kumagai H, Mehta HH, Xiang W, Liu J, et al. Peptides derived from small mitochondrial open reading frames: Genomic, biological, and therapeutic implications. Exp Cell Res. 2020 Aug 15;393(2):112056. https://doi.org/10.1016/j.yexcr.2020.112056 68 Южно-Российский онкологический журнал 2024. Т. 5, № 1. С. 60-70 Южно-Российский онкологический журнал 2024. Т. 5, № 1. С. 60-70 Кит О. И., Франциянц Е. М., Шихлярова А. И., Нескубина И. В.༈ Митохондриальная трансплантация – новые вызовы раку Южно-Российский онкологический журнал 2024. Т. 5, № 1. С. 60-70 Кит О. И., Франциянц Е. М., Шихлярова А. И., Нескубина И. В.༈ Митохондриальная трансплантация – новые вызовы раку 40. Picca A, Beli R, Calvani R, Coelho-Júnior HJ, Landi F, Bernabei R, et al. Older Adults with Physical Frailty and Sarcopenia Show Increased Levels of Circulating Small Extracellular Vesicles with a Specific Mitochondrial Signature. Cells. 2020 Apr 15;9(4):973. https://doi.org/10.3390/cells9040973 40. Picca A, Beli R, Calvani R, Coelho-Júnior HJ, Landi F, Bernabei R, et al. Older Adults with Physical Frailty and Sarcopenia Show Increased Levels of Circulating Small Extracellular Vesicles with a Specific Mitochondrial Signature. Cells. 2020 Apr 15;9(4):973. https://doi.org/10.3390/cells9040973 40. Picca A, Beli R, Calvani R, Coelho-Júnior HJ, Landi F, Bernabei R, et al. Older Adults with Physical Frailty and Sarcopenia Show Increased Levels of Circulating Small Extracellular Vesicles with a Specific Mitochondrial Signature. Cells. 2020 Apr 15;9(4):973. https://doi.org/10.3390/cells9040973 41. Sahinbegovic H, Jelinek T, Hrdinka M, Bago JR, Turi M, Sevcikova T, et al. Список источников Improvement of cognitive and motor performance with mitotherapy in aged mice. Int J Biol Sci. 2020;16(5):849–858. https://doi.org/10.7150/ijbs.40886 50. Fu A. Mitotherapy as a Novel Therapeutic Strategy for Mitochondrial Diseases. Curr Mol Pharmacol. 2020;1 https://doi.org/10.2174/1874467212666190920144115 51. Desdín-Micó G, Soto-Heredero G, Aranda JF, Oller J, Carrasco E, Gabandé-Rodríguez E, et al. T cells with dysfunctional mi- tochondria induce multimorbidity and premature senescence. Science. 2020 Jun 19;368(6497):1371–1376. https://doi.org/10.1126/science.aax0860 51. Desdín-Micó G, Soto-Heredero G, Aranda JF, Oller J, Carrasco E, Gabandé-Rodríguez E, et al. T cells with dysfunctional mi- tochondria induce multimorbidity and premature senescence. Science. 2020 Jun 19;368(6497):1371–1376. https://doi.org/10.1126/science.aax0860 52. Chen Y, Yang F, Chu Y, Yun Z, Yan Y, Jin J. Mitochondrial transplantation: opportunities and challenges in the treatment of obesity, diabetes, and nonalcoholic fatty liver disease. J Transl Med. 2022 Oct 22;20(1):483. https://doi.org/10.1186/s12967-022-03693-0 52. Chen Y, Yang F, Chu Y, Yun Z, Yan Y, Jin J. Mitochondrial transplantation: opportunities and challenges in the treatment of obesity, diabetes, and nonalcoholic fatty liver disease. J Transl Med. 2022 Oct 22;20(1):483. https://doi.org/10.1186/s12967-022-03693-0 53. Roushandeh AM, Kuwahara Y, Roudkenar MH. Mitochondrial transplantation as a potential and novel master key for treat- ment of various incurable diseases. Cytotechnology. 2019 Apr;71(2):647–663. https://doi.org/10.1007/s10616-019-00302-9 53. Roushandeh AM, Kuwahara Y, Roudkenar MH. Mitochondrial transplantation as a potential and novel master key for treat- ment of various incurable diseases. Cytotechnology. 2019 Apr;71(2):647–663. https://doi.org/10.1007/s10616-019-00302-9 54. McCully JD, Cowan DB, Emani SM, Del Nido PJ. Mitochondrial transplantation: From animal models to clinical use in hu- mans. Mitochondrion. 2017 May;34:127–134. https://doi.org/10.1016/j.mito.2017.03.004 55. Ramirez-Barbieri G, Moskowitzova K, Shin B, Blitzer D, Orfany A, Guariento A, et al. Alloreactivity and allorecogn geneic and allogeneic mitochondria. Mitochondrion. 2019 May;46:103–115. https://doi.org/10.1016/j.mito.201 56. Ventura-Clapier R, Moulin M, Piquereau J, Lemaire C, Mericskay M, Veksler V, et al. Mitochondria: a central ta differences in pathologies. Clin Sci (Lond). 2017 May 1;131(9):803–822. https://doi.org/10.1042/CS20160485 57. Beaudry KM, Devries MC. Sex-based differences in hepatic and skeletal muscle triglyceride storage and metabolism 1. Appl Physiol Nutr Metab. 2019 Aug;44(8):805–813. https://doi.org/10.1139/apnm-2018-0635 58. Demarest TG, McCarthy MM. Sex differences in mitochondrial (dys)function: Implications for neuroprotection. J Bioenerg Biomembr. 2015 Apr;47(1–2):173–188. https://doi.org/10.1007/s10863-014-9583-7 58. Demarest TG, McCarthy MM. Sex differences in mitochondrial (dys)function: Implications for neuroprotection. J Bioenerg Biomembr. 2015 Apr;47(1–2):173–188. https://doi.org/10.1007/s10863-014-9583-7 59. Yu Z, Hou Y, Zhou W, Zhao Z, Liu Z, Fu A. The effect of mitochondrial transplantation therapy from different gender on in- hibiting cell proliferation of malignant melanoma. Int J Biol Sci. 2021;17(8):2021–2033. Список источников Intercellular Mitochondrial Transfer in the Tumor Microenvironment. Cancers (Basel). 2020 Jul 4;12(7):1787. https://doi.org/10.3390/cancers12071787 41. Sahinbegovic H, Jelinek T, Hrdinka M, Bago JR, Turi M, Sevcikova T, et al. Intercellular Mitochondrial Transfer in the Tumor Microenvironment. Cancers (Basel). 2020 Jul 4;12(7):1787. https://doi.org/10.3390/cancers12071787 42. Roh JS, Sohn DH. Damage-Associated Molecular Patterns in Inflammatory Diseases. Immune Netw. 2018 Aug;18(4):e27. https://doi.org/10.4110/in.2018.18.e27 42. Roh JS, Sohn DH. Damage-Associated Molecular Patterns in Inflammatory Diseases. Immune Netw. 2018 Aug;18(4):e27. https://doi.org/10.4110/in.2018.18.e27 43. Elliott RL, Jiang XP, Head JF. Mitochondria organelle transplantation: introduction of normal epithelial mitochondria into human cancer cells inhibits proliferation and increases drug sensitivity. Breast Cancer Res Treat. 2012 Nov;136(2):347– 354. https://doi.org/10.1007/s10549-012-2283-2 43. Elliott RL, Jiang XP, Head JF. Mitochondria organelle transplantation: introduction of normal epithelial mitochondria into human cancer cells inhibits proliferation and increases drug sensitivity. Breast Cancer Res Treat. 2012 Nov;136(2):347– 354. https://doi.org/10.1007/s10549-012-2283-2 44. Rushande AM, Kuwahara Y, Roudkenar MH. Mitochondrial transplantation as a potential and novel master key for treatment of various incurable diseases. Cytotechnology. 2019 Apr;71(2):647–663. https://doi.org/10.1007/s10616-019-00302-9 44. Rushande AM, Kuwahara Y, Roudkenar MH. Mitochondrial transplantation as a potential and novel master key for treatment of various incurable diseases. Cytotechnology. 2019 Apr;71(2):647–663. https://doi.org/10.1007/s10616-019-00302-9 45. Fu A., Hou Y., Yu Z., Zhao Z., Liu X. Healthy mitochondria inhibit the metastatic melanoma in lungs. Int J Biol Sci. 2019;15:2707– 2718. https://doi.org/10.7150/ijbs.38104 45. Fu A., Hou Y., Yu Z., Zhao Z., Liu X. Healthy mitochondria inhibit the metastatic melanoma in lungs. Int J Biol Sci. 2019;15:2707– 2718. https://doi.org/10.7150/ijbs.38104 46. Chang JC, Chang HS, Wu YC, Cheng WL, Lin TT, Chang HJ, et al. Mitochondrial transplantation regulates antitumour activ- ity, chemoresistance and mitochondrial dynamics in breast cancer. J Exp Clin Cancer Res. 2019 Jan 23;38(1):30. https://doi.org/10.1186/s13046-019-1028-z 46. Chang JC, Chang HS, Wu YC, Cheng WL, Lin TT, Chang HJ, et al. Mitochondrial transplantation regulates antitumour activ- ity, chemoresistance and mitochondrial dynamics in breast cancer. J Exp Clin Cancer Res. 2019 Jan 23;38(1):30. https://doi.org/10.1186/s13046-019-1028-z 47. Sun C, Liu X, Wang B, Wang Z, Liu Y, Di C, et al. Endocytosis-mediated mitochondrial transplantation: Transferring normal human astrocytic mitochondria into glioma cells rescues aerobic respiration and enhances radiosensitivity. Theranostics. 2019;9(12):3595–3607. https://doi.org/10.7150/thno.33100 48. Al Amir Dache Z, Otandault A, Tanos R, Pastor B, Meddeb R, Sanchez C, et al. Blood contains circulating cell-free respira- tory competent mitochondria. FASEB J. 2020 Mar;34(3):3616–3630. https://doi.org/10.1096/fj.201901917RR 49. Zhao Z, Yu Z, Hou Y, Zhang L, Fu A. South Russian Journal of Cancer 2024. Vol. 5, No. 1. P. 60-70 Список источников https://doi.org/10.7150/ijbs.59581 59. Yu Z, Hou Y, Zhou W, Zhao Z, Liu Z, Fu A. The effect of mitochondrial transplantation therapy from different gender on in- hibiting cell proliferation of malignant melanoma. Int J Biol Sci. 2021;17(8):2021–2033. https://doi.org/10.7150/ijbs.59581 69 South Russian Journal of Cancer 2024. Vol. 5, No. 1. P. 60-70 Kit O. I., Frantsiyants E. M., Shikhlyarova A. I., Neskubina I. V.༈ Mitochondrial transplantation: new challenges for cancer Информация об авторах: ф р р Кит Олег Иванович – академик РАН, д.м.н., профессор, генеральный директор, ФГБУ «Национальный медицинский исследовательский центр онкологии» Министерства здравоохранения Российской Федерации, г. Ростов-на-Дону, Российская Федерация ORCID: https://orcid.org/0000-0003-3061-6108, SPIN: 1728-0329, AuthorID: 343182, ResearcherID: U-2241-2017, Scopus Author ID: 55994103100 Франциянц Елена Михайловна – д.б.н., профессор, заместитель генерального директора по научной работе, ФГБУ «Национальный меди- цинский исследовательский центр онкологии» Министерства здравоохранения Российской Федерации, г. Ростов-на-Дону, Российская Федерация д р ц ORCID: https://orcid.org/0000-0003-3618-6890, SPIN: 9427-9928, AuthorID: 462868, ResearcherID: Y-1491-2018, Scopus Author ID: 55890047700 Шихлярова Алла Ивановна – д.б.н., профессор, старший научный сотрудник лаборатории изучения патогенеза злокачественных опухо- лей, ФГБУ «Национальный медицинский исследовательский центр онкологии» Министерства здравоохранения Российской Федерации, г. Ростов-на-Дону, Российская Федерация Д у д р ц ORCID: https://orcid.org/0000-0003-2943-7655, SPIN: 6271-0717, AuthorID: 482103, ResearcherID: Y-6275-2018, Scopus Author ID: 6507723229 Нескубина Ирина Валерьевна ༈ – к.б.н., старший научный сотрудник лаборатории изучения патогенеза злокачественных опухолей, ФГБУ «Национальный медицинский исследовательский центр онкологии» Министерства здравоохранения Российской Федерации, г. Ростов-на-Дону, Российская Федерация Д у, д р ц //orcid.org/0000-0002-7395-3086, SPIN: 3581-8531, AuthorID: 794688, ResearcherID: AAG-8731-2019, Scopus Author ID: 6507509066 Вклад авторов: Кит О. И. – научное редактирование; Франциянц Е. М. – написание текста, анализ и интерпретация данных; Шихлярова А. И. – научное редактирование; Нескубина И. В. – техническое редактирование, оформление библиографии. Вклад авторов: Кит О. И. – научное редактирование; Франциянц Е. М. – написание текста, анализ и интерпретация данных; Шихлярова А. И. – научное редактирование; Нескубина И. В. – техническое редактирование, оформление библиографии. Вклад авторов: Кит О. И. – научное редактирование; Франциянц Е. М. – написание текста, анализ и интерпретация данных; Шихлярова А. И. – научное редактирование; Нескубина И. В. – техническое редактирование, оформление библиографии Вклад авторов: Кит О. И. – научное редактирование; Франциянц Е. М. – написание текста, анализ и интерпретация данных; Шихлярова А. И. – научное редактирование; Нескубина И. В. – техническое редактирование, оформление библиографии 70 70
https://openalex.org/W2597165347	https://zenodo.org/records/1062972/files/6467.pdf	English	null	Potential Role of Halophytic Macrophytes in Saline Effluent Treatment	Zenodo (CERN European Organization for Nuclear Research)	2,010	cc-by	4,693	Potential Role of Halophytic Macrophytes in Saline Effluent Treatment R. Hegedűs, É. Kerepeczki, D. Gál, F. Pekár, M. Oncsik Bíróné and Gy. Lakatos reactions supported by the significant wetland components [1]-[5]. There are some functional characteristics of macrophytes: plants uptake nutrients directly, provide good conditions for physical filtration reduce water flow and provide a huge surface area for microbial colonisation [6]. Nevertheless a limited efficiency in removing alkali and alkaline cations (e.g., Na, Ca, Mg) in wastewater has been observed in wetland treatment systems [7]-[9]. Associated with primary stresses caused by high salinity, higher plants also suffer from secondary stresses generated by cellular damages [10]. Salinity disrupts the integrity of cell membranes by inducing structural changes and by replacing Ca with Na in the plasma membrane, altering the K/Na ratio [11]. However there are some halophytes from natural saline lakes, which have adaptability to salinity. The protected processes by halophytes should prevent or alleviate the structural and functional damages caused at the cell level. They should also contribute to re-establishment of the homeostatic conditions required for nutrient uptake and intermediate distribution in the presence of an excess of Na+ and for an internal net flux of water allowing turgor maintenance at the cell level and transpiration at the whole plant level. Halophytes have a number of specific and important mechanisms to achieve crucial protective functions [12]. In the long term, salt accumulation in the plants may impact upon the efficiency of the wetlands in reducing the salt load [13]. Na removal efficiency in wetlands is variable ranging from -78 to 43% [14]-[15]. Sodium and other components of salinity are the most persistent components of recycled water and are among the most difficult removable pollutants from water, usually requiring the use of expensive cation exchange resins or reverse osmosis membranes. Abstract—The growth of the aquaculture industry has been associated with negative environmental impacts through the discharge of raw effluents into the adjacent receiving water bodies. Macrophytes from natural saline lakes, which have adaptability to the high salinity, can be suitable for saline effluent treatment. Eight emergent species from natural saline area were planted in an experimental gravel bed hydroponic mesocosm (GBH) which was treated with effluent water from an intensive fish farm using geothermal water. World Academy of Science, Engineering and Technology International Journal of Agricultural and Biosystems Engineering Vol:4, No:4, 2010 World Academy of Science, Engineering and Technology International Journal of Agricultural and Biosystems Engineering Vol:4, No:4, 2010 The financial support for the research work was provided by the Sustainaqua EC-project (COLL-CT-2006-030384). I. INTRODUCTION HE major environmental issue related to the intensive aquaculture production of Hungary is the treatment of nutrient and salt-enriched aquaculture effluents causing physical, chemical and biological changes in the environment. The utilization of saline geothermal water for aquaculture production potentially creates an additional impact if the effluent is higher in salinity than the receiving water bodies. T T Constructed wetland treatment systems could provide a simple and low-cost mechanism to treat aquaculture effluents through an integration of physical, biological and chemical This study was conducted to evaluate the potential role of halophytic macrophytes in purification of specific waste water like saline effluent. International Science Index, Agricultural and Biosystems Engineering Vol:4, No:4, 2010 waset.org/Pub Keywords—Gravel bed hydroponic system, halophytes, intensive fish farm, salt removal International Science Index, Agricultural and Biosystems Engineering Vol: Gy. Lakatos is with the Department of Applied Ecology, University of Debrecen, Hungary, 4032, Debrecen, Egyetem tér 1. (e-mail: lakgyu@delfin.unideb.hu) International Scholarly and Scientific Research & Innovation 4(4) 2010 R. Hegedűs is with the Research Institute for Fisheries, Aquaculture and Irrigation, Hungary, 5541, Szarvas, Anna-liget 8. (corresponding author; phone:+36-66-515-300/625; fax:+36-66-312-142; e-mail: hegedusr@haki.hu) É. Kerepeczki is with the Research Institute for Fisheries, Aquaculture and Irrigation, Hungary, 5541, Szarvas, Anna-liget 8. (e-mail: kerepecz@haki.hu) D. Gál is with the Research Institute for Fisheries, Aquaculture and Irrigation, Hungary, 5541, Szarvas, Anna-liget 8. (e-mail: gald@haki.hu) F. Pekár is with the Research Institute for Fisheries, Aquaculture and Irrigation, Hungary, 5541, Szarvas, Anna-liget 8. (e-mail: pekarf@haki.hu) M. Oncsik Bíróné is with the Research Institute for Fisheries, Aquaculture and Irrigation, Hungary, 5541, Szarvas, Anna-liget 8. (e-mail: oncsikm@haki.hu) Gy. Lakatos is with the Department of Applied Ecology, University of Debrecen, Hungary, 4032, Debrecen, Egyetem tér 1. (e-mail: lakgyu@delfin.unideb.hu) The financial support for the research work was provided by the Sustainaqua EC-project (COLL-CT-2006-030384). M. Oncsik Bíróné is with the Research Institute for Fisheries, Aquaculture and Irrigation, Hungary, 5541, Szarvas, Anna-liget 8. (e-mail: oncsikm@haki.hu) Potential Role of Halophytic Macrophytes in Saline Effluent Treatment In order to examine the applicability of the halophytes in treatment processes, we tested the relative efficacy of total nitrogen (TN), total phosphorus (TP), potassium (K), sodium (Na), magnesium (Mg) and calcium (Ca) removal for the saline wastewater treatment. Four of the eight species, which were Phragmites australis, Typha angustifolia, Glyceria maxima, Scirpus lacustris spp. tabernaemontani could survive and contribute the experimental treatment. International Science Index, Agricultural and Biosystems Engineering Vol:4, No:4, 2010 waset.org/Publ The macrophytes were observed throughout the experimental period (90 days) for general appearance and health and were sampled once in 30 days. The fresh and dry biomass of above-ground plant organs (leaves, stems) and below-ground organs (rhizomes, roots) were weighed separately. The plant parts were washed in tap water, rinsed with distilled water to remove metal precipitates or epiphytic microorganisms that might have bound to the surfaces, dried, and ground. To measure the dry weight, the biomass was first dried at 105ºC in a drying oven. The powdered samples of the macrophytes were analysed for phosphorus (P), sodium (Na), potassium (K), magnesium (Mg) and calcium (Ca) content using ICP-OES (Application note by Thermo Scientific: 40755), and N was determined by the Kjeldahl method. Water samples were analysed for pH, electrical conductivity (EC), total salt, total suspended solids (TSS), chemical oxygen demand (COD), total nitrogen (TN) and nitrogen forms (NH4- N, NO2-N, NO3-N), total phosphorus (TP) and orthophosphate phosphorus (PO4 3--P) and Na, K, Mg and Ca, chlorid (Cl-), sulphate (SO4 2-) and hydrogen-carbonate (HCO3 -) (Table II.) according to the Hungarian Standard Methods (MSZ). Statistical analyses were performed on the growth rate and element accumulation using SPSS software packages. A multiple-range test was used for testing significant differences between the means at the confidence interval of 95%. The data obtained was subjected to an analysis of variance (ANOVA) and the mean differences were compared by LSD tests. International Science Index, Agricultural and Biosystems Engineering Vol:4, No The average Na content of the samples was significantly higher (297±12.1 mg/L) than in the control water samples (33.1±6.5 mg/L). The Mg, Ca and K concentrations were also significantly higher (p<0.05) than those in the control (Table II.). II. MATERIALS AND METHODS The experiments were performed at the Research Institute for Fisheries, Aquaculture and Irrigation (HAKI), Szarvas, in south-eastern Hungary in 2009. The gravel bed hydroponic (GBH) mesocosm were constructed with dimensions of 200 cm wide, 200 cm long and 50 cm deep plastic tanks. Ten centimetres of 1-3 mm gravel layer were filled at the bottom of each unit. One tank was treated with effluent from an intensive African catfish farm and one tank was supplied with river water (Körös) as a control. At both of the tanks, the same scholar.waset.org/1307-6892/6467 International Scholarly and Scientific Research & Innovation 4(4) 2010 194 World Academy of Science, Engineering and Technology International Journal of Agricultural and Biosystems Engineering Vol:4, No:4, 2010 III. RESULTS AND DISCUSSION water flow rate (200 L/day) was applied. The water level was maintained at 0.1 m above the gravel bed surface. Wild plant species, used in the experiment, were collected from a natural saline area located in Homoródszentpál (Sanpaul), mid-eastern Romania. Eight emergent plant species, Aster tripolium spp. tripolium, Bolboschoenus maritimus, Glyceria maxima, Scirpus lacustris spp. tabernaemontani, Triglochin palustris, Phragmites australis, Typha angustifolia, Carex vulpina, which are either wetland or salt-tolerant species, were primarily selected for investigations (Table I). These macrophytes were planted in June and after acclimation the tested plots were fed with effluent water and the control plot A. Inlet water quality TABLE I LIST OF TESTED EMERGENT PLANTS Scientific name Common name Sign Bolboshoenus maritimus L. Alkali bulrush BM Carex vulpina L. Great prickly sedge CV Glyceria maxima Hartm. Reed mannagrass GM Scirpus lacustris L. ssp. tabernaemontani K.C.Gmel. Syme Soft-stem bulrush ST Triglochin palustris L. Marsh arrowgrass TP Aster tripolium L. spp. tripolium Sea startwort ATT Phragmites australis Cav. Common reed PA Typha angustifolia L. Narrow-leaved cattail TA International Science Index, Agricultural and Biosystems Engineering Vol:4, No:4, 2010 waset.org/Publication/6467 TABLE II PARAMETERS OF INLET WATER FOR EXPERIMENTAL WETLAND TREATMENTS Parameters Unit Effluent River water (control) Average (n=8) s.d. Average (n=8) s.d. Conductivity μS/cm 1357a 54.4 390b 50.4 CODCr mg/L 230a 34.7 26.9b 19.5 NH4-N mg/L 19.7a 4.03 0.130b 0.0745 NO2 --N mg/L 0.016a 0.013 0.017a 0.013 NO3 --N mg/L 0.135a 0.108 0.496a 0.341 TIN mg/L 18.0a 5.77 0.786b 0.274 ON mg/L 8.39a 6.46 1.32a 2.15 TN mg/L 28.0a 5.36 2.03a 2.23 PO4 3--P mg/L 1.29a 0.335 0.139b 0.034 TP mg/L 3.24a 0.418 0.294b 0.268 TSS mg/L 287a 307 28.6a 21.9 Total salt mg/L 961a 54.4 270b 59.9 Ca mg/L 22.7a 2.31 40.1b 3.65 K mg/L 7.88a 1.23 4.38b 0.959 Mg mg/L 11.6a 0.590 9.11b 1.020 Na mg/L 297a 12.1 33.1b 6.5 Cl- mg/L 28.2a 11.4 31.6a 7.93 SO4 2- mg/L 8.15a 3.28 21.8b 10.8 HCO3 - mg/L 968a 75.0 181b 16.8 Values within a row followed by the same letters are not statistically different at p< 0.05 by ANOVA TABLE II PARAMETERS OF INLET WATER FOR EXPERIMENTAL WETLAND TREATMENTS e Index, Agricultural and Biosystems Engineering Vol:4, No:4, 2010 waset.org/Publication/6467 A. Inlet water quality The average chemical oxygen demand (COD) of the samples was relatively high (230 mg/L). The total nitrogen content (TN) in the inlet effluent water was about 28.0 mg/L with a maximum peak (35.6 mg/L) measured in June (Table II). The concentration of NO3 --N ranged between 0.024– 0.355 mg/L, and the PO4 3--P concentration was 1.290±0.335 mg/L. The effluent contained notable concentrations of carbonates (968±75 mg/L), chlorides (28.2±11.4 mg/L) and alkaline metals such as Na, K, Ca, Mg. with river water. TABLE I LIST OF TESTED EMERGENT PLANTS Scientific name Common name Sign Bolboshoenus maritimus L. Alkali bulrush BM Carex vulpina L. Great prickly sedge CV Glyceria maxima Hartm. Reed mannagrass GM Scirpus lacustris L. ssp. tabernaemontani K.C.Gmel. Syme Soft-stem bulrush ST Triglochin palustris L. Marsh arrowgrass TP Aster tripolium L. spp. tripolium Sea startwort ATT Phragmites australis Cav. Common reed PA Typha angustifolia L. Narrow-leaved cattail TA TABLE I LIST OF TESTED EMERGENT PLANTS with river water. The macrophytes were observed throughout the experimental period (90 days) for general appearance and health and were sampled once in 30 days. The fresh and dry biomass of above-ground plant organs (leaves, stems) and below-ground organs (rhizomes, roots) were weighed separately. The plant parts were washed in tap water, rinsed with distilled water to remove metal precipitates or epiphytic microorganisms that might have bound to the surfaces, dried, and ground. To measure the dry weight, the biomass was first dried at 105ºC in a drying oven. The powdered samples of the macrophytes were analysed for phosphorus (P), sodium (Na), potassium (K), magnesium (Mg) and calcium (Ca) content using ICP-OES (Application note by Thermo Scientific: 40755), and N was determined by the Kjeldahl method. Water samples were analysed for pH, electrical conductivity (EC), total salt, total suspended solids (TSS), chemical oxygen demand (COD), total nitrogen (TN) and nitrogen forms (NH4- N, NO2-N, NO3-N), total phosphorus (TP) and orthophosphate phosphorus (PO4 3--P) and Na, K, Mg and Ca, chlorid (Cl-), sulphate (SO4 2-) and hydrogen-carbonate (HCO3 -) (Table II.) according to the Hungarian Standard Methods (MSZ). Statistical analyses were performed on the growth rate and element accumulation using SPSS software packages. A multiple-range test was used for testing significant differences between the means at the confidence interval of 95%. The data obtained was subjected to an analysis of variance (ANOVA) and the mean differences were compared by LSD tests. International Scholarly and Scientific Research & Innovation 4(4) 2010 B. Elemental composition of the wild plant species The initial element concentrations in the plant tissues varied. Ca and Mg accumulated mainly in the roots and Na in the above-ground organs. In the macrophytes, the initial concentrations (in percentage of the dry mass) of Na ranged from 0.547 to 4.42 % in the above-ground organ and 0.456- 0.911 % in the below-ground organs, Ca from 0.256 %- 1.280 % and Mg from 0,101 %-0.312 % in aerial parts and 0.394-1.115 % and 0.156-0.312 % in the below-ground scholar.waset.org/1307-6892/6467 International Scholarly and Scientific Research & Innovation 4(4) 2010 195 World Academy of Science, Engineering and Technology International Journal of Agricultural and Biosystems Engineering Vol:4, No:4, 2010 World Academy of Science, Engineering and Technology International Journal of Agricultural and Biosystems Engineering Vol:4, No:4, 2010 Fig. 3 Comparison of the mean above-ground (white) and below- ground (shaded) tissue Mg concentrations (% in DM) for the eight test species (see Table I. for full species names) Mg B C GM ST TP ATT P TA B C GM ST TP ATT P TA 0.4 0.3 0.2 0.1 0 0.1 0.2 0.3 (% Mg B C GM ST TP ATT P TA B C GM ST TP ATT P TA 0.4 0.3 0.2 0.1 0 0.1 0.2 0.3 (% tissues, respectively (Fig. 1-Fig. 3). Previous studies [16], [17] reported values of element concentrations in shoots of macrophytes lower than those found in present study. The order of the species in terms of Na content in the biomass was Triglochin > Aster > Scirpus > Bolboschoenus > Carex > Typha > Phragmites > Glyceria (Fig. 1). With regard to the Ca content, the order of the species was Typha > Carex > Aster > Scirpus = Triglochin > Bolboschoenus > Glyceria > Phragmites (Fig. 2.). Regarding the Mg levels in the biomass, the order was Triglochin > Typha > Carex > Aster > Scirpus > Bolboschoenus > Glyceria> Phragmites (Fig. 3). Fig. 1 Comparison of the mean above-ground (white) and below- ground (shaded) tissue Na concentrations (% in DM) for the eight test species (see Table I. for full species names) Fig. 2 Comparison of the mean above-ground (white) and below- ground (shaded) tissue Ca concentrations (% in DM) for the eight test species (see Table I. C. Plant growth and nutrient uptake Four out of the eight species, which were Bolboschoenus (BM), Carex (CV), Triglochin (TP) and Aster (ATT) showed unhealthy symptoms from the combined effect of high salt concentration and flood conditions. They appeared dry and yellowish and retardation was observed. Oxygen restriction, which is resulted by prolonged flood conditions as well as organic waste, caused the growth of these plants to suffer. The other species, which were Typha (TA), Phragmites (PA), Glyceria (GM) and Scirpus (ST), exhibited a stress tolerance and also may have a potential role in wastewater treatment. After 90 days growth, Typha was more productive (aerial biomass of 31.2 kg/m2) than the others like Scirpus (10.98 kg/m2), Glyceria (8.68 kg/m2) and Phragmites (2.24 kg/m2) in wet mass. The biomass values of the control tank were lower, the aerial organs of Typha: 0.886 kg/m2, the Scirpus: 0.261 kg/m2, the Phragmites: 0.611 kg/m2, and the Glyceria: 0,700 kg/m2. International Science Index, Agricultural and Biosystems Engineering Vol:4, No:4, 2010 waset.org/Public Fig. 1 Comparison of the mean above-ground (white) and below- ground (shaded) tissue Na concentrations (% in DM) for the eight test species (see Table I. for full species names) Ca B C GM ST TP ATT P TA B C GM ST TP ATT P TA 1.5 1 0.5 0 0.5 1 1.5 (%) x, Agricultural and Biosystems Engineering Ca The element uptake (i.e. the quantity of element removed by the plant) was influenced by the plant issue concentration and repartition of the organs in the harvested biomass. “Fig 4- Fig 9"-show the nutrient uptake in the biomass of the tested plants. The nutrient uptake rates of the aerial biomass were in the range of 0.218-1.38 g/m2/d, 0.019-0.176 g/m2/d and 0.154- 0.602 g/m2/d for N, P and K, respectively. Tanner [16] reported N uptake rates of eight macrophytes similar to those found in present study, the mean uptake rates were 0.744±0.072 g/m2/d. and observed lower uptake rates of P (0.104±0.007 g/m2/d). The rate of K (1.03±0.065 g/m2/d) was higher compared to the levels found in biomass in the present study. The N uptake rates differed among the tested species at p<0.05 and Typha showed the highest rate. In comparison, Klomjek [18] reported a lower N uptake rate of 0.061 g/m2/d and P uptake rate of 0.00024 g/m2/d at Typha species in a treatment wetland receiving a lower N loading rate. Fig. B. Elemental composition of the wild plant species for full species names) Ca B C GM ST TP ATT P TA B C GM ST TP ATT P TA 1.5 1 0.5 0 0.5 1 1.5 (%) Na B C GM ST TP ATT P TA B C GM ST TP ATT P TA 1.5 0.5 0.5 1.5 2.5 3.5 4.5 5.5 (%) rnational Science Index, Agricultural and Biosystems Engineering Vol:4, No:4, 2010 waset.org/Publication/6467 Fig. 1 Comparison of the mean above-ground (white) and below- ground (shaded) tissue Na concentrations (% in DM) for the eight test species (see Table I. for full species names) Na B C GM ST TP ATT P TA B C GM ST TP ATT P TA 1.5 0.5 0.5 1.5 2.5 3.5 4.5 5.5 (%) g Vol:4, No:4, 2010 waset.org/Publication/6467 Fig. 3 Comparison of the mean above-ground (white) and below- ground (shaded) tissue Mg concentrations (% in DM) for the eight test species (see Table I. for full species names) International Science Index, Agricultural and Biosystems Engineering Vol:4, No:4, 2010 waset.org/Publication/6467 ce Index, Agricultural and Biosystems Engineering Vol:4, No:4, 2010 waset.org/Publication/6467 International Scholarly and Scientific Research & Innovation 4(4) 2010 C. Plant growth and nutrient uptake 7 Uptake (g/m2/day) of Na and repartition among the plant organs, mean above-ground (white) and below-ground (shaded) and control (grey) (see Table I. for full species names) Fig. 7 Uptake (g/m2/day) of Na and repartition among the plant organs, mean above-ground (white) and below-ground (shaded) and control (grey) (see Table I. for full species names) Fig. 5 Uptake (g/m2/day) P and repartition among the plant organs, mean above-ground (white) and below-ground (shaded) and control (grey) (see Table I. for full species names) P TA ST P GM TA ST P GM 0.10 0.05 0.00 0.05 0.10 0.15 0.20 (g/m 2/day) ex, Agricultural and Biosystems Engineering Vol:4, No:4, 2 P Fig. 8 Uptake (g/m2/day) of Ca and repartition among the plant organs, mean above-ground (white) and below-ground (shaded) and control (grey) (see Table I. for full species names) Fig 9 Uptake (g/m2/day) of Mg and repartition among the plant Ca TA ST P GM TA ST P GM 0.5 0.4 0.3 0.2 0.1 0.0 0.1 0.2 0.3 0.4 0.5 (g/m2/day) Mg TA ST P GM TA ST P GM 0.10 0.05 0.00 0.05 0.10 0.15 0.20 (g/m2/day) Fig. 8 Uptake (g/m2/day) of Ca and repartition among the plant organs, mean above-ground (white) and below-ground (shaded) and control (grey) (see Table I. for full species names) Ca TA ST P GM TA ST P GM 0.5 0.4 0.3 0.2 0.1 0.0 0.1 0.2 0.3 0.4 0.5 (g/m2/day) Ca TA ST P GM TA ST P GM 0.5 0.4 0.3 0.2 0.1 0.0 0.1 0.2 0.3 0.4 0.5 (g/m2/day) Ca Fig. 5 Uptake (g/m2/day) P and repartition among the plant organs, mean above-ground (white) and below-ground (shaded) and control (grey) (see Table I. for full species names) Fig. 8 Uptake (g/m2/day) of Ca and repartition among the plant organs, mean above-ground (white) and below-ground (shaded) and control (grey) (see Table I. for full species names) Fig. 6 Uptake (g/m2/day) of K and repartition among the plant organs, mean above-ground (white) and below-ground (shaded) and control (grey), (see Table I. for full species names) K TA ST P GM TA ST P GM 0.3 0.2 0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 (g/m 2 /day) International Science In K TA ST P GM TA ST P GM 0 3 0.2 0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 (g/m 2 /day) K Fig. C. Plant growth and nutrient uptake 2 Comparison of the mean above-ground (white) and below- ground (shaded) tissue Ca concentrations (% in DM) for the eight test species (see Table I. for full species names) onal Science International Scholarly and Scientific Research & Innovation 4(4) 2010 scholar.waset.org/1307-6892/6467 196 World Academy of Science, Engineering and Technology International Journal of Agricultural and Biosystems Engineering Vol:4, No:4, 2010 Fig. 4 Uptake (g/m2/day) of N and repartition among the plant organs, mean above-ground (white) and below-ground (shaded) and control (grey) (see Table I. for full species names) N TA ST P GM TA ST P GM 0.6 0.4 0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 (g/m 2 /day) N TA ST P GM TA ST P GM 0.6 0.4 0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 (g/m 2 /day) The nutrient uptake rates were in the range of 0.036- 1.449 g/m2/d, 0.022-0.426 g/m2/d and 0.022-0.156 g/m2/d for Na, Ca and Mg, respectively. The Na uptake rate differed among the tested species at p<0.05 and Phragmites showed the lowest rate (0.036 g/m2/d), its Na uptake rate statistically differed from that of Glyceria (0.218 g/m2/d), Scirpus (0.522 g/m2/d) and Typha (1.44 g/m2/d) p<0.05 in aerial biomass. Fig. 7 Uptake (g/m2/day) of Na and repartition among the plant organs, mean above-ground (white) and below-ground (shaded) and control (grey) (see Table I. for full species names) Na TA ST P GM TA ST P GM 0.6 0.4 0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 (g/m2/day) Na TA ST P GM TA ST P GM 0.6 0.4 0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 (g/m2/day) Na Fig. 4 Uptake (g/m2/day) of N and repartition among the plant organs, mean above-ground (white) and below-ground (shaded) and control (grey) (see Table I. for full species names) ce Index, Agricultural and Biosystems Engineering Vol:4, No:4, 2010 waset.org/Publication/6467 International Science Index, Agricultural and Biosystems Engineering Vol:4, No:4, 2010 waset.org/Publication/6467 International Science Index, Agricultural and Biosystems Engineering Vol:4, No:4, 2010 waset.org/Publicatio A similar trend was observed for the P uptake rate, Typha was the most efficient plant in this category. Nonetheless, the P uptake rate showed difference among the different species, Phragmites and Glyceria at p<0.05. A higher K accumulation was observed in the aerial biomass, without significant differences between the species (p<0.05). Fig. C. Plant growth and nutrient uptake 9 Uptake (g/m2/day) of Mg and repartition among the plant Mg TA ST P GM TA ST P GM 0.10 0.05 0.00 0.05 0.10 0.15 0.20 (g/m2/day) Fig. 6 Uptake (g/m2/day) of K and repartition among the plant organs, mean above-ground (white) and below-ground (shaded) and control (grey), (see Table I. for full species names) Fig. 9 Uptake (g/m2/day) of Mg and repartition among the plant scholar.waset.org/1307-6892/6467 International Scholarly and Scientific Research & Innovation 4(4) 2010 197 World Academy of Science, Engineering and Technology International Journal of Agricultural and Biosystems Engineering Vol:4, No:4, 2010 organs, mean above-ground (white) and below-ground (shaded) and control (grey), (see Table I. for full species names) organs, mean above-ground (white) and below-ground (shaded) and control (grey), (see Table I. for full species names) organs, mean above-ground (white) and below-ground (shaded) and control (grey), (see Table I. for full species names) treat effluent from inland saline aquaculture”, Aquacultural Engineering, vol 34, pp.1-7, March, 2005. treat effluent from inland saline aquaculture”, Aquacultural Engineering, vol 34, pp.1-7, March, 2005. [14] C. J. Richardson, “Freshwater wetlands: transformers, filters or sinks”, In: Sharitz, R.R., and Gibbons, J.W. (Eds.), Freshwater Wetlands and Wildlife. US DOE Office of Scientific and Technical Information, Oak Ridge, TN, USA. 1989. Ca and Mg were accumulated mainly in the above-ground organs at significantly higher rate in Typha than the other species, with values of 0.426 g/m2/d and 0.156 g/m2/d, respectively. The Ca uptake rate of Typha statistically differed from Glyceria, Scirpus and Phragmites at p<0.05. Harvesting the above-ground biomass is therefore a possibility to remove accumulated elements. [15] R.H. Kadlec, and R. L. Knight, Treatment Wetlands. CRC Press, Florida, USA, 1996, pp. 893. [16] C. C. Tanner, “Plants for constructed wetland treatment systems, a comparison of the growth and nutrient uptake of eight emergent species,” Ecological Engineering, vol. 7, pp. 59-83, Dec.1996. [17] C. Bragato, H. Brix, and M. Malagoli, “Accumulation of nutrients and heavy metals in Phragmites australis (Cav.) Trin. ex Steudel and Bolboschoenus maritimus (L.) Palla in a constructed wetland of the Venice lagoon watershed,” Environmental Pollution, vol. 144, Issue 3, pp. 967-975, Dec. 2006. IV. CONCLUSION An understanding of the importance and sustainability of different nutrient removal processes is necessary to improve the longer-term capabilities of constructed wetland systems [19]. The feasibility of using salt-tolerant plants (halophytes) as biofilters may remove nutrients and salt components from the saline aquaculture effluents. Typha, Phragmites, Glyceria and Scirpus could survive and facilitate the experimental treatment, so the salt-tolerant plant species may provide a suitable alternative for constructed wetlands receiving effluent water loaded by salinity and plant nutrient. [18] P. Klomjek, and S. Nitisoravut, “Constructed treatment wetland: a study of eight plant species under saline conditions,” Chemosphere, vol. 58, pp. 585–593, Aug. 2004. International Science Index, Agricultural and Biosystems Engineering Vol:4, No:4, 2010 waset.org/Publication/6467 [19] C. C. Tanner, J. S. Clayton, and M. P. Upsdell, “Effect of loading rate and planting on treatment of dairy farm wastewaters in constructed wetlands. II. Removal of nitrogen and phosphorus,” Water Res., vol. 29, no. 1, pp. 27–34, Jan. 1995. e Index, Agricultural and Biosystems Engineering Vol:4, No:4, 2010 waset.org/Publication/6467 International Science Index, Agricultural and Biosystems Engineering Vol:4, No:4, 2010 waset.org/ REFERENCES [1] M. F. Schwartz, and C. E. Boyd, ”Constructed wetlands for treatment of channel catfish pond effluents,” Prog. Fish Cult., vol. 57, pp. 255–266, Oct. 1995. [2] T. Redding, S. Todd, and A. Midlen, “The treatment of aquaculture wastewaters - a botanical approach,” J. Environ. Man., vol. 50, pp. 283– 299. 1997. [3] J. J. Brown, E. P. Glenn, K. M. Fitzsimmons, and S. E. Smith, “Halophytes for the treatment of saline aquaculture effluent,” Aquaculture, vol. 175, pp. 255–268, Febr.1999. [4] Y.-F. Lin, S.-R. Jing, D.-Y. Lee, and T.-W. Wang, “Nutrient removal from aquaculture wastewater using a constructed wetlands system,” Aquaculture, vol. 209, no. 1-4, pp. 169–184, June 2002. [5] C. Schulz, J. Gelbrecht, and B. Rennert, “Treatment of rainbow trout effluents in constructed wetland with emergent plants and subsurface horizontal water flow,” Aquaculture, vol. 217, pp. 207–221 Apr. 2003. [6] H. Brix, “Functions of macrophytes in constructed wetlands,” Water Sci. Tech., vol. 4, pp. 71–78. 1994. [7] E. A. Kohler, V. L. Poole, Z. J. Reicher, and R. F. Turco, “Nutrient, metal, and pesticide removal during storm and nonstorm events by a constructed wetland on an urban golf course,” Ecol. Eng., vol. 23, pp. 285–298. 2004. [8] B. H. Gu, M. J. Chimney, J. Newman, and M. K. Nungesser, “Limnological characteristics of a subtropical constructed wetland in south Florida (USA),” Ecol. Eng., vol. 27, pp. 345–360. 2006. al Science [9] A. Samecka-Cymerman, D. Stepien, and A. J Kempers, “Efficiency in removing pollutants by constructed wetland purification systems in Poland”, J. Toxicol. Env. Heal., vol. 67, no. 4. pp. 265–275, Jun. 2004. [10] Z. K. Zhu, “Plant salt tolerance,” Trends Plant Sci., vol. 6, pp. 66–71. 2001. [11] P. J. C. Kuiper, “Functioning of plant cell membranes under saline conditions. Membrane lipid composition and ATPases” In: Staples, R.C., Toenniessen, (Eds.), Salinity Tolerance in Plants. John Wiley, New York, 1984. pp. 77–91. [12] R. Tipirdamaz, D. Gagneul, C. Duhazé, A. Aïnouche, C. Monnier, D. Özkum, and F. Larher, “Clustering of halophytes from an inland salt marsh in Turkey according to their ability to accumulate sodium and nitrogenous osmolytes,“ Environmental and Experimental Botany, vol 57, no. 1-2, pp. 139-153, Aug. 2006. [13] A. J. Lymbery, R.G. Doupé, T.Benett, and M.R. Starcevich, “Efficacy of subsurface-flow wetland using the estuarine sedge Juncus kraussii to scholar.waset.org/1307-6892/6467 International Scholarly and Scientific Research & Innovation 4(4) 2010 198
W2955517707.txt	https://www.microbiologyjournal.org/download/21185/	en		Psychrotrophic Microbiota in Milk and Fermented Milk Products	Journal of pure and applied microbiology	2,019	cc-by	3,922	Patil J Pure Appl Microbiol, 13(2), 1257-1266 \| June 2019 Article 5638 \| https://dx.doi.org/10.22207/JPAM.13.2.68 Print ISSN: 0973-7510; E-ISSN: 2581-690X Research Article OPEN ACCESS Psychrotrophic Microbiota in Milk and Fermented Milk Products Sunita Hanamant Patil Department of Microbiology, K.R.T. Arts, B.H. Commerce & A.M. Science College, Savitribai Phule Pune University, Nasik - 422 002, India. Abstract Milk and dairy products form a significant part of the human diet. Temperature control is considered as the key to extend shelf life for dairy products. Psychrotrophs grow at temperature such as 7oC or lower but had higher optimal growth temperature. Most psychrotrophs produce extracellular enzymes while growing in milk and deteriorate its quality. Six types of samples constituting buffalo milk, flavored milk, curd, shrikhand, lassi and butter were collected in different seasons to study the psychrotrophic microbiota. Sixty psychrotrophic bacteria were grouped into fifteen clusters of very similar isolates. One isolate from each of the fifteen clusters of closely related isolates was identified by phylogenetic analysis namely Stenotrophomonas maltophilia, Pseudomonas putida, Pseudomonas stutzeri, Enterobacter hormaechei, Macrococcus caseolyticus, Exiguobacterium acetylicum, Exiguobacterium indicum, Micrococcus luteus, Kocuria rosea, Staphylococcus equorum, Ornithinmicrobium humiphilum, Planococcus psychrotoleratus, Bacillus cereus and two isolates upto genus level namely Exiguobacterium spp. and Kocuria spp. Fatty acid profile of milk sample inoculated with lipolytic psychrotrophic bacterial isolate was determined using Hydrolytic Extraction Gas Chromatographic method. Keywords: Psychrotrophs, dairy products, spoilage, gas chromatography. *Correspondence: sunitahp26@gmail.com; 00919423912287 (Received: 18 April 2019; accepted: 25 May 2019) Citation: Sunita Hanamant Patil, Psychrotrophic Microbiota in Milk and Fermented Milk Products, J Pure Appl Microbiol., 2019; 13(2):1257-1266. doi: 10.22207/JPAM.13.2.68 © The Author(s) 2019. Open Access. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License which permits unrestricted use, sharing, 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. Journal of Pure and Applied Microbiology 1257 www.microbiologyjournal.org Patil J Pure Appl Microbiol, 13(2), 1257-1266 \| June 2019 \| DOI 10.22207/JPAM.13.2.68 INTRODUCTION Psychrotrophs grow in cold environments due to unique features like cold shock proteins, short and unsaturated fatty acids in membranes, enzymes with high specific activity, thermolability and genetic changes to thermal shifts (Margesin et al., 2007). Milk is an ideal and perfect medium for growth of bacteria and therefore it gets contaminated very easily and readily. It is extremely perishable in nature and its shelf life is limited to 3 to 4 hours depending upon the temperature of storage (Kumar, A. and Seth, R., 2008). Extension of shelf life from hours to months has been a prime objective of the dairy industry for many years to meet the demands for increasing distribution times and distances (Goff, H.D. and Griffiths, M.W., 2006). The growth of microorganisms in milk causes disintegration of fat, protein and lactose and will soon make the product unsuitable for drinking. The microbial changes in milk and milk products are produced as a result of fermentation of one or more of the milk constituents by the causative micro-organisms. These milk fermentations may be of normal or abnormal type. Prolonging the storage of raw milk before pasteurization results in increasing the psychrotrophic, lipolytic and proteolytic bacteria that produce enzymes and cause the milk to change, such that there will be problems in processing the milk and quality of the milk products. Law, B.A. reported that Farm refrigeration of cow’s milk has led to increased levels of psychrotrophic Gram negative rods (Law, B.A., 1979). Proteolysis by the psychrotrophic microorganisms results in coagulation of the milk and unclean and bitter flavor of the milk (Burdova et al., 2002). Psychrotrophic bacteria grow luxuriantly in dairy products and multiply at refrigeration temperature (Xin et al, 2017). The problem of maintaining good shelf life or achieving extended keeping quality has always been one of great concern to milk processors. Keeping quality of milk products depends on initial quality of the products, on care and techniques employed in processing and distribution and on storage temperatures. milk, 5 samples of flavored milk, 5 samples of curd, 9 samples of shrikhand, 9 samples of lassi and 2 samples of butter were collected from ten different dairies of Nasik district. Isolation and identification Isolation of psychrotrophs was carried out by streak plate technique using sterile milk agar plates. Plates were incubated at 7oC for 10 days. Colonies developed on milk agar were picked up, purified repeatedly and preserved on nutrient agar slants and sub-cultured after every two months. Isolates of different morphotypes were appropriately coded and studied for morphological and biochemical characters. Physiological studies of the isolates The effect of pH, temperature and salt concentration on growth of isolates was studied using the method given by Cruickshank et al. (1975). Log phase cultures of each isolate of a cell density 106 cells per ml were inoculated in 0.01 ml quantity in to 5 ml of sterile nutrient broth in triplicates adjusted to different pH values such as 4, 5, 6, 7, 8, 9, 10, 11 and 12. To study the effect of different temperatures on growth, cells were inoculated in 0.01 ml quantity in to 5 ml of sterile nutrient broth in triplicates. The sets were incubated at different temperatures such as 7oC for ten days, 15oC, 25oC, 37oC, 45oC and 55oC for 24 hrs. Each of isolates were inoculated in 0.01 ml quantity in to 5 ml of sterile nutrient broth in triplicates having different concentrations of NaCl such as 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6% 6.5%, 7%, 7.5% and 8% for observation of growth at different salt concentrations. All the sets were incubated at 7oC for ten days. After incubation tubes were observed for the growth. Molecular identification and phylogenetic analysis The Microseq 16S rRNA gene kit (Applied Biosystems Division) was used for PCR and sequencing. The amplification of the genomic DNA was carried out by PCR, upon optimization. (ABI 9700 geneamp PCR). The facility was availed from Molecular Diagnostic Centre, Pune. The sequence was pasted in the BLAST tool and program was run using default parameters against non-redundant databases to avoid redundancy in the obtained hits. Hits obtained were analyzed and used for identification of the isolates. MATERIAL AND METHODS Collection and coding of samples Forty different samples of milk and milk products constituting 10 samples of pasteurized Journal of Pure and Applied Microbiology 1258 www.microbiologyjournal.org Patil J Pure Appl Microbiol, 13(2), 1257-1266 \| June 2019 \| DOI 10.22207/JPAM.13.2.68 Analysis of fatty acids profile The 500 ml tryptone dextrose yeast extract broth media was inoculated at 1.0% (v/v) with cultures of lipolytic isolate and incubated at 7oC for 10 days along with the control flask (uninoculated pasteurized milk). After incubation of milk samples (Control and test) fatty acid profile was determined using Hydrolytic Extraction Gas Chromatographic method (AOAC Official Method 996.06). RESULTS AND DISCUSSION Isolation of psychrotrophic bacteria and coding of isolates A total of sixty psychrotrophic bacterial isolates were obtained from six different types of milk and fermented milk products which constitutes nineteen isolates from milk namely RAtM-1, RAtM-2, RAtM-3, RAtM-4, WSM-1, WSM2, WSM-3,WTM-1, WTM-2, RRM-1, SPM-1, SShM1, SNM-1, WAM-1, WVM-1, WVM-2, WVM-3, RGM-1 and RGM-2, five isolates from flavored milk namely SShFM-1, RShFM-1, RRFM-1, RPFM-1 and WPFM-1, five isolates from curd namely SAtC-1, SGC-1, SSC-1, WSC-1and RSC-1, fourteen isolates from shrikhand namely RRS-1, RTS-1, WTS-1, WTS2, WShS-1, WShS-2, WAtS-1, RVS-1, RVS-2, WVS-1, WVS-2, RSS-1,WSS-1and WSS-2, fifteen isolates from lassi namely WTL-1, RShL-1, SShL-1,SNL-1, RGL-1, RGL-2, SGL-1, SGL-2, SSL-1, WRL-1, WRL-2, RAL-1, RAL-2, RAL-3 and RAL-4 and two isolates from butter samples namely RVB-1 and RSB-1. Microbiological examination of milk, flavored milk, curd, shrikhand, butter and lassi Santana et al. (2004) reported that the psychrotrophic bacteria in the refrigerated milk include 69% Gram negative bacilli that means 45% of the total proteolytic microorganisms in the dairy process. Table 1. Characterization of psychrotrophic bacteria S.N. Gram nature, motility, morphology No. of Isolates 1 2 19 1 3 4 5 6 7 Gram negative motile rods Gram negative motile short rod Gram positive nonmotile cocci Gram positive motile short rods Gram positive motile rods Gram positive nonmotile short rods Gram positive motile Cocci 22 9 5 3 1 Table 2. Utilization of Carbohydrates by isolates Table 3. Enzymatic characteristics S.N. Sugar Positive Isolates S.N. Enzyme 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 36 01 21 34 29 15 20 17 41 31 21 03 08 18 12 34 05 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Glucose Lactose Mannitol Sucrose Arabinose Xylose Galactose Cellobiose Maltose Trehalose Mannose Raffinose Rhamnose Ribose Sorbitol Fructose Dulcitol Journal of Pure and Applied Microbiology 1259 Catalase Oxidase Gelatinase Amylase Protease Lipase Lecithinase Phosphatase Urease Nitrate Reductase Arginine Hydrolase Ornithin Decarboxylase Indole test Methyl Red test Voges-Proskauer’s test Citrate Utilization test Positive Isolates 60 44 28 14 29 50 05 18 09 24 03 07 00 12 08 31 www.microbiologyjournal.org Patil J Pure Appl Microbiol, 13(2), 1257-1266 \| June 2019 \| DOI 10.22207/JPAM.13.2.68 During cold storage after milk collection, psychrotrophic bacterial populations dominate the microflora and their extracellular enzymes, mainly proteases and lipases contribute to the spoilage of dairy products (Elionora, H.Z. and Malka, H., 2007). Bashir et al. (2010) used citrate test, methyl red test and Voges-Proskauer’s test for identification of psychrotrophic bacteria isolated from freezer (-20°C). Physiological characteristics of the isolates The isolates showed variable response of growth towards temperature, pH and salt concentrations. All sixty isolates showed maximum growth at pH 7. None of the sixty isolates showed growth at pH 4. Only one isolate showed growth at a very high pH value i.e. pH 12. All sixty isolates showed growth at 7oC, 15oC and 25oC. Forty one isolates showed growth at 37oC. Out of sixty isolates, only five isolates showed growth at 45oC. None of the sixty isolates grew at 55oC temperature. All sixty isolates showed growth at mesophilic temperature as well as at 7oC, an important distinguishing characteristic feature of psychrotrophic bacteria. Psychrotrophic bacterial isolates showed variable response of the growth to different salt concentrations ranging from 0.5% to 8%. All sixty isolates showed growth at a salt concentration in a range of 0.5% to 3%. None of the isolates grew at 8% salt concentration. It can also be seen that twelve isolates tolerated maximum 3.0% salt concentration, eleven isolates tolerated maximum 3.5% salt concentration, five isolates tolerated maximum 4% salt concentration, eight isolates tolerated maximum 4.5% salt concentration, five isolates tolerated maximum 5% salt concentration, two isolates tolerated maximum 5.5% salt concentration, six isolates tolerated maximum 6% salt concentration, two isolates tolerated maximum 7% salt concentration and nine isolates tolerated maximum 7.5% salt concentration. Forming clusters of isolates based on morphological, cultural, biochemical and physiological characters On the basis of maximum similarities of characteristics studied, sixty isolates of Fig. 1. Phylogenetic analysis of RAL-4 Fig. 2. Phylogenetic analysis of WAM-1 and SPM-1 Journal of Pure and Applied Microbiology 1260 www.microbiologyjournal.org Patil J Pure Appl Microbiol, 13(2), 1257-1266 \| June 2019 \| DOI 10.22207/JPAM.13.2.68 psychrotrophic bacteria were grouped into fifteen clusters of very similar isolates and labelled as 1 to 15. Isolates of each of the fifteen clusters were obtained from different sources. Molecular identification and phylogenetic analysis One isolate from each of the fifteen clusters of closely related isolates was identified by phylogenetic analysis and sequencing 16S rRNA gene. The results of molecular identification of such fifteen representative isolates are presented in Table 5 and Fig. 1 to 11. As shown in Fig. 1, isolate namely RAL4 which was identified as Stenotrophomonas maltophilia (Acc. No.: GQ 982892) (100% similarity) forms cluster with the clade of Stenotrophomonas maltophilia (Acc. No.: HQ631975, JQ281541 and JQ266282) indicating that this strain (RAL-4) is identified as Stenotrophomonas maltophilia. As shown in Fig. 2, isolate namely WAM-1, which was identified as Pseudomonas putida (Acc. No.: GQ982897) (100% similarity) forms cluster with the clade of five isolates namely Pseudomonas spp. HaNA23 (Acc. No.: HM352366), Pseudomonas putida strain (Acc.No. JN696723), Pseudomonas putida strain (Acc. No.: JN696718), Pseudomonas putida strain (Acc. No.: AB680123) and Pseudomonas spp. R-26428 (Acc. No.: AM944713). It can be also seen from the phylogenetic analysis that strain SPM-1, which was identified as Pseudomonas stutzeri (Acc. No.:GQ982903) (99% similarity) by analysis of 16S rRNA gene sequence analysis forms cluster with the clade of isolates of Pseudomonas stutzeri. As shown in Fig. 3, isolate SAtC-1 (GQ982904) identified as Enterobacter hormaechei (100% similarity). As shown in Fig. 4, isolate SGL-1 (GQ982900) was identified as Macrococcus caseolyticus (100% similarity) forms clade with Macrococcus caseolyticus. Phylogenetic analysis revealed that an isolate namely WSM-2 (Acc.No.: GQ982891) which was identified as Exiguobacterium acetylicum (98% similarity) by analysis of 16S rRNA gene sequence did not cluster with clades of Exiguobacterium acetylicum (HM047519 and JN544145) instead forms separate clade indicating that probably this strain might be a different species of Exiguobacterium acetylicum on the basis of genotypic analysis. It can be seen from the phylogenetic analysis that an isolate namely RRFM-1 (Acc. No.: GQ982902) which was identified as Exiguobacterium indicum (99% similarity) by analysis of 16S rRNA gene sequence did not cluster with clade of Exiguobacterium indicum (Acc. No.: NR042347). The strain found to be very distant from the other strains of Exiguobacterium indicum in the phylogenic tree. Phylogenetic analysis revealed that Exiguobacterium spp. ((Acc. No.: GQ982893) formed a separate cluster from that of the known Exiguobacterium type strain viz. Exiguobacterium acetylicum (Acc. No.: GQ982891) and Exiguobacterium indicum ((Acc. No.: Fig. 3. Phylogenetic analysis of SAtC-1 Fig. 4. Phylogenetic analysis of SGL-1 Journal of Pure and Applied Microbiology 1261 www.microbiologyjournal.org Patil J Pure Appl Microbiol, 13(2), 1257-1266 \| June 2019 \| DOI 10.22207/JPAM.13.2.68 GQ982902). It was also seen from the phylogenetic analysis that strain WShS-2 which was identified as Exiguobacterium spp. ((Acc. No.: GQ982893) by analysis of 16S rRNA gene sequence did not cluster with the clade of Exiguobacterium acetylicum (Acc. No.: GQ982891) and Exiguobacterium indicum ((Acc. No.: GQ982902) but forms clade with Exiguobacterium aurantiacum (Acc. No.: HM030747) and Exiguobacterium mexicanum ((Acc. No.: JF505980) indicating that probably this strain is complex of Exiguobacterium aurantiacum and Exiguobacterium mexicanum (Fig. 5). As shown in Fig. 6, isolate RRM-1(GQ 982894) identified as Micrococcus luteus (100% similarity). Phylogenetic analysis reveals that an isolate namely WSS-2, which was identified as Kocuria rosea (Acc. No.: GQ 982895) (100% similarity) by analysis of 16S rRNA gene sequence did not forms cluster with clade of Kocuria rosea (Acc. No.: JQ684249) indicating that probably this strain is different from the Kocuria rosea (Acc. No.: JQ684249). Phylogenetic analysis also reveals that an isolate namelySShFM-1 identified as Kocuria spp. (Acc. No.: GQ982898) (100% similarity) forms cluster with clade of different species of Kocuria (Fig. 7). As shown in Fig. 8, SSL-1 (GQ982896) identified as Staphylococcus equorum (100% Fig. 5. Phylogenetic analysis of WSM-2, RRFM-1 and WShS-2 Fig. 6. Phylogenetic analysis of RRM-1 Fig. 7. Phylogenetic analysis of WSS-2 and SShFM-1 Journal of Pure and Applied Microbiology 1262 www.microbiologyjournal.org Patil J Pure Appl Microbiol, 13(2), 1257-1266 \| June 2019 \| DOI 10.22207/JPAM.13.2.68 similarity) and forms cluster with clade of Staphylococcus equorum. As shown in Fig. 9, RAtM-3 (GQ982899) was identified as Ornithinmicrobium humiphilum (100% similarity). Phylogenetic analysis revealed that an isolate namely WVS-1 which was identified as Planococcus psychrotoleratus (Acc. No.: GQ982901) (96% similarity) indicating that this strain (WVS-1) is identified as Planococcus psychrotoleratus. It can be also seen from phylogenetic analysis that strain WVS-1 did not cluster with the clade of Planococcus psychrotoleratus, indicating that probably this strain might be a different or new species of Planococcus (Fig. 10) As shown in Fig. 11, isolate namely SNM-1 (Acc. No.: JN230858) is identified as Bacillus cereus and this strain SNM-1shown 99% similarity with Bacillus cereus (Acc.No.: GU81593). Delbes et al. (2007) using the 16S rRNA gene, shown that culturable bacterial flora in raw milk were highly diversified. These organisms are one of the most prevalent groups found in the form of biofilms on dairy equipments causing spoilage of conventionally pasteurized liquid milk. Stenotrophomonas maltophilia (formerly Pseudomonas and Xanthomonas maltophilia) is a ubiquitous bacterium. It is increasingly prevalent in hospitals as an opportunistic human pathogen causing nosocomial infections (Berg et al., 1999). It is important to note that some heat resistant strains of B.cereus exhibiting psychrotrophic characteristics have been reported to grow and produce toxins in milk and dairy products at Fig. 8. Phylogenetic analysis of SSL-1 Fig. 9. Phylogenetic analysis of RAtM-3 Fig. 10. Phylogenetic analysis of WVS-1 Journal of Pure and Applied Microbiology 1263 www.microbiologyjournal.org Patil J Pure Appl Microbiol, 13(2), 1257-1266 \| June 2019 \| DOI 10.22207/JPAM.13.2.68 Fig. 11. Phylogenetic analysis of SNM-1 refrigeration temperatures (Guinebretiere et al., 2010). B.cereus causes problems to the food industry both by deteriorating the products and by endangering people’s life upon consuming them (Ghelardi et al., 2002). The storage and transportation of raw milk at low temperatures promote the growth of psychrotrophic bacteria and the production of thermo-stable enzymes, which pose great threats to the quality and shelflife of dairy products (Yuan et al. 2017) Analysis of fatty acids profile Analysis of fatty acid profile of milk sample inoculated with Stenotrophomonas maltophilia (RAL-4) showed variations in saturated fatty acids, monounsaturated fatty acids, polyunsaturated fatty acids and trans fatty acids. Increase in short chain as well as middle length chain saturated fatty acids was observed which are responsible for development of rancid, soapy, unclean or bitter flavors. According to Chen et al., (2003) and Huis Veld J (1996), the hydrolysis of as little as 1% of milk triglycerides could lead to rancid off flavors. Jaeger et al., (1994) reported that increased levels of short chain fatty acids (C4 - C8) give rise mainly Table 4. Clustering of isolates Cluster 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Isolates Sample RAtM-1, WVM-1, WTM-2, WPFM-1, WTS-2, SGL-2, RAL-4 (07) RAtM-4 , WAM-1, RVS-2, RAL-1 (04) SPM-1, WTM-1, SShM-1, WSC-1, WRL-2, RAL-2, RAL-3, RSB-1 (08) SAtC-1 (01) SGL-1 (01) WSM-2, WShS-1, WRL-1, WVM-2 (04) WSM-1, RRFM-1 (02) WVM-3, WSM-3, WShS-2 (03) RAtM-2, RRM-1, RGM-1, SGC-1, WVS-2, SNL-1, RGL-2, RVB-1 (08) RGM-2, WSS-2, WAtS-1 (03) SShFM-1 (01) RSC-1 , SSC-1, WSS-1, RSS-1, RRS-1, SShL-1, RGL-1, SSL-1, WTL-1 (09) RAtM-3, RTS-1, RShL-1 (03) WVS-1 (01) SNM-1, RPFM-1, RShFM-1, RVS-1, WTS-1 (05) Journal of Pure and Applied Microbiology Milk, Flavored milk, Shrikhand, Lassi Milk, Shrikhand, Lassi Milk, Curd, Lassi, Butter Curd Lassi Milk, Shrikhand, Lassi Milk, Flavored Milk Milk, Shrikhand Milk, Curd, Shrikhand, Lassi, Butter Milk, Shrikhand Flavored Milk Curd, Shrikhand, Lassi Milk, Shrikhand, Lassi Shrikhand Milk, Flavored Milk, Shrikhand 1264 www.microbiologyjournal.org Patil J Pure Appl Microbiol, 13(2), 1257-1266 \| June 2019 \| DOI 10.22207/JPAM.13.2.68 Table 5. Identification of bacterial isolates Code of Isolate Phylum Order Family Genus Species RAL-4 Proteobacteria Xanthomonadales Xanthomonadaceae Stenotrophomonas maltophilia WAM-1 Proteobacteria Pseudomonadales Pseudomonadaceae Pseudomonas putida SPM-1 Proteobacteria Pseudomonadales Pseudomonadaceae Pseudomonas stutzeri SAtC-1 Proteobacteria Enterobacteriales Enterobacteriaceae Enterobacter hormaechei SGL-1 Firmicutes Bacillales Staphylococcaceae Macrococcus caseolyticus WSM-2 Firmicutes Bacillales Bacillaceae Exiguobacterium acetylicum RRFM-1 Firmicutes Bacillales Bacillaceae Exiguobacterium indicum WShS-2 Firmicutes Bacillales Bacillaceae Exiguobacterium spp. RRM-1 Actinobacteria Actinomycetales Micrococcaceae Micrococcus luteus WSS-2 Actinobacteria Actinomycetales Micrococcaceae Kocuria rosea SShFM-1 Actinobacteria Actinomycetales Micrococcaceae Kocuria spp. SSL-1 Firmicutes Bacillales Staphylococcaceae Staphylococcus equorum RAtM-3 Actinobacteria Actinomycetales Intrasporangiaceae Ornithinmicrobium humiphilum WVS-1 Firmicutes Bacillales Planococcaceae Planococcus psychrotoleratus SNM-1 Firmicutes Bacillales Bacillaceae Bacillus cereus & TUV Lab., Pune for Hydrolytic Extraction Gas Chromatography. to rancid flavors, while the middle length chains (C10 - C12) give rise to most of the soapy, unclean or bitter flavors. Funding None CONCLUSION Although the milk and milk products are preserved primarily by refrigeration, yet the Psychrotrophic bacteria can responsible for their spoilage. On the basis of morphological, cultural, biochemical and physiological characteristics, sixty psychrotrophic bacterial isolates were grouped into fifteen clusters of closely related isolates. Psychrotrophic bacteria producing protease and lipase enzymes emphasizes the spoilage potential from them in dairy products because proteolysis is a main factor limiting shelf life of milk and milk products due to flavour and texture changes. Spoilage of milk and milk products may be avoided or prevented by controlling proteolytic and lipolytic psychrotrophic organisms or protease or lipase enzymes produced by them in milk and milk products. Data availability All datasets generated or analyzed during this study are included in the manuscript. Ethics Statement This article does not contain any studies with human participants or animals performed by any of the authors. REFERENCES 1. 2. 3. ACKNOWLEDGEMENTS The author is grateful to Dr. Sonali Sanghavi of Molecular Diagnostic Center, Pune, for base sequencing of the bacterial cultures Journal of Pure and Applied Microbiology 4. 1265 Bashir A., Imran J., Aamer A.S., Abdul H., Fariha H. Psychrotrophic bacteria isolated from -20°C freezer. African Journal of Biotechnology, 2010; 9: 718-724. Berg G., Roskot N., Smalla K. Genotypic and phenotypic relationships between clinical and environmental isolates of Stenotrophomonas maltophilia. Journal of Clinical Microbiology, 1999; 37: 3594–3600. Burdova O., Baranova M., Laukova A., Rozanska, H. and Rola J.G. Hygiene of pasteurized milk depending on psychrotrophic microorganisms. Bulletin of Veterinary Institute in Pulawy. 2002; 46: 325-329. Chen L., Daniel R.M. and Coolbear T. Detection and impact of protease and lipase activities in milk and milk powders. International Dairy Journal, 2003; 13: www.microbiologyjournal.org Patil J Pure Appl Microbiol, 13(2), 1257-1266 \| June 2019 \| DOI 10.22207/JPAM.13.2.68 5. 6. 7. 8. 9. 10. 11. 255-275. Cruickshank R., Dugid J.P., Shwain R.H.A. ‘Medical Microbiology’- A guide to the laboratory diagnosis and control of infection. 1975 11th edn. Delbes C.I., Mandjee A. and Montel M.C. Monitoring bacterial communities in raw milk and cheese by culture-dependent and independent 16S rRNA gene-based analyses. Applied and Environmental Microbiology, 2007; 73: 1882-1891. Elionora H.Z. and Malka H. Culturable psychrotrophic bacterial communities in raw milk their proteolytic and lipolytic traits. Applied and Environmental Microbiology, 2007; 73: 7162-7168. Ghelardi E., Celandroni F., Salvetti S., Barsoti C., Baggiani A., Senesi S. Identification and characterization of toxigenic Bacillus cereus isolates responsible for two food-poisoning outbreaks. FEMS Microbiology Letters, 2002; 208: 129-134. Goff H.D. and Griffiths M.W. Major advances in fresh milk and milk products. Journal of Dairy Science, 2006; 89: 1163-1173. Guinebretiere M.H., Velge P., Couvert O., Carlin F., Debuyser M.L., Christophe N.T. Ability of Bacillus cereus group to cause food poisoning varies according to phylogenetic affiliation (Groups I to VII) rather than species affiliation. Journal of Clinical Microbiology, 2010; 48(9): 3388-3391. Journal of Pure and Applied Microbiology 12. 13. 14. 15. 16. 17. 18. 1266 Huis Veld J.H.J. Microbial and biochemical spoilage of foods: an overview. International Journal of Food Microbiology, 1996; 33: 1-18. Jaeger K.E., Ransac S., Dijkstra B.W., Colson C., Hauvel M.V. and Misset O. Bacterial lipases. FEMS Microbiology Reviews, 1994; 15: 29-63. Kumar A. and Seth R. Chemical Quality of Spray Dried Skim Milk Powder. Indian Journal of Dairy Science, 2008; 61: 27-30. Law B.A. Enzymes of psychrotrophic bacteria and their effects on milk and milk products. Journal of Dairy Research, 1979; 46: 573-588. Margesin R., Neuner G. and Storey KB. Cold loving microbes, plants and animals fundamental and applied aspects. Naturwissenschaften, 2007; 94: 77-99. Santana E., Beloti V., Muller E., Ferreira M., Moraes L., Pereira M. and Gusmao V. Milk contamination in different points of the dairy process. Ciencias Agrarias Londrina, 2004; 5: 349-358. Xin L., Meng Z., Zhang L., Cui Y., Han X., Yi H. The diversity and proteolytic properties of psychrotrophic bacteria in raw cows’ milk from North China. Int. Dairy J., 2017; 66: 34–4110.1016/j.idairyj.2016.10.014. Yuan L., Sadiq F.A., Liu T.J., et al. Psychrotrophic bacterial populations in Chinese raw dairy milk. LWT, 2017; 84: 409-418. www.microbiologyjournal.org
https://openalex.org/W2997779362	https://periodicos.ufsc.br/index.php/emtese/article/download/1806-5023.2020v17n1p104/42314	Portuguese	null	A filosofia da socialidade de Raimo Tuomela: um resumo	Em Tese	2,020	cc-by	9,840	1 Publicação original em inglês, PREYER, G.; PETER, G., The Philosophy of Sociality of Raimo Tuomela. Studia z Historii Filozofii, Band 9, Nr. 4, 2018, International Journal of Advances in Philosophy, Vol. 2, No. 1, 2018. RESUMO Este estudo fornece uma visão geral da filosofia da socialidade e ontologia social de Raimo Tuomela. É mostrado como os assuntos de sua análise da fundação do domínio social estão conectados. Assim, a visão geral auxilia no estudo de sua obra ao comparar e contrastar sua abordagem com outros fundadores da análise da intencionalidade coletiva (Bratman, Gilbert e Searle), e seus tópicos relacionados, por exemplo, I-mode e We-mode, compromisso coletivo, grupos sociais, cooperação e instituições, e a análise contemporânea em curso. Conclui-se que o núcleo-duro da ontologia social de Tuomela é a sociologia da associação. PALAVRAS-CHAVE: Filosofia da Sociologia, Ontologia Social, Intencionalidade Coletiva, Teoria da Corporação, Decisão e Teoria dos Jogos, Teoria dos Sistemas Sociais, Instituições, Cooperação, Individualismo Metodológico e Holismo, Teoria Sociológica. A FILOSOFIA DA SOCIALIDADE DE RAIMO TUOMELA: Um Resumo1 The philosophy of sociality of Raimo Tuomela Hugo NERI Universidade de São Paulo, Filosofia, São Paulo Hugo.munhoz@usp.br https://orcid.org/0000-0001-6065-4661 Veridiana CORDEIRO Doutoranda Universidade de São Paulo, Sociologia, São Paulo Veridiana.cordeiro@usp.br https://orcid.org/0000-0001-9666-4386 Veridiana CORDEIRO Doutoranda Universidade de São Paulo, Sociologia, São Paulo Veridiana.cordeiro@usp.br https://orcid.org/0000-0001-9666-4386 Mais informações da obra no final do artigo Tradução Tradução Em Tese, Florianópolis, v. 17, n. 1, p. 104-126, jan/jun., 2020. Universidade Federal de Santa Catarina. ISSN 1806-5023. DOI: https://doi.org/10.5007/1806-5023.2020v17n1p104 3 (Ver a visão de Tuomela em Tuomela & K. Miller 1988, Tuomela 1984, 1995, 2000, 2002, 2013). Ver também obras de autores centrais, como Gilbert 2006, 2014, Bratman 2014, Searle 2010, Miller e Pettit 1993, 2003, pesquisas recentes no campo, por exemplo, os volumes da série Springer Studies in the Philosophy of Sociality and Chant, Hindriks, Preyer eds. 2014, Preyer, Peter eds. 2017, bem como artigos em várias revistas como Synthese, Economics and Philosophy, e ProtoSociology. 2 A ontologia social é o estudo dos elementos fundamentais do domínio social e, portanto, uma análise dos compromissos ontológicos dos estudos do domínio social, isto é, das melhores teorias sociais, científicas e explicativas, e suas ontologias postuladas. 3 ABSTRACT The study gives an overview of Raimo Tuomela’s philosophy of sociality and social ontology. It is shown how the subjects of his analysis of the foundation of the social domain are connected. Thereby the overview is a help for the study of his work in progress, and comparing and contrasting his account with the other founders of the analysis of collective intentionality (Bratman, Gilbert, and Searle), and their related topics, for example, I-mode and we-mode, collective commitment, social groups, cooperation, and institutions, and the ongoing contemporary analysis. It is concluded that the hard core of Tuomela’s social ontology is a sociology of membership. KEYWORDS: Philosophy of sociology; social ontology; collective intentionality; theory of corporation; decision and game theory; theory of social system; institutions; cooperation; methodological individualism and holism; sociological theory. Em Tese, Florianópolis, v. 17, n. 1, p. 104-126, jan/jun., 2020. Universidade Federal de Santa Catarina. ISSN 1806-5023. DOI: https://doi.org/10.5007/1806-5023.2020v17n1p104 1 Introdução Desde o final da década de 1970, Raimo Tuomela lidou com a fundação do domínio social, enfatizando a intencionalidade coletiva, a cooperação e outros assuntos relacionados como a ação coletiva, os grupos sociais, as instituições, a responsabilidade de grupo e os compromissos. Ao mesmo tempo, seu extenso trabalho teórico é uma contribuição para a ontologia social2 e para a filosofia analítica da socialidade (sociality). Desta maneira, o trabalho de Tuomela é relevante não apenas para filósofos e sociólogos, seus estudos também se sobrepõem com problemas da filosofia da mente, da psicologia e da ciência cognitiva. Do ponto de vista filosófico, Tuomela ampliou os conceitos de filosofia prática e de teoria da ação. É também aceito na literatura que Tuomela iniciou a análise do conceito de intencionalidade coletiva3. Além disso, ele defende uma análise fundamental dos recursos conceituais e dos pressupostos filosóficos do domínio social. Ele ampliou seus estudos anteriores para um trabalho integrado de uma "Teoria coletiva de aceitação da socialidade e da ontologia social", que reúne a análise da intencionalidade coletiva, dos grupos sociais, da cooperação, da instituição social, da responsabilidade coletiva e da evolução sociocultural (Tuomela, 2007). Do ponto de vista sociológico, o trabalho de Tuomela é relevante por conta de seus impactos sobre a teoria dos sistemas sociais e a teoria sociológica em geral. Seu trabalho também pode ser aplicado ao campo da inteligência artificial de múltiplos agentes. Sua abordagem está baseada em um conceito abrangente de racionalidade que implica na racionalidade teleológica (com referência a fins), a valorativa e, também, a expressiva (Tuomela, 2000, pp. 299-300). Do ponto de vista teórico, Tuomela argumenta que a teoria da escolha racional baseada na ação, preferências e grau de crença, somada à teoria dos jogos cooperativos são ferramentas matemáticas poderosas, mas essas propostas são conceitual e teoricamente muito fracas e precisam de modificações – por exemplo, a teoria da escolha racional não considera seriamente os conceitos de objetivos, razão e compromissos. 105 Em Tese, Florianópolis, v. 17, n. 1, p. 104-126, jan/jun., 2020. Universidade Federal de Santa Catarina. ISSN 1806-5023. DOI: https://doi.org/10.5007/1806-5023.2020v17n1p104 Vamos delinear a seguir sua filosofia da socialidade e da ontologia social. Para tanto, é instrutivo começar com sua análise construtivista da filosofia da socialidade, começando pelo conceito-chave de aceitação coletiva e sua análise das We-attitudes e I- attitudes4. Ambos estão em sua consideração aplicada à caracterização das características constitutivas dos grupos sociais. 1. A razão de grupo – uma razão unificada como condição de participação em atividades constitutivas de grupo; 2. A condição de coletividade – algo equivalente à expressão: necessariamente “estar no mesmo barco" e; 3. Compromisso coletivos – os compromissos coletivos são geralmente baseados nas intenções conjuntas dos membros. Assim, a intenção conjunta implica um compromisso coletivo. Tais compromissos podem, em alguns casos, ser as razões dos membros para desempenhar seus papeis na ação do grupo em jogo. 1 Introdução A análise conceitual do ponto de vista compartilhado também é útil para a análise da autoridade, cooperação, normas sociais e instituições do domínio social. Considera-se aqui a análise de Tuomela (2013) da organização do grupo igualitário e hierárquico ao longo da distinção entre grupos autônomos e não autônomos que podem estar relacionados a diferentes aspectos da aceitação coletiva. Finalmente, alguns pontos são feitos sobre a filosofia da sociedade de Tuomela que teria como núcleo uma sociologia de pertencimento a grupos (membership). Isso ocorre porque o conceito básico da análise de todas as características do domínio social é uma teoria da associação. Ao final, apresenta-se uma conclusão sobre sua visão. 4 N.T. A decisão de não tradução dos conceitos compostos por I- e We- como I-mode ou I-attitudes é deliberada. Acreditamos que sua tradução colaboraria negativamente para a compreensão geral do texto. Em Tese, Florianópolis, v. 17, n. 1, p. 104-126, jan/jun., 2020. Universidade Federal de Santa Catarina. ISSN 1806-5023. DOI: https://doi.org/10.5007/1806-5023.2020v17n1p104 2 A filosofia das práticas sociais O que há de novo na filosofia das práticas sociais de Tuomela é que essas atividades são constituídas pela intencionalidade coletiva, basicamente como atividades em we-mode que são compartilhadas. Ele chama essa visão de "o amplo programa do construtivismo social", pois uma parte central dos sistemas de comunicação é construída coletivamente pelos conceitos de aceitação coletiva. Esta é uma questão da ontologia do domínio social como um objeto da análise construtivista. No entanto, ele também defende o "programa estreito do construtivismo", isto é, a intencionalidade coletiva em we-mode compartilhado como sendo basal para a construção conceitual e para a manutenção das instituições sociais. A intencionalidade coletiva em sentido forte é caracterizada por: 4 N.T. A decisão de não tradução dos conceitos compostos por I- e We- como I-mode ou I-attitudes é deliberada. Acreditamos que sua tradução colaboraria negativamente para a compreensão geral do texto. 106 Em Tese, Florianópolis, v. 17, n. 1, p. 104-126, jan/jun., 2020. Universidade Federal de Santa Catarina. ISSN 1806-5023. DOI: https://doi.org/10.5007/1806-5023.2020v17n1p104 O programa construtivista é pressuposto para a análise das atividades conceituais e do seguimento das regras como um comportamento governado por padrões. Neste sentido, Tuomela concorda com W. Sellars e L. Wittgenstein. O conceito básico é orientado para um padrão coletivo de comportamento, que não é analisado satisfatoriamente por Sellars. (Tuomela 2002, pp. 40-52). Ao contrário de uma interpretação holística e comunitária do seguimento das regras, supõe-se que o pensamento e a fala sejam possíveis solitariamente. Isso é enfatizado por Tuomela, porque atividades conceituais totalmente desenvolvidas, que exigem atos de fala, exigem um contexto social. O "amplo programa de construtivismo social" deve defender a "Teoria Coletiva de Aceitação da Socialidade". Portanto, a tarefa é mostrar como a "aceitação coletiva", analisada apropriadamente pelo we-mode é ontologicamente a característica básica do domínio social. A aceitação coletiva está baseada na construção performativa de entidades sociais pelos membros de grupos e instituições sociais. Ela é também autorreferencial, por exemplo, o euro é considerado como dinheiro somente se a moeda for aceita entre os membros de um domínio social específico5. Essas características do domínio social também são discutidas, por exemplo, por Barnes (1983), Bloor (1997), Kusch (1997), Searle (1995, 2001, 2010) e Tuomela (2013). Tuomela contrasta o we-mode com o I-mode. O we-mode basicamente significa pensar e atuar como um membro de um grupo. 7 Veja Tuomela 2007, 211, sobre aceitação coletiva e socialidade, 187-199. p 6 N.T. O bicondicional aparece na literatura pela expressão se e somente se. 7 We-mode ↔ 6coletividade ↔ socialidade coletiva ↔ (ampla) institucionalidade7. 5 Tuomela 2002, em seu "relato central das práticas sociais", 89-99. 6 Em Tese, Florianópolis, v. 17, n. 1, p. 104-126, jan/jun., 2020. Universidade Federal de Santa Catarina. ISSN 1806-5023. DOI: https://doi.org/10.5007/1806-5023.2020v17n1p104 2 A filosofia das práticas sociais Entretanto, a intencionalidade coletiva não é suficiente para a análise das práticas sociais institucionalizadas e para a comunicação, pois os componentes institucionalizados do domínio social também são estruturados por normas sociais, como expectativas de expectativas, sanções, autoridade e regulamentos de comunicação. Assim, "teoria da aceitação coletiva da socialidade" contribui com a análise de instituições como a linguagem, o dinheiro, o casamento, a propriedade e as organizações formais. A estrutura analítica do núcleo da filosofia de Tuomela de socialidade e ontologia social conecta cautelosamente, por equivalências, os seguintes níveis analíticos: 107 107 Em Tese, Florianópolis, v. 17, n. 1, p. 104-126, jan/jun., 2020. Universidade Federal de Santa Catarina. ISSN 1806-5023. DOI: https://doi.org/10.5007/1806-5023.2020v17n1p104 Os itens do we-mode (intenções, crenças, grupos, cooperação e instituições) pressupõem a aceitação coletiva. O fundamento dessa estrutura é a associação em grupos no domínio social, porque a aceitação coletiva dos membros como membros é uma condição constitutiva. 8 Sobre intenções conjuntas, Tuomela 2002, cap. 2, 2007, cap. 4, 2000, capítulo 3, e especialmente 2013, cap. 3. Em Tese, Florianópolis, v. 17, n. 1, p. 104-126, jan/jun., 2020. Universidade Federal de Santa Catarina. ISSN 1806-5023. DOI: https://doi.org/10.5007/1806-5023.2020v17n1p104 3 Aceitação coletiva O conceito-chave da filosofia de socialização de Tuomela é a aceitação coletiva do we-mode. Este conceito tem para ele relevância paradigmática na filosofia e ontologia social. A aceitação coletiva envolve um acordo sobre as we-attitudes, ou seja, uma atitude particular de membros de grupos sociais para um conteúdo de atitude p, envolvendo o compromisso coletivo dos membros do grupo enquanto membros para satisfazerem conjuntamente p. A maioria dos compromissos coletivos são do nível de grupo sociais e intencionais, mas eles não precisam ser normativos em sentido moral. A aceitação coletiva é analisada linguisticamente dado que o conceito requer que os membros possuam um tipo particular de atitude coletiva em relação a uma sentença ou proposição em questão. Assim, a aceitação coletiva envolve expectativas instanciadas e expectativas de expectativas dos membros do domínio social e pode aparecer com intensidades diferentes. A unidade central da intencionalidade coletiva é a intenção conjunta, isto é, os membros de um grupo social compartilham como grupo o conteúdo de uma intenção x. Uma intenção típica pode ser expressada da seguinte maneira: "nós, como grupo, fazemos x"8. Assim, as intenções conjuntas no we-mode não podem ser reduzidas às intenções do I-mode. O conteúdo de tais intenções conjuntas é o seguinte: "Os participantes procuram conjuntamente, enquanto um grupo, que x (x = estado do caso ou uma ação conjunta) aconteça". As we-intentions são muitas vezes "intenções de objetivo", isto é, intenções de que algum estado de coisas, possivelmente coletivo (por exemplo, que haja paz na Síria), seja feito ou mantido. A distinção entre metas de grupo é uma estratégia dos participantes, porque a comunidade de intenções coletivas é um acordo assumido entre as partes. Pode ser que o acordo sobre ações coletivas seja o de atingir o objetivo privado subjacente de alguns Em Tese, Florianópolis, v. 17, n. 1, p. 104-126, jan/jun., 2020. Universidade Federal de Santa Catarina. ISSN 1806-5023. DOI: https://doi.org/10.5007/1806-5023.2020v17n1p104 Em Tese, Florianópolis, v. 17, n. 1, p. 104-126, jan/jun., 2020. Universidade Federal de Santa Catarina. ISSN 1806-5023. DOI: https://doi.org/10.5007/1806-5023.2020v17n1p104 108 participantes. Mas em todo caso, os objetivos coletivos pressupõem a cooperação coletiva e a coordenação das ações como meio. 9 (Tuomela 2007, Cap. 4, 5. Os capítulos devem ser lidos em conjunto com Tuomela 2002, Cap. 4). Em Tese, Florianópolis, v. 17, n. 1, p. 104-126, jan/jun., 2020. Universidade Federal de Santa Catarina. ISSN 1806-5023. DOI: https://doi.org/10.5007/1806-5023.2020v17n1p104 3 Aceitação coletiva Para tanto, Tuomela introduz a metáfora do "quadro de avisos" da aceitação coletiva para descrever a característica conceitual geral de formar intenções e crenças conjuntas, por exemplo, o comitê organizador se comunica em um quadro negro "Os membros do grupo g vão limpar o parque no próximo sábado. Quem participará, inscreva-se aqui9". A "vontade" expressa uma intenção, e não uma previsão. Essa abordagem tem as seguintes vantagens: Isso explica a aceitação coletiva e compartilhada de atitudes (intenções, crenças e outras atitudes voluntárias) e exemplifica a aceitação com sendo o pressuposto de uma tarefa compartilhada. Se um número suficiente de membros se juntarem para limpar o parque, então existe uma intenção categórica, não condicional e uma intenção conjunta entre os membros, que assinaram a lista: 1. Os membros se comprometeram com o objetivo em questão; 2. Essa abordagem não assume uma intenção prévia de formar a intenção conjunta de limpar o parque; 3. Os membros podem ter diferentes funções e funções de status social simétrico, assimétrico e complementar, para realizar a tarefa; 4. Existe uma forte intenção epistêmica, porque os membros podem verificar o conteúdo da intenção; e 5. Essa abordagem também é aplicável à formação da intenção do I-mode. Ao revisar sua versão de aceitação coletiva em relação a grupos igualitários e hierárquicos, Tuomela analisou grupos sociais estruturados com diferentes papéis e posições de status em que nem todos os membros precisam participar da aceitação coletiva (Tuomela 2013, capítulo 5, especialmente pp.127-130.). Ele também discute outras abordagens sobre a formação de atitudes grupais no capítulo 5 do referido livro. A análise do domínio social pelo "Modelo de Aceitação" é "filosófica (conceitual, metafísica)" e da "teoria do design". (Tuomela 2002, 158). Tuomela enfatiza que a aceitação coletiva envolve um componente "processual" (Tuomela 2013, 125). A orientação dos possíveis participantes da "formação voluntária de atitudes grupais" é "qual atitude (por exemplo, desejo ou crença, como o caso presente pode exigir) devemos aceitar para nosso grupo nesta situação?" (Tuomela 2013, 125.) Portanto, a "Teoria Coletiva de Aceitação da Socialidade" leva à análise das We e I- attitudes dos membros dos grupos sociais. (Tuomela 2007, Cap. 4, 5. Os capítulos devem ser lidos em conjunto com Tuomela 2002, Cap. 4 Em Tese, Florianópolis, v. 17, n. 1, p. 104-126, jan/jun., 2020. Universidade Federal de Santa Catarina. ISSN 1806-5023. DOI: https://doi.org/10.5007/1806-5023.2020v17n1p104 109 10 N.T. A expressão “acredita que p” ou “uma ação que p” é frequentemente empregada em trabalhos de lógica. Para melhor compreensão ‘p’ é um substituto para acreditam que “real é dinheiro”, acreditam que “a grama é verde”, e assim por diante. 1. Socialidade coletiva – as características sociais coletivas das atividades, e 2. “mera” coletividade – dado que existem muitas ações sociais que não são realizadas coletivamente, por exemplo, como alguém pensa sobre os outros. 3. Há também atividades coletivas que não são sociais, por exemplo, quando as pessoas estão andando na rua abrindo seus guarda-chuvas para não ficarem molhadas (M. Weber). 4 We- e I-attitudes Tuomela aplica a ferramenta conceitual de aceitação coletiva a vários tipos de práticas sociais. As práticas sociais e a comunicação podem ser de diferentes tipos, por exemplo, trabalhar em uma empresa comercial, participar do tráfego rodoviário, de reuniões e de festas de Natal. Todos esses eventos comunicativos estão conectados por um conceito unificado de atitude coletiva. Essa atitude pode ser analisada por meio do we-mode compartilhado. Segue a seguir uma definição de We-attitude: A acredita que p e acredita que os outros no grupo acreditam que p, e tudo isso é mutuamente acreditado no grupo, se todos os membros tiverem uma crença desse tipo, estamos lidando com um tipo (fraco) de crença de grupo de que p10. O conteúdo deste modo é um terreno social compartilhado que os participantes têm para a ação social coletiva pretendida, isto é, os membros do grupo em we-mode atuam juntos como um agente único. Mas isso não implica que o grupo seja, de fato, um agente ou uma pessoa em sentido forte, mas apenas em sentido metafórico. (Tuomela, 2002, pp. 128-129; Tuomela, 2013, capítulo 2). A característica distintiva do we-mode e o I-mode é que no primeiro há a aceitação coletiva do grupo e o compromisso conjunto em contraste com um compromisso privado, no caso do I-mode. Com relação a essa distinção, sempre levamos em consideração condições de interação assimétricas. Pode ser que eu esteja comprometido com uma atitude privada sem ser comprometido coletivamente e vice- versa. Vale enfatizar que Tuomela faz a distinção entre: 1. Socialidade coletiva – as características sociais coletivas das atividades, e 1. Socialidade coletiva – as características sociais coletivas das atividades, e 1. Socialidade coletiva – as características sociais coletivas das atividades, e 2. “mera” coletividade – dado que existem muitas ações sociais que não são realizadas coletivamente, por exemplo, como alguém pensa sobre os outros. 3. Há também atividades coletivas que não são sociais, por exemplo, quando as pessoas estão andando na rua abrindo seus guarda-chuvas para não ficarem molhadas (M. Weber). 2. “mera” coletividade – dado que existem muitas ações sociais que não são realizadas coletivamente, por exemplo, como alguém pensa sobre os outros. 3. Há também atividades coletivas que não são sociais, por exemplo, quando as pessoas estão andando na rua abrindo seus guarda-chuvas para não ficarem molhadas (M. Weber). 3. 4 We- e I-attitudes Há também atividades coletivas que não são sociais, por exemplo, quando as pessoas estão andando na rua abrindo seus guarda-chuvas para não ficarem molhadas (M. Weber). O predicado "coletivo" aplica-se a um conjunto de pessoas, mas o predicado "social" é instanciado para atitudes que levam em conta as atitudes dos outros. O 10 N.T. A expressão “acredita que p” ou “uma ação que p” é frequentemente empregada em trabalhos de lógica. Para melhor compreensão ‘p’ é um substituto para acreditam que “real é dinheiro”, acreditam que “a grama é verde”, e assim por diante. 110 Em Tese, Florianópolis, v. 17, n. 1, p. 104-126, jan/jun., 2020. Universidade Federal de Santa Catarina. ISSN 1806-5023. DOI: https://doi.org/10.5007/1806-5023.2020v17n1p104 componente conceitual relevante das atitudes coletivas é o objetivo coletivo compartilhado. Tuomela faz as distinções típicas-ideais entre: componente conceitual relevante das atitudes coletivas é o objetivo coletivo compartilhado. Tuomela faz as distinções típicas-ideais entre: 1. Objetivos coletivos, que se baseiam em um objetivo mútuo (we-want), e 2. Objetivos coletivos intencionados. 1. Objetivos coletivos, que se baseiam em um objetivo mútuo (we-want), e 2. Objetivos coletivos intencionados. 2. Objetivos coletivos intencionados. O primeiro tipo é o mais fraco e o segundo tipo é um conceito forte por implicar em um objetivo coletivo intencionado, embora não haja intenção conjunta planejada. O primeiro tipo é o mais fraco e o segundo tipo é um conceito forte por implicar em um objetivo coletivo intencionado, embora não haja intenção conjunta planejada. 1. Os participantes têm uma intenção conjunta básica de atingir conjuntamente um objetivo específico. O terceiro tipo significa que cada participante pretende contribuir com a ação conjunta e os participantes formam a intenção de alcançar o (mesmo) objetivo. Esta intenção é realizada pelos participantes como membros de um grupo, e eles estão mutuamente comprometidos. Essa é uma versão forte de um compromisso coletivo. 2. Objetivos atribuídos aos membros de um coletivo social, por exemplo, objetivos das organizações. 2. Objetivos atribuídos aos membros de um coletivo social, por exemplo, objetivos das organizações. O quarto tipo mostra os membros como sendo a base da atribuição de objetivos uma vez que a atribuição é instanciada para os membros de uma coletividade. Se alguém é um membro de uma coletividade, esse alguém satisfaz as condições particulares que tornam possível o seu pertencimento ao grupo. A estrutura de uma coletividade inclui: 3. Um sistema de decisões que corrige os objetivos dos membros. Em Tese, Florianópolis, v. 17, n. 1, p. 104-126, jan/jun., 2020. Universidade Federal de Santa Catarina. ISSN 1806-5023. DOI: https://doi.org/10.5007/1806-5023.2020v17n1p104 4 We- e I-attitudes Isso implica que nem todos os membros têm objetivos coletivos fixos, mas são obrigados a acompanhar os objetivos da coletividade; 4. Uma coletividade pode perseguir um objetivo, embora os membros tenham apenas uma intenção fraca. Esta é a característica estatística do conceito de metas coletivas. Esses assuntos também dizem respeito ao sistema de autoridade. Tuomela analisa a intencionalidade coletiva forte em termos de pensamento e raciocínio em we-mode que está conectado às atividades dos membros do grupo em we- mode, com base na razão do grupo, na satisfação da condição da coletividade e no compromisso coletivo. A intencionalidade coletiva está ligada à cooperação, que é um exemplo significativo de estados coletivos intencionais mentais e de ação. Ambos estão 111 Em Tese, Florianópolis, v. 17, n. 1, p. 104-126, jan/jun., 2020. Universidade Federal de Santa Catarina. ISSN 1806-5023. DOI: https://doi.org/10.5007/1806-5023.2020v17n1p104 Em Tese, Florianópolis, v. 17, n. 1, p. 104-126, jan/jun., 2020. Universidade Federal de Santa Catarina. ISSN 1806-5023. DOI: https://doi.org/10.5007/1806-5023.2020v17n1p104 111 baseados em um "nós", ou seja, um grupo social cujos membros se envolvem em raciocínio e ação coletiva. baseados em um "nós", ou seja, um grupo social cujos membros se envolvem em raciocínio e ação coletiva. 11 Sobre o conceito de grupo social, Tuomela 2007, capítulo 1, 2013, cap. 2. Em Tese, Florianópolis, v. 17, n. 1, p. 104-126, jan/jun., 2020. Universidade Federal de Santa Catarina. ISSN 1806-5023. DOI: https://doi.org/10.5007/1806-5023.2020v17n1p104 5 Grupos Sociais Um grupo social faz parte de um sistema de ação social/comunicação, ou seja, um sistema social com diferentes ambientes. Os grupos não são uma pessoa, não têm um corpo, mas são compostos de membros. Assim, não possuem intencionalidade intrínseca, mas apenas extrínseca. Na abordagem mais recente de Tuomela, os grupos sociais são vistos como agentes do grupo funcional aos quais os predicados, ações e responsabilidades dos estados mentais podem ser atribuídos aos membros do grupo. Um raciocínio baseado em grupo é: "Ao operar como membros do grupo, queremos X e levamos isso para exigir que juntos façamos Y e, portanto, o façamos como um grupo" (Tuomela, 2013, p. 99). Os grupos consistem basicamente dos membros do grupo, de suas inter-relações e também das ferramentas materiais necessárias, especialmente no caso das organizações. Portanto, a versão de Tuomela do agente do grupo refere-se à unidade de pertencimento ao grupo. Assim, o domínio social é constituído por membros e não-membros de grupos, suas funções de status, papéis e expectativas. A adesão ao grupo pressupõe o conhecimento mútuo dessa associação11. A caracterização substancial da socialidade de Tuomela é que o domínio social é baseado no ponto de vista do grupo social, isto é, a perspectiva compartilhada dos membros dos grupos. O we-mode pressupõe um grupo social no sentido forte. Portanto, a afiliação de grupo (membership) é um pressuposto para a condição constitutiva. Mas isso pressupõe também compromissos coletivos e seu acesso público. A intencionalidade coletiva forte (we-mode) define que atuar intencional e conjuntamente como um grupo deve satisfazer "razões de grupo impostas", "compromissos coletivos" e a "condição de coletividade". Assim, o núcleo duro da filosofia da socialidade de Tuomela é a "teoria coletiva de aceitação da socialidade", ou seja, é mostrar como tal "teoria" funciona em conjunto com o ponto de vista compartilhado e os conceitos de grupo. O we-mode implica em conceitos grupais dos membros dos grupos sociais. O equivalente linguístico para isso é a expressão (auto)referencial coletiva, por exemplo, "nós", "nosso" ou "um de nós". O que não é trivial, dado que o ponto de vista do grupo 112 Em Tese, Florianópolis, v. 17, n. 1, p. 104-126, jan/jun., 2020. Universidade Federal de Santa Catarina. ISSN 1806-5023. DOI: https://doi.org/10.5007/1806-5023.2020v17n1p104 Em Tese, Florianópolis, v. 17, n. 1, p. 104-126, jan/jun., 2020. Universidade Federal de Santa Catarina. ISSN 1806-5023. Em Tese, Florianópolis, v. 17, n. 1, p. 104-126, jan/jun., 2020. Universidade Federal de Santa Catarina. ISSN 1806-5023. DOI: https://doi.org/10.5007/1806-5023.2020v17n1p104 12 Tuomela 2007, capítulo 3, analisou soluções deste problema. 5 Grupos Sociais No caso normal, a aceitação coletiva significa que: 1. Cada participante concorda com a intenção e é verdade que ele próprio tem a intenção de fazer x; 2. Existe uma crença compartilhada sobre isso que os membros solteiros do grupo coincidem com o acordo deles; 3. Os participantes estão comprometidos com o seu acordo – os acordos coletivos podem variar em sua força. A condição de coletividade é uma versão da "regra de ouro" aplicada aos grupos sociais. O membro do grupo na atitude do I-mode está comprometido com seus objetivos e crenças privadas. A condição de coletividade toma como base o "ponto de vista compartilhado completo" como condição de pertencimento ao grupo (Tuomela, 2007). O I- mode da atitude compartilhada ocorre em uma versão diferente. Uma pessoa que está em uma conexão de grupo examina um objetivo particular p em uma situação sabendo que outros também examinam esse mesmo objetivo. Este é um exemplo simples para uma atitude de conformidade que é compartilhada pelo grupo. O we-mode é diluído no I-mode. Neste caso, o we-mode representa um I-mode. Portanto, esse caso é relevante porque qualquer coletividade, que tem algumas propriedades de identidade intersubjetiva, sempre se baseia em um conhecimento compartilhado de crenças, podendo ter uma we-attitude. Por exemplo, pessoas que aguardam a partida de um trem em uma estação podem ter crenças em we-mode "porque o trem esperado está atrasado". No caso das intenções adequados de we-mode, o grupo não é necessário, diferentemente do I-mode correspondente, dado que os motivos são contingentes. No I-mode pode acontecer uma circularidade, por exemplo, "Eu quero, se você quiser ...". Nesses casos, ocorre um dilema de coordenação: eu quero x (uma das minhas opções de fazer alguma coisa) se você quiser fazer x, mas você quer x se eu quiser x e assim por diante12. 5 Grupos Sociais DOI: https://doi.org/10.5007/1806-5023.2020v17n1p104 112 está conectado simultaneamente com a aceitação de objetivos, valores, crenças e normas do grupo aos quais os membros estão comprometidos. Estes são compromissos autovinculantes enquanto membros de grupos. Portanto, o fundamento da perspectiva de um grupo social é a autovinculação de seus membros. Tuomela chama isso de "atitude em we-mode". Ao contrário, o I-mode é a atitude de uma pessoa privada. Um ponto crucial para sua teoria é que as we-attitudes não devem ser reduzidas às I-attitudes. Tuomela analisa os modos de compartilhamento pelo conceito de intenções coletivas e crenças mútuas compartilhadas. A descrição ideal-típica das we-attitudes de uma pessoa no contexto dos grupos sociais é a seguinte: 1. A atitude existe apenas se o grupo tiver essa atitude; 2. É pressuposto que as atitudes são compartilhadas entre os membros; 3. É assumido que a atitude compartilhada mútua é compartilhada factualmente. As atitudes compartilhadas são uma base social dos membros de grupos para a ação coletiva intencional correspondente e os participantes da cooperação são comprometidos coletivamente com o objetivo correspondente, que eles não têm à sua disposição voluntariamente. Tuomela nomeia esta abordagem de "teoria de metas coletivas de cooperação". Assim, ela está ligada à análise das we-attitudes e grupos sociais conjuntamente a sua análise da cooperação. Está ligada também às crenças coletivas, em que "nós, os membros do grupo g, acreditamos que p". (Tuomela, 2002: 41) P é a crença aceita pelos membros de g. A crença tem um benefício para o grupo, e os membros estão comprometidos com essa crença idealmente. Já uma atitude ou ação do we-mode pressupõe satisfazer a condição de coletividade. Como afirma Tuomela: Os membros enquanto membros do grupo social que trabalham juntos em um objetivo comum, realizando x, têm uma intenção conjunta, que satisfaz a condição verdadeira de razões quase-conceituais para a intenção conjunta de interpretação convencional, se e somente se a satisfação de um membro for simultaneamente a satisfação de cada membro do grupo. (Tuomela 2002, pp. 29-36). Aqui há o pressuposto geral de que os membros do grupo têm um conhecimento compartilhado sobre a condição de coletividade. O objetivo coletivo do we-mode e o símbolo instanciado da intenção coletiva dos membros solteiros estão, necessariamente, 113 113 Em Tese, Florianópolis, v. 17, n. 1, p. 104-126, jan/jun., 2020. Universidade Federal de Santa Catarina. ISSN 1806-5023. DOI: https://doi.org/10.5007/1806-5023.2020v17n1p104 relacionados com a "aceitação coletiva" (Tuomela, 2007, pp. 47-51). Em Tese, Florianópolis, v. 17, n. 1, p. 104-126, jan/jun., 2020. Universidade Federal de Santa Catarina. ISSN 1806-5023. DOI: https://doi.org/10.5007/1806-5023.2020v17n1p104 6 Autoridade Para Tuomela, a aceitação coletiva entre os membros dos grupos e as atitudes de grupo são o núcleo conceitual de sua filosofia da socialidade e da ontologia social. A análise leva à definição da função de autorização e dos membros autorizados de grupos 114 114 Em Tese, Florianópolis, v. 17, n. 1, p. 104-126, jan/jun., 2020. Universidade Federal de Santa Catarina. ISSN 1806-5023. DOI: https://doi.org/10.5007/1806-5023.2020v17n1p104 sociais. A autorização é fundamental para os motivos grupais, ação em grupo, crença de grupo e a atitude aceita em geral. (Tuomela, 2007, pp. 129-134) A "estrutura de poder normativo" dos grupos sociais é baseada na autorização de alguns membros do grupo. O "relato posicional de atitudes grupais" de Tuomela significa que existe uma diferenciação dentro de um sistema social, grupo ou organização entre membros operacionais e não operacionais em relação à construção de atitudes e os membros operacionais são internamente autorizados para tipos de solução de tarefa determinada (Tuomela, 2007, pp. 129-130; 2013, pp. 130-136). Do ponto de vista sociológico, é de particular relevância os modos compartilhados de tomadores de decisão dos grupos sociais como membros operacionais por serem significativos para a autovinculação de um grupo social como o todo. E ao mesmo tempo são responsáveis pela crença do grupo, ou seja, eles levam em consideração o grupo, os membros operacionais aceitam p e estão comprometidos com tal preposição. Os membros operacionais são determinados por funções de status, que definem funções e tarefas de um grupo formalmente em corporações ou informalmente em grupos que não possuem condições de associação formalizados em manuais ou cartas corporativas. Uma pessoa operacional para um grupo g pode ser um membro do grupo ou, em alguns casos, um não-membro. Em geral, os membros do grupo autorizam a operação de um grupo em we-mode para qualquer tomada de decisão em g ou para ação, por exemplo, na realização das decisões do grupo em nome do grupo. Uma pessoa pode ser operativa em um sentido (por exemplo, para decisão) sem ser operativa em outro sentido (por exemplo, ação). Uma pessoa não-operativa (membro) pode, portanto, ser uma que diz respeito à tomada de decisão ou ação. Aqui, dois aspectos devem ser enfatizados: 1. Em um grupo we-mode, todos os membros podem ser membros operacionais (por decisão e ação) se nenhum ter sido especialmente autorizado para uma tarefa, e; e; 2. 6 Autoridade A autorização significa que os membros não operacionais são obrigados a obedecer apenas se os membros decidirem manter sua autorização em vigor – a autorização deve ser para o uso e benefício do grupo e seus membros e só se justifica se esta função for realizada. A distinção entre membros operacionais e não-operacionais divide as intenções do grupo porque nem todos os membros decidem sobre o que é a intenção do grupo ou não. 115 115 Em Tese, Florianópolis, v. 17, n. 1, p. 104-126, jan/jun., 2020. Universidade Federal de Santa Catarina. ISSN 1806-5023. DOI: https://doi.org/10.5007/1806-5023.2020v17n1p104 Tuomela acompanha a visão de J. Raz de que a autoridade é uma questão de "preempção"13 porque a diretriz autorizada exclui e substitui o próprio julgamento do destinatário14. Com a "conta posicional", o grupo é estruturado em membros operacionais e não operacionais com diferentes funções de status, papéis sociais, e tarefas. Autoridade é instanciar os membros operacionais. Esses membros decidem e / ou agem para o grupo, isto é, em nome do grupo ou de outras instâncias. Pode ser que suas decisões também sejam motivadas psicologicamente, mas são decisões independentes de desejo e razões dos membros em princípio. Os analistas são o conceito de atitude de grupo como uma atitude de adesão. A atitude de grupo tem uma função autorizada para o raciocínio relevante prático em grupos (Tuomela 2000: 141-156). Os membros autorizados compartilham atitudes particulares em we-mode como membros do grupo, ou seja, a intenção conjunta formada por uma razão preventiva é uma razão de grupo autorizada. A análise da autoridade de Tuomela relaciona-se com sua "visão posicional de grupos e instituições sociais (incluindo organizações)", ou seja, funcionando como um membro de grupo posicionalmente. A comunicação nestas funções de status baseia-se no pensamento e no raciocínio do I- e we-mode. A autoridade é baseada em pessoas detentores de certas posições, que são autorizadas a realizar ações, isto é, a agir e a falar por uma unidade social. Tuomela continuou a análise de autoridade com respeito a unidades sociais hierárquicas (organizações formais). Analysans é a função dos líderes autorizados "interna" e "externamente". O primeiro é intrinsecamente cooperativo dado que a ação em questão é um componente intrínseco do grupo ou da ação de pertencimento. Essas ações intragrupo/membro são feitas em we-mode enquanto um "membro completo de um agente de grupo", que são intrinsecamente cooperativas. p p 14 J. Raz 1986, 1975, sobre o poder normativo, 98-104. 13 N.T. Direito de preferência em um contrato de compra e venda. 14 p p J. Raz 1986, 1975, sobre o poder normativo, 98-104. 13 N.T. Direito de preferência em um contrato de compra e venda. 14 6 Autoridade A autoridade é dada pelos membros, isto é, pela aceitação coletiva. O poder imposto ocorre sobre os membros enquanto membros. O segundo é uma cooperação não-autônoma nos modos de organização formal. Eles existem como um sistema de comunicação, que exige uma unificação de ações. O discurso e as ações dos membros deste tipo de sistemas sociais são os "membros de um corpo coletivo, para adotar uma metáfora apropriada". (Tuomela, 2013, p.22). No caso de líderes externos autorizados, existe um domínio de um grupo sobre o outro. Neste caso, a inter-relação entre os indivíduos e sua observação não deve se 116 Em Tese, Florianópolis, v. 17, n. 1, p. 104-126, jan/jun., 2020. Universidade Federal de Santa Catarina. ISSN 1806-5023. DOI: https://doi.org/10.5007/1806-5023.2020v17n1p104 116 aplicar estruturalmente. Em ambos os casos, os líderes podem dar novas diretivas e objetivos15. Todo sistema de autoridade como sistema de comunicação e decisão, bem como a diferenciação operativa-não-operativa para o sistema orientado a tarefas pode ser de várias camadas estruturado por hierarquias. Sobre a diferença entre grupos autônomos e não autônomos, basicamente um grupo autônomo é governado pelo próprio grupo, ou seja, coletivamente por seus membros ou por pessoas autorizadas por seus membros. Em contrapartida, um grupo não autônomo é governado por uma autoridade externa (por exemplo, outro grupo ou pessoa que possui os poderes de um ditador ou algo do tipo) ou por um interno não autorizado pelo grupo. Em um grupo não-autônomo, seu ethos (ou seja, seus objetivos constitutivos ou mais centrais, crenças, normas, padrões, práticas, etc.) é determinado por outros que não os membros do grupo (em contraste com a forma como eles são geralmente determinados em um sistema autônomo grupo). Esta é uma característica importante porque o ethos é altamente relevante para explicar as atividades dos membros dos grupos. As atividades dos operários e outros membros pressupõem, em geral, que eles atuem como membros adequados do grupo de acordo com as obrigações normativas e as recomendações que o ethos do grupo impõe. Se em um grupo não-autônomo o ditador (independentemente de ser um membro do grupo) muda o ethos, a explicação original não é válida (porque se refere ao velho e não ao novo ethos). 15 (Tuomela 2013, em líderes externos, capítulo 2, 4. 6). 15 (Tuomela 2013, em líderes externos, capítulo 2, 4. 6). 6 Autoridade É claro que, no caso de um grupo autônomo, os membros operacionais para a tomada de decisões podem mudar coletivamente o ethos, mas o explanandum geralmente será diferente do que é no caso não autônomo – é diferente porque atua como um membro do grupo. Portanto, a distinção autônoma/não autônoma é uma característica importante em uma consideração sobre as atividades do grupo. Este é o link para as características da cooperação em sistemas sociais, grupos e organizações. Este passo não é um passo contingente na filosofia da socialidade de Tuomela, porque os grupos hierárquicos e autoritários de modos de organização formal, que funcionam como agente de grupo, são uma característica importante do domínio social. Um agente de grupo não é um agente intrinsecamente intencional, mas extrínseco em relação a atitudes e ações conjuntas de membros de um grupo. É uma concepção fracamente coletivista de grupos como agentes intencionais. 117 117 Em Tese, Florianópolis, v. 17, n. 1, p. 104-126, jan/jun., 2020. Universidade Federal de Santa Catarina. ISSN 1806-5023. DOI: https://doi.org/10.5007/1806-5023.2020v17n1p104 Em Tese, Florianópolis, v. 17, n. 1, p. 104-126, jan/jun., 2020. Universidade Federal de Santa Catarina. ISSN 1806-5023. DOI: https://doi.org/10.5007/1806-5023.2020v17n1p104 16 Tuomela, 2000, ver, 6-7, 2007, cap. 7. 3. Tese da Proximidade Dadas e das Preferências Finais: Ceteris paribus, "o quão mais próximas e altas forem as preferências dadas e as preferências finais de uma pessoa, maior é a chance de ela cooperar de forma racional a longo prazo". (Tuomela, 2000, p. 15); 2. Tese da Commonality – Ceteris paribus, há uma (definição de) situação interna em que é preferível ter e iniciar uma cooperação bem-sucedida para a coesão de preferências dos participantes. (Tuomela, 2000, pp. 12-14); 1. Tese Básica de Cooperação – dois ou mais participantes cooperam se e somente se eles têm um objetivo em comum e agem juntos para atingir seus objetivos. (Tuomela, 2000, pp. 12-14); Em Tese, Florianópolis, v. 17, n. 1, p. 104-126, jan/jun., 2020. Universidade Federal de Santa Catarina. ISSN 1806-5023. DOI: https://doi.org/10.5007/1806-5023.2020v17n1p104 1. Como G-cooperation, que é "baseada em objetivos coletivos compartilhados" como "cooperação plena" (institucional e não institucional) e 17 Tuomela 2000, Capt. 6, "Apêndice: Conceitos de Instituição como Conceitos Reflexivos". 7 Cooperação 17). A "Teoria da aceitação coletiva da socialidade" pressupõe que um tipo relativo de cooperação fraca também é suficiente para uma aceitação coletiva produzindo e mantendo eventos sociais coletivos e instituições sociais. As normas e o acordo social são atividades cooperativas. Rituais e cerimônias são casos extremos dessas atividades. As preferências cooperativas são fatores em componentes básicos do "controle social", cujos tipos de situações sociais de interações dentro dos participantes têm um conhecimento compartilhado mútuo sobre atividades coletivas. O objeto é a análise do componente semi-motivacional do controle social. Os componentes são as determinações estruturais ou situacionais. A ideia é exemplificada para interações simples de duas pessoas, ou seja, os participantes poderiam estar avaliando o componente do benefício de seu resultado mútuo expresso em: a) O controle absoluto de suas próprias ações, b) em relação às ações dos outros participantes, e c) no seu controle condicional e interativo. A correlação relevante entre as preferências dos participantes da sua cooperação pode ser medida e depende, em particular, do componente do controle social. As I- e G- cooperation pressupõem preferências correspondentes entre os participantes. A interpretação de Tuomela, nos casos de conflitos em relação ao bem público, os objetivos coletivos com a condição de coletividade, ou seja, os objetivos coletivos pretendidos são determinados pelo objetivo mútuo dos participantes e são fixados, portanto, por compromissos coletivos. Em Tese, Florianópolis, v. 17, n. 1, p. 104-126, jan/jun., 2020. Universidade Federal de Santa Catarina. ISSN 1806-5023. DOI: https://doi.org/10.5007/1806-5023.2020v17n1p104 7 Cooperação Existem abordagens filosóficas, matemáticas, teórico-práticas e experimentais, que estudam o dilema de ação coletiva na análise e na pesquisa sobre cooperação. Há também pesquisas biológicas (etnológicas) de cooperação entre animais. As abordagens econômicas e teórico-práticas assumem que a cooperação é baseada na decisão individual. Tuomela tenta corrigir essas abordagens assumindo que razões grupais são necessárias para a explicação da maioria das situações. A teoria dos jogos, por exemplo, não considerou significativamente o quadro de referência institucional, portanto, o dilema dos prisioneiros não é o exemplo paradigmático da análise da cooperação16. Uma característica estrutural do domínio social é construída pela aceitação coletiva enquanto uma "atividade fracamente cooperativa". Tuomela enfatiza a função de "construção social coletiva" em sua filosofia de socialidade. A cooperação é um exemplo instrutivo para a análise da intencionalidade coletiva, uma vez que atuar em conjunto exige intenções conjuntas, crenças compartilhadas e objetivos coletivos. Tuomela analisou características básicas e tipos de cooperação, fazendo distinções auto- explicativas entre a cooperação: 1. Como G-cooperation, que é "baseada em objetivos coletivos compartilhados" como "cooperação plena" (institucional e não institucional) e 1. Como G-cooperation, que é "baseada em objetivos coletivos compartilhados" como "cooperação plena" (institucional e não institucional) e 2. Como I-cooperation – que é a cooperação como coação, ou seja, "com base em objetivos privados compatíveis" (institucional e não institucional). 2. Como I-cooperation – que é a cooperação como coação, ou seja, "com base em objetivos privados compatíveis" (institucional e não institucional). A distinção é feita a partir dos objetivos (conteúdo intencional), que os participantes estão orientados a alcançar (Tuomela, 2000, p.10). As principais teses de Tuomela para cooperação são: 16 Tuomela, 2000, ver, 6-7, 2007, cap. 7. 118 Em Tese, Florianópolis, v. 17, n. 1, p. 104-126, jan/jun., 2020. Universidade Federal de Santa Catarina. ISSN 1806-5023. DOI: https://doi.org/10.5007/1806-5023.2020v17n1p104 4. Tese da Recompensa – Ceteris paribus, se os participantes esperam por uma recompensa maior ao cooperar do que no caso de não cooperar, então um agente racional coopera. (Tuomela, 2000, pp. 16-17); 5. Tese Institucional – a cooperação e a sua organização é fundamental para as instituições (Tuomela, 2000, p. 17). 4. Tese da Recompensa – Ceteris paribus, se os participantes esperam por uma recompensa maior ao cooperar do que no caso de não cooperar, então um agente racional coopera. (Tuomela, 2000, pp. 16-17); 5. Tese Institucional – a cooperação e a sua organização é fundamental para as instituições (Tuomela, 2000, p. 1. Elas são práticas sociais regidas por governantes e estabelecidas por membros autorizados. Por isso, têm fortes sanções que regulam o comportamento dos membros. A principal característica das organizações sociais enquanto organização é que existem relações de poder baseadas na autoridade. 8 Instituições As instituições como artefato social são "coletivamente feitas pelo homem". O conceito de instituição é reflexivo, constituído por um sistema de normas fundamentado basicamente na aceitação coletiva17. As instituições envolvem a atividade we-mode e não o I-mode de seus membros. Eles definem "regras básicas", expectativas de expectativas para seus membros. As atividades de seus membros são determinadas por funções 119 Em Tese, Florianópolis, v. 17, n. 1, p. 104-126, jan/jun., 2020. Universidade Federal de Santa Catarina. ISSN 1806-5023. DOI: https://doi.org/10.5007/1806-5023.2020v17n1p104 119 sociais (status). Tuomela descreve coletivamente as instituições (comportamento coletivo regido por padrões), que também é uma abordagem para análise das "organizações sociais". O trabalho das instituições também depende de rotinas e comportamentos não intencionais (Tuomela, 2013, pp. 215-216). As instituições tipicamente têm um nível de autoridade de modo que os membros autorizados fornecem dispositivos aos membros em geral. Os membros autorizados estabeleceram regras e normas, que são válidas para os membros em questão, e envolvem direitos e deveres. Essas normas regulam o comportamento dos membros das instituições, que se baseiam em expectativas mútuas. Portanto, o mútuo conhecimento das normas é uma característica particular das instituições. Isso não é válido em outros sistemas sociais em geral. Em "Teoria Coletiva de Aceitação da Socialidade", Tuomela analisou as instituições da seguinte maneira: 1. Elas são práticas sociais regidas por governantes e estabelecidas por membros autorizados. Por isso, têm fortes sanções que regulam o comportamento dos membros. A principal característica das organizações sociais enquanto organização é que existem relações de poder baseadas na autoridade. 2. Elas conferem um novo status conceitual e social a algumas entidades e eventos, por exemplo, membros, atividades, comunicação ou objetos. 3. Eles conferem uma nova função deôntica e de status para seus membros. Isso acompanha a institucionalização do sistema de comunicação de autoridade. 4. Elas implicam no status social de organização e na posição de função de seus membros além de um sistema de tarefa correta. 5. Eles não precisam apenas do I-mode, mas envolvem idealmente a intencionalidade do we-mode, isto é, os membros estão comprometidos com o status conceitual, o que cria o status da associação. 6. As competências e habilidades linguísticas são necessárias para participar da comunicação institucional e da atuação dos membros das instituições. Em Tese, Florianópolis, v. 17, n. 1, p. 104-126, jan/jun., 2020. Universidade Federal de Santa Catarina. ISSN 1806-5023. DOI: https://doi.org/10.5007/1806-5023.2020v17n1p104 al, a visão das instituições de Tuomela é a seguinte: 1. Elas são constituídos por aceitação construtiva / performativa, que é uma "aceitação coletiva reflexiva", 2. As ações e a comunicação são reguladas por normas, possibilitando comportamentos conceitualmente novos, que não existem em situações pré- institucionais e 3. Os membros têm crenças mais ou menos corretas sobre, por exemplo, os regulamentos institucionais, empresa e escola, como um imperativo funcional. 120 120 4. Uma das principais características das instituições é que eles estabelecem a ordem social, que acompanha a satisfação das necessidades básicas das pessoas. 4. Uma das principais características das instituições é que eles estabelecem a ordem social, que acompanha a satisfação das necessidades básicas das pessoas. Mas os conflitos entre interesses individuais e coletivos não são para excluir na provisão processo institucionalizado, por exemplo, dilemas de coordenação, por exemplo, de que lado da estrada pessoas devem dirigir e situações de conflito total. No entanto, a dinâmica das práticas sociais e das instituições sociais requer uma análise particular. Tuomela e W. Balzer implementam a análise com um modelo matemático geral. O modelo acompanha a teoria de estruturação de A. Giddens. Mas ao contrário de Giddens, o analysandum do modelo são atividades sociais repetidas e a manutenção das estruturas sociais relevantes por atitudes comuns. A partir da teoria do modelo, resulta também uma troca científica para a pesquisa de inteligência distributiva e simulação computacional de sistemas sociais. (Tuomela, 2002). A aplicação do "Modelo de Aceitação Coletiva", portanto, a intencionalidade coletiva na forma de atitude /we- attitude compartilhada, basicamente a intenção ou crença coletiva, mostra que as instituições sociais são coleções de estrutura normativas reguladoras Em Tese, Florianópolis, v. 17, n. 1, p. 104-126, jan/jun., 2020. Universidade Federal de Santa Catarina. Em Tese, Florianópolis, v. 17, n. 1, p. 104-126, jan/jun., 2020. Universidade Federal de Santa Catarina. ISSN 1806-5023. DOI: https://doi.org/10.5007/1806-5023.2020v17n1p104 Em Tese, Florianópolis, v. 17, n. 1, p. 104-126, jan/jun., 2020. Universidade Federal de Santa Catarina. ISSN 1806-5023. DOI: https://doi.org/10.5007/1806-5023.2020v17n1p104 9 Pertencimento e Ontologia social Tuomela é um defensor de uma teoria dos sistemas sociais, porque grupos, comunicação de decisões, cooperação, instituições e organizações fazem parte de um sistema de comunicação social de ação. A filosofia de Tuomela de socialidade e ontologia social tem como núcleo duro a sociologia do pertencimento (Preyer, 2017). A função de status da associação e sua indicação são o ponto de vista compartilhado, que não pode ser caracterizado por um único indivíduo. A indicação dessa função de status é observador-dependente em princípio. Searle fez uma frutuosa distinção entre objetividade e subjetividade ontológica, e objetividade e subjetividade epistemológica. (Searle, 2010) O domínio social não faz parte do mundo físico. Mas isso não significa que julgamentos objetivos sobre este domínio não são possíveis. Esses julgamentos são observadores dependentes dos membros do domínio e dos participantes da comunicação. Isso implica que we-attitudes pressupõem um quadro social de referência em sentido forte. Portanto, essas atitudes pressupõem o pertencimento e os compromissos coletivos, que instanciam os membros enquanto membros e, ao mesmo tempo, seu acesso público, ou seja, a observação dos membros Em Tese, Florianópolis, v. 17, n. 1, p. 104-126, jan/jun., 2020. Universidade Federal de Santa Catarina. ISSN 1806-5023 DOI: https://doi org/10 5007/1806-5023 2020v17n1p104 121 em suas funções de status. Esta é a distinção ontológica entre o domínio social e o restante da natureza. De acordo com Tuomela, Searle (1995, 1998, 2001, 2010) e outros, o próprio domínio social não faz parte da ontologia do mundo físico, isto é, não é uma propriedade natural, não é constituída de indivíduos atomísticos, e não é um ser ontologicamente local, mas é um ser artificial. Em princípio, os sociólogos estariam de acordo. Portanto, é ontologicamente subjetivo. Mas o domínio é um objetivo epistemológico porque isso não significa que seja uma ficção porque é reproduzir por funções de status objetivo e suas indicações, que estabelecem um modo particular de observação da indicação da função de status. Há uma ontologia social somente se uma pessoa que funciona como um membro do grupo e uma decisão de adesão é estabelecida no sistema de comunicação dos membros dos sistemas sociais. Para os membros como membros é válido que os motivos do grupo substituam os motivos individuais deles ("we-moders"). Os grupos sociais não são agentes e também não são uma pessoa no sentido literal da palavra, mas o domínio social é constituído ontologicamente por membros do grupo. 9 Pertencimento e Ontologia social Tuomela faz a distinção conceitual entre uma pessoa solitária e o membro de um grupo. O status ontológico de um grupo social é caracterizar, portanto, que grupos como sistemas sociais são supervenientes a seus membros. Em Tese, Florianópolis, v. 17, n. 1, p. 104-126, jan/jun., 2020. Universidade Federal de Santa Catarina. p p j j ISSN 1806-5023. DOI: https://doi.org/10.5007/1806-5023.2020v17n1p104 10 Conclusão Resumindo, o núcleo duro da filosofia de socialização de Tuomela é que a aceitação coletiva é analisada apropriadamente como we-mode dos membros de um grupo enquanto membros de um grupo. Analysans é a distinção entre o pensamento, o sentimento e a atuação do we-mode, sua relação conceitual e funcional com o pensamento e o sentimento do I-mode como pessoa privada. A intencionalidade coletiva está relacionada a uma das principais características da cooperação, como atuação conjunta elas estão baseadas em um we-mode como condição constitutiva. Elas são uma característica geral da socialidade e estão ligadas à manutenção das instituições. A característica básica das instituições sociais é que elas são coleções de sistemas envolvendo sistemas sociais regulatórios normativos. Mas o domínio social também é determinado por condições de interação assimétricas. Os membros operacionais e não operacionais (grupos autônomos e não autônomos) em relação à ordem de autoridade Em Tese, Florianópolis, v. 17, n. 1, p. 104-126, jan/jun., 2020. Universidade Federal de Santa Catarina. ISSN DOI h //d i / / 122 que divide o domínio social dos grupos sociais, cooperações, instituições e organizações formais. Mas os membros do grupo que compartilharam o conceito de we-mode têm prioridade parcial no domínio da socialidade, que não é redutível ao conceito do I-mode porque a intencionalidade coletiva "é necessária para a compreensão da vida social". Essa é uma ressonância fraca de uma sociologia durkheimiana moderada. É concluir que a decisão de pertencimento e a sua especificação para funções, funções de status, tarefas, obrigações e compromissos são a decisão básica e seleção pelas quais o domínio social é constituído e sua auto-observação enquanto tal. A partir da observação sociológica da intencionalidade coletiva, é frutífero recorrer à distinção entre membros e não membros de qualquer domínio social como a principal distinção teórica, bem como o que é necessário para a estrutura e continuação da comunicação. É uma condição de participação no domínio social que os membros trabalham em conjunto em diferentes posições sociais. Isso contribui para o seu sucesso reprodutivo. Mas os membros do domínio social também estão lutando uns com os outros, competindo no sistema econômico, político e científico; são egoístas e ambiciosos. Enquanto isso, temos evidências suficientes de que a luta e a regulamentação sobre o controle do fluxo de recursos são um universal evolutivo (Eisenstadt, 1995c, pp. 344-345). 10 Conclusão A análise da cooperação é relevante para uma sociologia do pertencimento porque é uma condição necessária dos sistemas sociais que os membros em diferentes posições sociais estão dispostos a cooperar na consecução de um objetivo coletivo. Os sistemas sociais têm poder de aprimoramento e o aumento da vantagem seletiva pela decisão de adesão resolve problemas de tarefas cooperativas. Estas são negações limitadas, pelo que o domínio social é capaz de aprender em geral. Se considerarmos que a análise da aceitação coletiva entre os membros do domínio social e as atitudes grupais são o núcleo conceitual e rígido da filosofia da socialidade, então devemos concluir que todos os sistemas sociais são auto-constituídos pela decisão de pertencimento e sua implementação. Isso deixa clara sua eficiência, mas também a sua ameaça de extinção. Referências BARNES, B. Social Life as Bootstrapped Induction. Sociology, 17, 534-545, 1983. BLOOR, D. Wittgenstein, Rules, and Institution. London. New York: Routledge, 1997. R, D. Wittgenstein, Rules, and Institution. London. New York: Routledge, 199 123 Em Tese, Florianópolis, v. 17, n. 1, p. 104-126, jan/jun., 2020. Universidade Federal de Santa Catarina. ISSN 1806-5023. DOI: https://doi.org/10.5007/1806-5023.2020v17n1p104 BRATMAN, M. B. Shared Agency: A Planning Theory of Acting together. New York: Oxford University Press, 2014. EISENSTADT, S. N. General Introduction: The Scope and Development of Political Sociology. In: EISENSTADT (ed.), Political Sociology. New York: Basic Books, 1971. EISENSTADT, S. N. Institutionalization and Change. In Power, Trust, and meaning: Essays in Sociological Theory. Chicago, London: The University of Chicago Press, 1995a. EISENSTADT, S. N. Charisma and Institution Building: Max Weber and Modern Sociology, 167-201. In: Power, Trust, and meaning: Essays in Sociological Theory. Chicago, London: The University of Chicago Press, 1995b. EISENSTADT, S. N. Action, Resources, Structure, and Meaning. In: Power, Trust, and Meaning: Essays in Sociological Theory. Chicago, London: The University of Chicago Press, 1995c. GILBERT, M. A. Theory of Political Obligations: Membership and Commitments. Oxford: Oxford University Press, 2006. GILBERT, M. Joint Commitment: How we make the Social World. New York: Oxford University Press, 2014. HABERMAS, J. Theory of communicative Actions (2 vol.). Frankfurt a. M./Berlin: Suhrkamp Verlag, 1981. Em Tese, Florianópolis, v. 17, n. 1, p. 104-126, jan/jun., 2020. Universidade Federal de Santa Catarina. ISSN 1806-5023. DOI: https://doi.org/10.5007/1806-5023.2020v17n1p104 HABERMAS, J. Theory of communicative Actions (2 vol.). Frankfurt a. M./Berlin: Suhrkamp Verlag, 1981. KUSCH, M. The Sociophilosophy of Folk Psychology. Studies in History and Philosophy of Science, 28, 1-25, 1997. PETTIT, P. The Common Mind. New York: Oxford University Press, 1993. PETTIT, P. Groups with Minds of Their Own. In: SCHMITT, F. (ed.), Socializing Metaphysics. Lanham: Rowman and Littlefield, 2003. PREYER, G. Transformation in der modernen Theorie und Soziologie des Rechts. In: A. AARNIO et al. (org.) Positivität, Normativität und Instittionalität des Rechts. Festschrift für Werner Kraweitz zum 80. Geburtstag. Berlin: Duncker & Humblot, 2013. PREYER, G. Soziologische Theorie der Gegenwartsgesellschaft (3) Vols., Vol 1: Mitgliedschaftstheoretische Untersuchungen, Vol. 2: Lebenswelt, System, Gesellschaft, Vol. 3.: Mitgliedschaft und Evolution. Wiesbaden: Springer/VS Verlag Sozialwissenschaften, 2017. PREYER, G., Peter, G. (eds.). Social Ontology and Collective Intentionality: Critical Essays on the Philosophy of Raimo Tuomela with his Responses. Leiden: Springer Publisher, 2017. PROTOSOCIOLOGY. Vol. 16: Understanding the Social: New Perspectives from Epistemology I, 2002. 124 124 Em Tese, Florianópolis, v. 17, n. 1, p. 104-126, jan/jun., 2020. Universidade Federal de Santa Catarina. ISSN 1806-5023. DOI: https://doi.org/10.5007/1806-5023.2020v17n1p104 PROTOSOCIOLOGY. Vol. 18-19: Understanding the Social: Philosophy of Sociality II, 2003. PROTOSOCIOLOGY. Vol. 18-19: Understanding the Social: Philosophy of Sociality II, 2003. RAZ, J. Practical Reasons and Norms. London: Hutchinson, 1975. RAZ, J. The Morality of Freedom. Oxford: Clarendom Press, 1986. SEARLE, J. The Construction of Social Reality. London: Allen Lane, Penguin Press, 1995. SEARLE, J. Mind, Language and Society: Philosophy in the Real World. New York: Basic Books, 1998. SEARLE, J. R. Rationality in Action. Cambridge. Mass.: MIT Press, 2001. SEARLE, J. R. Making the Social World: The Structure of Human Civilization. Oxford: Oxford University Press, 2010. THIBAUT J., KELLY, H. The Social Psychology of Groups. New York: Wiley, 1959. TUOMELA, R. MILLER, K. We-Intention. In: Philosophical Studies, 53 1988, 367-389, 1988. TUOMELA, R. Importance of Us: A Philosophical Study of Basic Social Notions. Stanford: Stanford University Press, 1995. TUOMELA, R. Cooperation: A Philosophical Study. Dordrecht: Kluwer (Springer), 2000. TUOMELA, R. The Philosophy of Social Practices: A Collective Acceptance Vie., Cambridge: Cambridge University Press, 2002. TUOMELA, R. The Philosophy of Sociality: The Shared Point of View. Oxford: Oxford UP, 2007. TUOMELA, R. Individualism and Collectivism in Social Science. In: Peter, G., Krausse, K. (ed.), Selbstbeobachtung der modernen Gesellschaft und die neuen Grenzen des Sozialen. Wiesbaden: Springer/VS Verlag Sozialwissenschaften, 2012. TUOMELA, R. Collective Acceptance, Social Institutions, and Social Reality. In: Preyer, G. (eds.) Neuer Mensch und kollektive Identität in der Kommunikationsgesellschaft. Wiesbaden: Springer/VS Verlag, 2009. TUOMELA, R. Social Ontology: Collective intentionality and Group Agents. New York: Oxford University Press, 2013. WILSON, K. V., Bixenstine, V.E. Forms of Social Control in Two-Person Choice Games. Behavioural Science, 7, 92-102, 1962. 125 Em Tese, Florianópolis, v. 17, n. 1, p. 104-126, jan/jun., 2020. Universidade Federal de Santa Catarina. ISSN 1806-5023. DOI: https://doi.org/10.5007/1806-5023.2020v17n1p104 AUTORES Gehard Preyer, Prof. Dr. phil. habil. Gehard Preyer, Prof. Dr. phil. habil. Gehard Preyer, Prof. Dr. phil. habil. y , p Professor of Sociology. Goethe-University Frankfurt am Main Editor-In-Chief, ProtoSociology. An International Journal of Interdisciplinary Research and Project. Georg Peter, Dr. phil. Georg Peter, Dr. phil. ProtoSociology, An International Journal of Interdisciplinary Research and Project, Goethe-University Frankfurt am Main, D-60054 Frankfurt a. M., Germany. Georg Peter, Dr. phil. ProtoSociology, An International Journal of Interdisciplinary Research and Project, Goethe-University Frankfurt am Main, D-60054 Frankfurt a. M., Germany. PUBLISHER PUBLISHER Universidade Federal de Santa Catarina. Programa de Pós-Graduação em Sociologia Política. Publicado no Portal de Periódicos UFSC. As ideias expressadas neste artigo são de responsabilidade de seus autores, não representando, necessariamente, a opinião dos editores ou da universidade. APROVAÇÃO DE COMITÊ DE ÉTICA EM PESQUISA Não houve comitê de ética. LICENÇA DE USO Os autores cedem à Em Tese os direitos exclusivos de primeira publicação, com o trabalho simultaneamente licenciado sob a Licença Creative Commons Attribution 4.0 Internacional (CC BY). Estra licença permite que terceiros remixem, adaptem e criem a partir do trabalho publicado, atribuindo o devido crédito de autoria e publicação inicial neste periódico. Os autores têm autorização para assumir contratos adicionais separadamente, para distribuição não exclusiva da versão do trabalho publicada neste periódico (ex.: publicar em repositório institucional, em site pessoal, publicar uma tradução, ou como capítulo de livro), com reconhecimento de autoria e publicação inicial neste periódico. TRADUTORES Hugo Neri, Dr. Universidade de São Paulo, Filosofia, São Paulo Hugo.munhoz@usp.br Hugo.munhoz@usp.br https://orcid.org/0000-0001-6065-4661 Em Tese, Florianópolis, v. 17, n. 1, p. 104-126, jan/jun., 2020. Universidade Federal de Santa Catarina. ISSN 1806-5023. DOI: https://doi.org/10.5007/1806-5023.2020v17n1p104 HISTÓRICO 126 Em Tese, Florianópolis, v. 17, n. 1, p. 104-126, jan/jun., 2020. Universidade Federal de Santa Catarina. ISSN 1806-5023. DOI: https://doi.org/10.5007/1806-5023.2020v17n1p104
https://openalex.org/W4386638412	https://www.researchsquare.com/article/rs-3292422/latest.pdf	English	null	Languages with more speakers tend to be harder to (machine-)learn	Research Square (Research Square)	2,023	cc-by	18,838	Version of Record: A version of this preprint was published at Scienti¦c Reports on October 28th, 2023. See the published version at https://doi.org/10.1038/s41598-023-45373-z. Languages with more speakers tend to be harder to (machine-)learn Authors: Alexander Koplenig1, Sascha Wolfer1 1Leibniz Institute for the German Language (IDS), Mannheim, Germany. Corresponding author. Email: koplenig@ids-mannheim.de Keywords: License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Additional Declarations: No competing interests reported. Additional Declarations: No competing interests reported. Version of Record: A version of this preprint was published at Scienti¦c Reports on October 28th, 2023. See the published version at https://doi.org/10.1038/s41598-023-45373-z. Abstract Computational language models (LMs), most notably exemplified by the widespread success of OpenAI's ChatGPT chatbot, show impressive performance on a wide range of linguistic tasks, thus providing cognitive science and linguistics with a computational working model to empirically study different aspects of human language. Here, we use LMs to test the hypothesis that languages with more speakers tend to be easier to learn. In two experiments, we train several LMs – ranging from very simple n-gram models to state-of-the-art deep neural networks – on written cross-linguistic corpus data covering 1293 different languages and statistically estimate learning difficulty. Using a variety of quantitative methods and machine learning techniques to account for phylogenetic relatedness and geographical proximity of languages, we show that there is robust evidence for a relationship between learning difficulty and speaker population size. However, contrary to expectations derived from previous research, our results suggest that languages with more speakers tend to be harder to learn. 1 1 Introduction It has long been taken for granted that there is no relationship between the structure of a language and the environment in which it is spoken1,2 leading to long-standing and largely unquestioned assumptions in modern linguistics that all languages are equally complex3–11 and equally difficult to learn.12 Yet, depending on how you count, there are between 6000 and 8000 different languages and language varieties on the planet13–15 that vary widely in their structural properties.16,17 A growing body of cross-linguistic research has begun to document that the natural and social environments in which languages are being used and learned drive this diversity18–21, that language structure is influenced by socio-demographic factors such as the estimated number of speakers18,21–23 and that the long-held belief in a principle of "invariance of language complexity"24 may be incorrect.25 In this article, we examine another long-held assumption that, to our knowledge, has never been systematically tested: the assumption that all languages are equally difficult to learn. The main obstacle to such an endeavour was already pointed out by a pioneer of modern linguistics, Henry Sweet, in 1899: “it is practically impossible for any one who has not an equally perfect knowledge of all languages to test this”.12 In this context, cognitive scientists and computational linguists have pointed out that computational language models (LMs), most notably exemplified by the widespread success of OpenAI's ChatGPT chatbot, provide a computational working model for empirically studying various aspects of human language.26,27 Recent research27–30 shows that computational models can learn core structures that are present in natural language from observed training input alone, something that was long thought to be impossible without innate linguistic knowledge.31 In this sense, we train LMs on written text data in different languages. The LM learns to make predictions about subsequent linguistic material by finding a In this article, we examine another long-held assumption that, to our knowledge, has never been systematically tested: the assumption that all languages are equally difficult to learn. Introduction The main obstacle to such an endeavour was already pointed out by a pioneer of modern linguistics, Henry Sweet, in 1899: “it is practically impossible for any one who has not an equally perfect knowledge of all languages to test this”.12 In this context, cognitive scientists and computational linguists have pointed out that computational language models (LMs), most notably exemplified by the widespread success of OpenAI's ChatGPT chatbot, provide a computational working model for empirically studying various aspects of human language 26,27 Recent research27–30 In this article, we examine another long-held assumption that, to our knowledge, has never been systematically tested: the assumption that all languages are equally difficult to learn. The main obstacle to such an endeavour was already pointed out by a pioneer of modern linguistics, Henry Sweet, in 1899: “it is practically impossible for any one who has not an equally perfect 2 short encoding of the training material to which it is exposed.28,32 With increasing input, the LM gets better at predicting subsequent data.32 We measure how fast the LM learns to make optimal predictions and treat this as a measure of learning difficulty. We then statistically analyse this measure across different languages to test the above assumption. Recent research using LMs in this way has indirectly suggested that languages with more speakers may be easier (i.e. faster) to learn: in a large-scale quantitative cross-linguistic analysis, Ref.25 trained an LM on more than 6500 documents in over 2000 different languages and statistically inferred the entropy rate of each document, which can be seen as an index of the underlying language complexity.28,33–35 The results showed that documents in languages with more speakers tended to be more complex. Furthermore, documents that were more complex tended to be easier and faster for the LM to learn. These findings indirectly suggest that we should expect documents in languages with more speakers to be easier for the LM to learn. In this article, we first use part of the data used by Ref.25 to explicitly test this hypothesis. Since the LM used by Ref.25 is rather simple, we train two more sophisticated LMs that use machine learning and deep learning on the data and compare the results. We then discuss some potential limitations of the multilingual text collection used by Ref.25. Introduction To rule out that the results are driven by these limitations, we create two fully balanced and parallel multilingual corpora, which we use to train seven different LMs – ranging from very simple n-gram models to state-of-the-art deep neural networks – and measure how difficult it is for each LM to build an adequate probabilistic representation of the input. Importantly, previous research21,36–42 has shown that cross-linguistic (and cross-cultural) studies that seek to analyse potential statistical associations between language features and external factors must take into account Galton's problem, which refers to the potential confounding of 3 linguistic and cultural similarities by phylogenetic relatedness and geographical proximity. To address this issue, we take a comprehensive approach, using both established analytical methods41 and novel quantitative techniques developed in the field of econometrics that leverage machine learning43,44 and spatial autoregressive models.45 In a series of tests, we show that there is stable evidence for an association between learning difficulty and speaker population size across LMs – but in the opposite direction to that expected from previous research, suggesting that languages with more speakers tend to be harder to (machine-)learn. We argue that this finding challenges the popular linguistic niche hypothesis18, which suggests that languages with larger communities of speakers should be easier, not harder, to learn. Results First study. We first highlight some key points so readers can interpret our analyses more easily, see the Methods section for in-depth details. In our first series of quantitative analyses, we use part of a large-scale database of written multilingual texts comprising a variety of different text types compiled by Ref.25. In total, we analyse 3853 documents contained in 40 different multilingual corpora covering 1293 different languages and ranging in length from a few tens to several hundreds of millions of words. Figure 1 illustrates how learning difficulty is assessed using a shape parameter, b, which quantifies how difficult it is for an LM to learn to make optimal predictions46 (see Supplementary Figure 1 for a further illustration). Since lower b-values are indicative of higher learning difficulty, we should expect a positive statistical relationship between b and speaker population size, if indeed languages with more speakers tend to be easier to learn. First study. We first highlight some key points so readers can interpret our analyses more easily, see the Methods section for in-depth details. In our first series of quantitative analyses, we use part of a large-scale database of written multilingual texts comprising a variety of different text types compiled by Ref.25. In total, we analyse 3853 documents contained in 40 different First study. We first highlight some key points so readers can interpret our analyses more easily, see the Methods section for in-depth details. In our first series of quantitative analyses, we use part of a large-scale database of written multilingual texts comprising a variety of different text types compiled by Ref.25. In total, we analyse 3853 documents contained in 40 different multilingual corpora covering 1293 different languages and ranging in length from a few tens to several hundreds of millions of words. Figure 1 illustrates how learning difficulty is assessed using a shape parameter b which quantifies how difficult it is for an LM to learn to make 4 Figure 1 \| Illustration of measuring learning difficulty in Study 1. Circles represent observed bits-per-symbol Figure 1 \| Illustration of measuring learning difficulty in Study 1. Circles represent observed bits-per-symbol that are needed (on average) to encode/predict symbols based on increasing amounts of training data for different (hypothetical) documents in different (hypothetical) languages, each with a source entropy of 5. Results Lines represent fitted values based on an ansatz function that has three parameters: the limiting entropy rate h, which quantifies how difficult it is to predict, a proportionality constant and a parameter b, which quantifies how difficult it is to learn to predict by describing the shape of the curve and can thus be used to quantify learning difficulty (see Methods for details). The blue circles illustrate a document for which learning is more difficult: to learn to make optimal predictions, the LM needs comparatively more training data and convergence to the underlying source entropy is rather slow. The orange circles, on the other hand, represent a document that is easier to learn – convergence is much faster: after comparatively little training data, the LM has already generated an adequate representation of the statistical structure of the input that can be used to make optimal predictions. The fitted lines show that this difference in learning difficulty can be quantified by b, where higher values indicate faster convergence and thus lower learning difficulty. Figure 1 \| Illustration of measuring learning difficulty in Study 1. Circles represent observed bits-per-symbol Figure 1 \| Illustration of measuring learning difficulty in Study 1. Circles represent observed bits-per-symbol Figure 1 \| Illustration of measuring learning difficulty in Study 1. Circles represent observed bits-per-symbol that are needed (on average) to encode/predict symbols based on increasing amounts of training data for different (hypothetical) documents in different (hypothetical) languages, each with a source entropy of 5. We first focus on prediction by partial matching (PPM)47, which is based on a variable-order Markov LM. To this end, we use estimates for b measured for both words and characters as information encoding units provided by Ref.25. On both levels (words/characters), we run 5 separate multilevel mixed-effects linear regressions (LMER) with b as the outcome. We include fixed effects for the (log of) speaker population size and, to account for document- and corpus- specific characteristics, the entropy rate h, the text length L and the interaction between h and L. To account for the potential non-independence of data-points described above, we include (crossed) random intercepts for the following groups: corpus, language family, language, macro area, country and writing script. In addition, we include random slopes (i.e. we allow the effect of population size to vary across different groups) for all groups except language (since population size does not vary within languages). Results Given the absence of clear theoretical or empirical reasons to determine which covariates to include, we adopted a multi-model inference approach48 by subsetting the full model, i.e. we generated a set of models with all possible covariate subsets, which were then fitted to the data. In total, we ran 4860 different sub-models (see Methods for details). As a means of selecting between models, we use Akaike’s information criterion (AIC)49 where lower values indicate a more apt model. A comparison of reduced models without a fixed effect (and potential random slopes) for speaker population size with full models where speaker population size is included reveal that in all 2430 possible model pairs, for both words and characters, the model that includes speaker population size has a lower AIC (median difference between reduced and full models ΔAICmed = 46.55 for words and ΔAICmed = 71.16 for characters, see Supplementary Table 1 for numerical results). This result clearly points towards a statistical association between learning speed and population size. However, Figure 2a shows that all β- coefficients, βLMER, estimated for speaker population size are negative for both words and characters indicating that larger population sizes are associated with lower values of b and thus higher learning difficulty. To account for the uncertainty in the model selection process, we compute a frequentist model averaging (FMA) estimator50 (see Methods for details), 𝛽𝛽𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 𝐹𝐹𝐿𝐿𝐹𝐹= -𝛽𝐹𝐿𝐹 separate multilevel mixed-effects linear regressions (LMER) with b as the outcome. We include fixed effects for the (log of) speaker population size and, to account for document- and corpus- specific characteristics, the entropy rate h, the text length L and the interaction between h and L. To account for the potential non-independence of data-points described above, we include (crossed) random intercepts for the following groups: corpus, language family, language, macro area, country and writing script. In addition, we include random slopes (i.e. we allow the effect of population size to vary across different groups) for all groups except language (since population size does not vary within languages). Given the absence of clear theoretical or empirical reasons to determine which covariates to include, we adopted a multi-model inference approach48 by 6 speakers tend to be harder for PPM to learn. Figure 2b shows the estimated β-coefficients and 95% confidence intervals for the best models, i.e. the models with the lowest AIC for both symbolic levels. Results In both cases, an increase in speaker population size predicts a decrease in b and thus higher learning difficulty (both parametric p-values < 0.05). Figure 2 \| Multilevel mixed-effects linear regression results (Study 1) for three different LMs – PPM, PAQ Figure 2 \| Multilevel mixed-effects linear regression results (Study 1) for three different LMs – PPM, PAQ and LSTMcomp. (a), (c) and (e). Distribution of the estimated impact of speaker population size, βLMER, per LM and per symbol for a total of 2430 models that include a fixed effect (and potential random slopes) for speaker population size. To control for the potential non-independence of data points due to phylogenetic relatedness and geographic proximity, all models additionally include fixed covariates, random intercepts and random slopes (see Methods for details). (b), (d) and (f). Estimated βLMER (vertical line) and 95% confidence interval (horizontal line) for the model with the lowest AIC per LM and per symbol (see Supplementary Table 1 for numerical results and model specifications). Olive colour – words as information encoding units. Pink colour – characters as information encoding units. and LSTMcomp. (a), (c) and (e). Distribution of the estimated impact of speaker population size, βLMER, per LM and per symbol for a total of 2430 models that include a fixed effect (and potential random slopes) for speaker population size. To control for the potential non-independence of data points due to phylogenetic relatedness and geographic proximity, all models additionally include fixed covariates, random intercepts and random slopes (see Methods for details). (b), (d) and (f). Estimated βLMER (vertical line) and 95% confidence interval (horizontal line) for the model with the lowest AIC per LM and per symbol (see Supplementary Table 1 for numerical results and model specifications). Olive colour – words as information encoding units. Pink colour – characters as information encoding units. To test whether these results are specific to PPM, whose LM is relatively simple25, we trained two further LMs on all written data from Ref.25: (i) PAQ51, which employs several machine learning techniques for prediction52 and (ii) LSTMcomp53, which uses a deep learning model54 for prediction (see Methods for details). 7 The results obtained from these algorithms strongly support the results obtained from PPM. Results For PAQ, all models that include speaker population size as a covariate have a lower AIC than 7 reduced models for words (ΔAICmed = 36.87) and 2422 out of all 2430 full models have a lower AIC than reduced models (99.67%) for characters (ΔAICmed = 37.76). For LSTMcomp, all models that include speaker population size as a covariate have a lower AIC than reduced models for both words and characters (ΔAICmed = 62.81 for characters and ΔAICmed = 54.84 for words). Figure 2c,e shows that for both algorithms, the estimated β-coefficients for speaker population size were consistently negative for both characters and words in all models. For PAQ, 𝛽𝛽 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 𝐹𝐹𝐿𝐿𝐹𝐹 = - 0.043 for words and 𝛽𝛽 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 𝐹𝐹𝐿𝐿𝐹𝐹 = -0.035 for characters. For LSTMcomp, 𝛽𝛽 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 𝐹𝐹𝐿𝐿𝐹𝐹 = -0.059 for words and 𝛽𝛽 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 𝐹𝐹𝐿𝐿𝐹𝐹 = -0.023 for characters. Figure 2d,f visualises the estimated β-coefficients and 95% confidence intervals for the best models per symbol. For both LMs and on both levels (words/characters), the confidence intervals do not include zero. Supplementary Table 1 provides numerical details and shows that all four estimates are statistically significant at p < 0.005. In Supplementary Table 2, we show that the results hold when documents from comparable corpora are excluded and only fully parallel corpora are considered (see Methods for details). To further estimate the potential effect of speaker population size on learning difficulty while controlling for potential confounding due to translation effects55 and pluricentism56 in addition to covariation due to phylogenetic relatedness and geographic proximity, we generated three sets of potential control variables. The small set, consisting of a total of Nc = 225 candidates, includes these variables: (i) A set of indicator variables for the levels (categories) of corpus, language family, writing script, macro area and the Expanded Graded Intergenerational Disruption Scale (EGIDS)57; (ii) To control for geographical proximity, third-order B-spline basis functions for both latitude and longitude58,43; 8 (iii) Several other continuous environmental variables in addition to h and L, such as the number of countries in which a language is spoken, geographical range, altitude and climate (see Methods for details). (iii) Several other continuous environmental variables in addition to h and L, such as the number of countries in which a language is spoken, geographical range, altitude and climate (see Methods for details). Results (iii) In addition to the variables of the small set, the medium set (Nc = 274) includes first order/two- way interactions of the basis functions of (ii). In addition to the variables of the medium set, the big set (Nc = 2226) includes first-order interactions between the indicators of (i). To estimate the effect of speaker population size on learning difficulty in such a high- dimensional setting, we use a technique called double selection43, which uses the lasso machine learning technique59 to select the relevant control variables from the candidate set (see Methods for details). Figure 3a shows that all selected models have high predictive power60 explaining between 62.90% and 94.47% of the total variance in learning difficulty (median out-of-sample 𝑅𝑅𝑂𝑂𝑂𝑂 2 = 89.23%) and between 77.95% and 79.68% of the total variance of speaker population size (median 𝑅𝑅𝑂𝑂𝑂𝑂 2 = 78.82%). Note that the use of standard parametric tests may be questioned in this study, as the sample of languages for which we have available documents cannot be considered a random sample of the population of all languages.61,62 To address this issue, we used the selected relevant controls as input for Freedman-Lane permutation tests63 to compute non-parametric p- values (see Methods for details). Figure 3b,c,d shows that the β-coefficient for speaker population, βDS, size remains negative in all scenarios and passes the permutation test at p < 0.05 in all but one case. The exception is PPM on the level of words for the big candidate set. Supplementary Table 3, which contains numerical results, shows that in this case, p = 0.07. In Supplementary Table 4, we show that the results also hold when only fully parallel corpora are considered. Again, βDS remains negative in all scenarios and passes the permutation test at p < 0.05 in all but one case. To further assess the robustness of these findings, we employ a more Supplementary Table 3, which contains numerical results, shows that in this case, p = 0.07. In Supplementary Table 4, we show that the results also hold when only fully parallel corpora are considered. Again, βDS remains negative in all scenarios and passes the permutation test at p < 0.05 in all but one case. Results (b) Candidate set: small (number of control covariates Nc 10 = 225), (c) Candidate set: medium (Nc = 274), (d) Candidate set: big (Nc = 2226). p < 0.05, * p < 0.01, *** p < 0.005. Statistical significance is determined based on non-parametric permutation tests (see Methods for details and Supplementary Table 3 for numerical results). Olive colour – words as information encoding units. Pink colour – characters as information encoding units. = 225), (c) Candidate set: medium (Nc = 274), (d) Candidate set: big (Nc = 2226). p < 0.05, * p < 0.01, *** p < 0.005. Statistical significance is determined based on non-parametric permutation tests (see Methods for details and Supplementary Table 3 for numerical results). Olive colour – words as information encoding units. Pink colour – characters as information encoding units. Second study. While the results presented in the previous section indicate that texts in languages with more speakers tend to be harder to learn, it is important to rule out that the results are mainly driven by several limitations inherent in the multilingual corpus collection used25: First, most of the texts in the database are rather short (median length = 150188 words; first quartile Q1 = 3385; third quartile Q3 = 234838). This can be a problem as LMs, especially more complex ones, typically require a lot of training input in order to achieve good performance.27,64 Secondly, Second study. While the results presented in the previous section indicate that texts in languages with more speakers tend to be harder to learn, it is important to rule out that the results are mainly driven by several limitations inherent in the multilingual corpus collection used25: First, most of the texts in the database are rather short (median length = 150188 words; first quartile Q1 = 3385; third quartile Q3 = 234838). This can be a problem as LMs, especially more complex ones, typically require a lot of training input in order to achieve good performance.27,64 Secondly, learning difficulty as defined by Ref.46 ultimately rests on an ansatz function that cannot be proven analytically.65 Thirdly, the database is unbalanced at the language level: while there are more than 100 languages with at least 10 available data points, i.e. training documents, there are less than four available data points for most languages (~84%). Results To further assess the robustness of these findings, we employ a more 9 9 computationally intensive technique known as cross-fit partialing-out (or double machine learning)44 to estimate the effect of speaker population size on learning difficulty and compute parametric p-values. This method has a less restrictive sparsity requirement and provides an additional validation of our results: Supplementary Table 5 shows that that the β-coefficient for speaker population is again negative and significant (at p < 0.005) in all cases. In Supplementary Table 6, we adopt a Bayesian perspective by using the lasso-selected controls as input for Bayesian linear regression models and show that, consistent with the results presented here, the probability of the coefficient of speaker population size being negative was estimated to be 1 across all compressors and both symbolic levels. Figure 3 \| Double-selection lasso linear regression results (Study 1). (a) Prediction performance: Out-of- sample R2 of learning difficulty against Out-of-sample R2 of speaker population size. In both cases, the out-of- sample R2 is computed for a sample distinct from the sample for which the control variables were selected by the lasso for the three different candidate sets. (b), (c) and (d) Bars – estimated coefficients, βDS, for the effect of speaker population size per LM and per symbol. (b) Candidate set: small (number of control covariates Nc Figure 3 \| Double-selection lasso linear regression results (Study 1). (a) Prediction performance: Out-of- Figure 3 \| Double-selection lasso linear regression results (Study 1). (a) Prediction performance: Out-of- sample R2 of learning difficulty against Out-of-sample R2 of speaker population size. In both cases, the out-of sample R2 is computed for a sample distinct from the sample for which the control variables were selected by the lasso for the three different candidate sets. (b), (c) and (d) Bars – estimated coefficients, βDS, for the effec of speaker population size per LM and per symbol. (b) Candidate set: small (number of control covariates N sample R2 of learning difficulty against Out-of-sample R2 of speaker population size. In both cases, the out-of- sample R2 is computed for a sample distinct from the sample for which the control variables were selected by the lasso for the three different candidate sets. (b), (c) and (d) Bars – estimated coefficients, βDS, for the effect of speaker population size per LM and per symbol. Results This reflects the fact that for languages spoken by a small number of people, there are only very few documents available electronically.66 This unbalancedness precludes the use of several approaches that have been discussed and successfully used in the literature41, but require balanced data as input. learning difficulty as defined by Ref.46 ultimately rests on an ansatz function that cannot be proven analytically.65 Thirdly, the database is unbalanced at the language level: while there are more than 100 languages with at least 10 available data points, i.e. training documents, there are less than four available data points for most languages (~84%). This reflects the fact that for languages spoken by a small number of people, there are only very few documents available electronically.66 This unbalancedness precludes the use of several approaches that have been discussed and successfully used in the literature41, but require balanced data as input. In consideration of these issues, we used the Parallel Bible Corpus67, which is available in a very fine-grained parallel structure (in terms of book, chapter and verse) and that provides additional information regarding the genealogical classification of languages. We created two fully balanced and parallel multilingual training corpora: (i) a New Testament (NT) version consisting of 5000 parallel verses available in 504 different languages and (ii) an Old Testament (OT) version consisting of 15000 parallel verses available in 138 different languages. While the Methods 11 11 section provides detailed information, we again would like to highlight a few key points here to facilitate the interpretation of our analyses: per version (NT/OT), we randomly assigned each available verse to one of ten folds. Per language, we then conducted a 10-fold rotation estimation where each fold served once as the test data and the remaining folds were used as training data resulting in 9 (data points per rotation) x 10 (folds) = 90 data points per version and language. section provides detailed information, we again would like to highlight a few key points here to facilitate the interpretation of our analyses: per version (NT/OT), we randomly assigned each available verse to one of ten folds. Per language, we then conducted a 10-fold rotation estimation where each fold served once as the test data and the remaining folds were used as training data resulting in 9 (data points per rotation) x 10 (folds) = 90 data points per version and language. Results For example, if fold 10 serves as the test data, we will train an LM on one of the remaining folds (randomly selected). We then computed the cross entropy H, i.e. the average number of bits per verse required to encode/predict each byte of fold 10 as a measure of the quality of the language model.68 Next, we added another of the remaining folds to the training data, re-trained the LM and calculated H again. This process was repeated until all nine folds have been used as training data. Per version, we then use all the resulting data points to fit an LMER with H as the outcome and a fixed effect for f, the number of folds used for training. We include (crossed) random intercepts for the test fold and for language. Crucially, we include random slopes per language, i.e. we allow the relationship between H and f to be different for each language. As illustrated in Figure 4, this random slope can be used as a measure of learning difficulty: the slope measures how much additional input improves the quality of the LM – for a language that is easy to learn, the LM has already generated an adequate representation of the input after the first (few) folds resulting in a slope that is comparatively less steep. For a language that is difficult to learn, the LM needs more input to learn to predict. The LM should therefore improve its quality more with more input, resulting in a comparatively steeper slope. In other words, the random slope parameter we are analysing here modulates the general relationship between H and f for all languages. The language-specific value of this random slope parameter is then indicative of a stronger or weaker relationship between H and f for that language. section provides detailed information, we again would like to highlight a few key points here to facilitate the interpretation of our analyses: per version (NT/OT), we randomly assigned each available verse to one of ten folds. Per language, we then conducted a 10-fold rotation estimation where each fold served once as the test data and the remaining folds were used as training data resulting in 9 (data points per rotation) x 10 (folds) = 90 data points per version and language. Results that are required (on average) to encode/predict the test data as a function of the number of training folds, f. We fit an LMER with the log of f as a fixed effect and random intercepts for the test fold and language. We include random slopes per language, which are represented by the different lines in the figure. The pink line corresponds to the estimated fixed effect of f, here a value of -9.00. Using the LMER, we obtained language- specific best linear unbiased predictions (BLUPs) of the random slopes, represented by the value of µ for each language. These BLUPs capture the interactions between f and language, with higher values of µ indicating faster learning and lower values indicating slower learning. As illustrated by the orange circles/line in the figure, some languages have higher values of µ, indicating that they are easier to learn. For these languages, the LM achieves better prediction quality with fewer training data points, resulting in a flatter slope of the regression line. The blue circles/line, on the other hand, represent languages that are more difficult to learn. These languages require more training data for the LM to achieve better levels of prediction quality, resulting in steeper slopes of the regression line. We fit an LMER with the log of f as a fixed effect and random intercepts for the test fold and language. We include random slopes per language, which are represented by the different lines in the figure. The pink line corresponds to the estimated fixed effect of f, here a value of -9.00. Using the LMER, we obtained language- specific best linear unbiased predictions (BLUPs) of the random slopes, represented by the value of µ for each language. These BLUPs capture the interactions between f and language, with higher values of µ indicating faster learning and lower values indicating slower learning. As illustrated by the orange circles/line in the figure, some languages have higher values of µ, indicating that they are easier to learn. For these languages, the LM achieves better prediction quality with fewer training data points, resulting in a flatter slope of the regression line. The blue circles/line, on the other hand, represent languages that are more difficult to learn. These languages require more training data for the LM to achieve better levels of prediction quality, resulting in steeper slopes of the regression line. Results For example, if fold 10 serves as the test data, we will train an LM on one of the remaining folds (randomly selected). We then computed the cross entropy H, i.e. the average number of bits per verse required to encode/predict each byte of fold 10 as a measure of the quality of the language model.68 Next, we added another of the remaining folds to the training data, re-trained the LM and calculated H again. This process was repeated until all nine folds have been used as training data. Per version, we then use all the resulting data points to fit an LMER with H as the outcome and a fixed effect for f, the number of folds used for training. We include (crossed) random intercepts for the test fold and for language. Crucially, we include random slopes per language, i.e. we allow the relationship between H and f to be different for each language. As illustrated in Figure 4, this random slope can be used as a measure of learning difficulty: the slope measures how much additional input improves the quality of the LM – for a language that is easy to learn, the LM has already generated an adequate representation of the input after the first (few) folds resulting in a slope that is comparatively less steep. For a language that is difficult to learn, the LM needs more input to learn to predict. The LM should therefore improve its quality more with more input, resulting in a comparatively steeper slope. In other words, the random slope parameter we are analysing here modulates the general relationship between H and f for all languages. The language-specific value of this random slope parameter is then indicative of a stronger or weaker relationship between H and f for that language. 12 Figure 4 \| Illustration of measuring learning difficulty in Study 2. Circles represent observed cross entropies H Figure 4 \| Illustration of measuring learning difficulty in Study 2. Circles represent observed cross entropies H g \| g g y y p p that are required (on average) to encode/predict the test data as a function of the number of training folds, f. that are required (on average) to encode/predict the test data as a function of the number of training folds, f. Results 13 As information encoding units, we estimate on two levels: on the level of words and, instead of estimating on the level of characters, we tokenize our text into sub-word units by byte pair encoding (BPE)69,70 which plays an important role in many state-of-the-art natural language 13 model applications71,72 and provides strong baseline results on a multilingual corpus73. In total, we trained seven different LMs on the data – ranging from very simple n-gram models to state- of-the-art deep neural networks (Table 1). Table 1 \| Language models used in Study 2. The language models used in Study 2 are listed below along with their implementation techniques, source, and time (in seconds) required to train each model on a median length document. The first three models are relatively simple, while the remaining four are more complex. The first four models were trained on 231480 documents while the last three were trained on 115740 documents due to the significant increase in training time, i.e. we only used the first five folds as test folds for the last three LMs, resulting in 45 data points per version and language, whereas all ten folds were used as test folds for the first four LMs, resulting in 90 data points per version and language. Further details on training and implementation are provided in the Methods section. LM Technique/Algorithm Source Time PPM2 N-gram modelling74, prediction by partial matching47, number of previous symbols: 2, memory: 2000 megabytes Ref.75,76 0.1 PPM6 N-gram modelling74, prediction by partial matching47, number of previous symbols: 6, memory: 2000 megabytes 0.1 LZMA Dictionary encoding77, dictionary size 1536 megabytes Ref.76 0.2 PAQ Context mixing52,78, gated linear network79, ~1.7 million weights, parameters ~3800 Ref.51,80 60.7 LSTMcomp Long short term memory54, parameters ~ 0.5 million Ref.53 149.2 NNCPsmall Transformer64, parameters ~ 2.24 million Ref.81 146.0 NNCPlarge Transformer64, parameters ~ 6.45 million 431.9 Per LM, per version (NT/OT) and per symbolic level (words/BPE), we estimated language- Table 1 \| Language models used in Study 2. The language models used in Study 2 are listed below along with their implementation techniques, source, and time (in seconds) required to train each model on a median length document. The first three models are relatively simple, while the remaining four are more complex. The first four models were trained on 231480 documents while the last three were trained on 115740 documents due to the significant increase in training time, i.e. Results For the NT version, 𝛽𝛽 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 𝐹𝐹𝐿𝐿𝐹𝐹= -0.0022 for words and 𝛽𝛽 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 𝐹𝐹𝐿𝐿𝐹𝐹= -0.0007 for BPE for PPM2 as LM, 𝛽𝛽 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 𝐹𝐹𝐿𝐿𝐹𝐹= -0.0019 for words and 𝛽𝛽 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 𝐹𝐹𝐿𝐿𝐹𝐹= -0.0016 for BPE for PPM6, 𝛽𝛽 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 𝐹𝐹𝐿𝐿𝐹𝐹= -0.0019 for words and 𝛽𝛽 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 𝐹𝐹𝐿𝐿𝐹𝐹= -0.0011 for BPE for LZMA, 𝛽𝛽 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 𝐹𝐹𝐿𝐿𝐹𝐹= -0.0014 for words and 𝛽𝛽 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 𝐹𝐹𝐿𝐿𝐹𝐹= -0.0009 for BPE for PAQ, 𝛽𝛽 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 𝐹𝐹𝐿𝐿𝐹𝐹= -0.0008 for words and 𝛽𝛽 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 𝐹𝐹𝐿𝐿𝐹𝐹= -0.0011 for BPE for LSTMcomp, 𝛽𝛽 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 𝐹𝐹𝐿𝐿𝐹𝐹= 0.0002 for words and 𝛽𝛽 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 𝐹𝐹𝐿𝐿𝐹𝐹= -0.0001 for BPE for NNCPsmall and 𝛽𝛽 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 𝐹𝐹𝐿𝐿𝐹𝐹= -0.0005 for words and 𝛽𝛽 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 𝐹𝐹𝐿𝐿𝐹𝐹= -0.0007 for BPE for NNCPsmall. For the OT version, 𝛽𝛽 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 𝐹𝐹𝐿𝐿𝐹𝐹= -0.0032 for words and 𝛽𝛽 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 𝐹𝐹𝐿𝐿𝐹𝐹= -0.0010 for BPE for PPM2, 𝛽𝛽 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 𝐹𝐹𝐿𝐿𝐹𝐹= -0.0033 for words and 𝛽𝛽 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 𝐹𝐹𝐿𝐿𝐹𝐹= -0.0020 for BPE for PPM6, 𝛽𝛽 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 𝐹𝐹𝐿𝐿𝐹𝐹= -0.0028 for words and 𝛽𝛽 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 𝐹𝐹𝐿𝐿𝐹𝐹= -0.0014 for BPE for LZMA, 𝛽𝛽 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 𝐹𝐹𝐿𝐿𝐹𝐹= -0.0024 for words and 𝛽𝛽 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 𝐹𝐹𝐿𝐿𝐹𝐹= -0.0011 for BPE for PAQ, 𝛽𝛽 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 𝐹𝐹𝐿𝐿𝐹𝐹= - 0.0021 for words and 𝛽𝛽 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 𝐹𝐹𝐿𝐿𝐹𝐹= -0.0015 for BPE for LSTMcomp, 𝛽𝛽 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 𝐹𝐹𝐿𝐿𝐹𝐹= -0.0013 for words and 𝛽𝛽 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 𝐹𝐹𝐿𝐿𝐹𝐹= -0.0016 for BPE for NNCPsmall and 𝛽𝛽 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 𝐹𝐹𝐿𝐿𝐹𝐹= -0.0015 for words and 𝛽𝛽 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 𝐹𝐹𝐿𝐿𝐹𝐹= -0.0017 for BPE for NNCPsmall. Figure 5c,d visualizes the estimated β-coefficients and 95% confidence intervals for the models with the lowest AIC per version, LM and symbolic level. There is a significant negative impact of population size on learning difficulty (p < 0.005) for all language separate LMERs with learning difficulty, µ, as the outcome. Analogous to Study 1, we created a maximum model that contains a fixed effect of the log of speaker population size and (crossed) random effects and slopes for writing script, macro area, country, language family, language subfamily and sub-branch. We then computed LMERs for all possible covariate subsets (1456 models). Figure 5a,b shows that with the exception of the small transformer model for the NT version, all computed FMA estimates for all N = 728 models that include a fixed effect for speaker population size (and potential random slopes) are negative for both corpus versions and both symbolic levels. Results we only used the first five folds as test folds for the last three LMs, resulting in 45 data points per version and language, whereas all ten folds were used as test folds for the first four LMs, resulting in 90 data points per version and language. Further details on training and implementation are provided in the Methods section. LM Technique/Algorithm Source Time PPM2 N-gram modelling74, prediction by partial matching47, number of previous symbols: 2, memory: 2000 megabytes Ref.75,76 0.1 PPM6 N-gram modelling74, prediction by partial matching47, number of previous symbols: 6, memory: 2000 megabytes 0.1 LZMA Dictionary encoding77, dictionary size 1536 megabytes Ref.76 0.2 PAQ Context mixing52,78, gated linear network79, ~1.7 million weights, parameters ~3800 Ref.51,80 60.7 LSTMcomp Long short term memory54, parameters ~ 0.5 million Ref.53 149.2 NNCPsmall Transformer64, parameters ~ 2.24 million Ref.81 146.0 NNCPlarge Transformer64, parameters ~ 6.45 million 431.9 14 Per LM, per version (NT/OT) and per symbolic level (words/BPE), we estimated language- specific random slopes, which serve as measures of learning difficulty as shown in Figure 4. To test for a potential relationship between population size and learning difficulty, we first ran 14 separate LMERs with learning difficulty, µ, as the outcome. Analogous to Study 1, we created a maximum model that contains a fixed effect of the log of speaker population size and (crossed) random effects and slopes for writing script, macro area, country, language family, language subfamily and sub-branch. We then computed LMERs for all possible covariate subsets (1456 models). Figure 5a,b shows that with the exception of the small transformer model for the NT version, all computed FMA estimates for all N = 728 models that include a fixed effect for speaker population size (and potential random slopes) are negative for both corpus versions and both symbolic levels. Results For the NT version, 𝛽𝛽 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 𝐹𝐹𝐿𝐿𝐹𝐹= -0.0022 for words and 𝛽𝛽 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 𝐹𝐹𝐿𝐿𝐹𝐹= -0.0007 for BPE for PPM2 as LM, 𝛽𝛽 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 𝐹𝐹𝐿𝐿𝐹𝐹= -0.0019 for words and 𝛽𝛽 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 𝐹𝐹𝐿𝐿𝐹𝐹= -0.0016 for BPE for PPM6, 𝛽𝐿𝐿𝐿𝐿𝐹𝐿𝐹𝛽𝐿𝐿𝐿𝐿𝐹𝐿𝐹𝛽𝐿𝐿𝐿𝐿𝐹𝐿𝐹 speaker population size (and potential random slopes) are negative for both corpus versions and both symbolic levels. For the NT version, 𝛽𝛽 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 𝐹𝐹𝐿𝐿𝐹𝐹= -0.0022 for words and 𝛽𝛽 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 𝐹𝐹𝐿𝐿𝐹𝐹= -0.0007 for BPE for PPM2 as LM, 𝛽𝛽 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 𝐹𝐹𝐿𝐿𝐹𝐹= -0.0019 for words and 𝛽𝛽 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 𝐹𝐹𝐿𝐿𝐹𝐹= -0.0016 for BPE for PPM6, 𝛽𝛽 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 𝐹𝐹𝐿𝐿𝐹𝐹= -0.0019 for words and 𝛽𝛽 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 𝐹𝐹𝐿𝐿𝐹𝐹= -0.0011 for BPE for LZMA, 𝛽𝛽 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 𝐹𝐹𝐿𝐿𝐹𝐹= -0.0014 for words and 𝛽𝛽 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 𝐹𝐹𝐿𝐿𝐹𝐹= -0.0009 for BPE for PAQ, 𝛽𝛽 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 𝐹𝐹𝐿𝐿𝐹𝐹= -0.0008 for words and 𝛽𝛽 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 𝐹𝐹𝐿𝐿𝐹𝐹= -0.0011 for BPE for LSTMcomp, 𝛽𝛽 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 𝐹𝐹𝐿𝐿𝐹𝐹= 0.0002 for words and 𝛽𝛽 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 𝐹𝐹𝐿𝐿𝐹𝐹= -0.0001 for BPE for NNCPsmall and 𝛽𝛽 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 𝐹𝐹𝐿𝐿𝐹𝐹= -0.0005 for words and 𝛽𝛽 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 𝐹𝐹𝐿𝐿𝐹𝐹= -0.0007 for BPE for NNCPsmall. For the OT version, 𝛽𝛽 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 𝐹𝐹𝐿𝐿𝐹𝐹= -0.0032 for words and 𝛽𝛽 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 𝐹𝐹𝐿𝐿𝐹𝐹= -0.0010 for BPE for PPM2, 𝛽𝛽 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 𝐹𝐹𝐿𝐿𝐹𝐹= -0.0033 for words and 𝛽𝛽 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 𝐹𝐹𝐿𝐿𝐹𝐹= -0.0020 for BPE for PPM6, 𝛽𝛽 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 𝐹𝐹𝐿𝐿𝐹𝐹= -0.0028 for words and 𝛽𝛽 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 𝐹𝐹𝐿𝐿𝐹𝐹= -0.0014 for BPE for LZMA, 𝛽𝛽 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 𝐹𝐹𝐿𝐿𝐹𝐹= -0.0024 for words and 𝛽𝛽 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 𝐹𝐹𝐿𝐿𝐹𝐹= -0.0011 for BPE for PAQ, 𝛽𝛽 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 𝐹𝐹𝐿𝐿𝐹𝐹= - 0.0021 for words and 𝛽𝛽 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 𝐹𝐹𝐿𝐿𝐹𝐹= -0.0015 for BPE for LSTMcomp, 𝛽𝛽 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 𝐹𝐹𝐿𝐿𝐹𝐹= -0.0013 for words and 𝛽𝛽 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 𝐹𝐹𝐿𝐿𝐹𝐹= -0.0016 for BPE for NNCPsmall and 𝛽𝛽 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 𝐹𝐹𝐿𝐿𝐹𝐹= -0.0015 for words and 𝛽𝛽 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 𝐹𝐹𝐿𝐿𝐹𝐹= -0.0017 for BPE for NNCPsmall. Figure 5c,d visualizes the estimated β-coefficients and 95% confidence intervals for the models with the lowest AIC per version, LM and symbolic level. There is a significant negative impact of population size on learning difficulty (p < 0.005) for all language models in the NT version, except for the two transformer models (see Supplementary Table 7 for numerical results and model specifications). Here, only the coefficient for the larger transformer 15 15 on the BPE level was statistically significant (p < 0.005), while both coefficients for the small transformer LM were deemed non-significant. Results Given that transformers are known to require large amounts of training data to perform well64, we attribute the lack of significance in the coefficients based on the smaller transformer to the limited size of the training data used for the NT version. This assumption is consistent with the fact that all βLMER-values at both symbolic levels were negative at p < 0.005 for the OT version, where the amount of training data is three times larger. Supplementary Table 7 shows that these results are corroborated by the ΔAIC-values. In addition, we show in Supplementary Table 8 that these results are fully supported by lasso linear regressions similar to those presented in Supplementary Tables 3, 4, 5. on the BPE level was statistically significant (p < 0.005), while both coefficients for the small transformer LM were deemed non-significant. Given that transformers are known to require large amounts of training data to perform well64, we attribute the lack of significance in the coefficients based on the smaller transformer to the limited size of the training data used for the NT version. on the BPE level was statistically significant (p < 0.005), while both coefficients for the small transformer LM were deemed non-significant. Given that transformers are known to require large amounts of training data to perform well64, we attribute the lack of significance in the coefficients based on the smaller transformer to the limited size of the training data used for the NT version. This assumption is consistent with the fact that all βLMER-values at both symbolic levels were negative at p < 0.005 for the OT version, where the amount of training data is three times larger. Supplementary Table 7 shows that these results are corroborated by the ΔAIC-values. In addition, we show in Supplementary Table 8 that these results are fully supported by lasso linear regressions similar to those presented in Supplementary Tables 3, 4, 5. 16 16 Figure 5 \| Multilevel mixed-effects linear regression results (Study 2). (a) and (c) Results for NT (N = 504). (b) and (d) Results for OT (N = 138). (a) and (b) Distribution of the estimated impact of speaker population size, βLMER, per LM and per symbol for a total of 2430 models that include a fixed effect (and potential random slopes) for speaker population size. Results (c) and (d) Estimated βLMER (vertical line) and 95% confidence interval (horizontal line) for the model with the lowest AIC per LM and per symbol (see Supplementary Table 7 for numerical results and model specifications). Olive colour – words as information encoding units. Blue colour – byte-pair encoding. To further investigate the relationship between language learning difficulty and population size, we proceed by explicitly modelling the degree of covariation due to descent from a common ancestor. To this end, we conduct a Phylogenetic Generalised Least Squares (PGLS) regression with learning difficulty as the outcome and speaker population size as a covariate.41 We use a phylogenetic tree provided by Ref.82 that was generated using language taxonomies from Ethnologue.83 Figure 6 presents the results, which are in close agreement with the LMER results (Figure 5c,d): with the exception of the small transformer model for the NT version, all βPGLS- coefficients are negative at p < 0.005 (see Supplementary Table 9 for numerical details). The information-theoretic approach where we calculate ΔAIC between reduced models that do not include population size and full models, again supports these results. Excluding the small transformer model for the NT version, speaker population size explains the median amount of variance in learning difficulty of 𝑅𝑅𝑚𝑚𝑚𝑚𝑚𝑚 2 = 7.59% (Q1 = 5.29%, Q3 = 8.42%). For the OT version, the results for all LMs are more pronounced (𝑅𝑅𝑚𝑚𝑚𝑚𝑚𝑚 2 = 22.00%, Q1 = 18.49%, Q3 = 25.33%, no exclusion of the small transformer model). 17 Figure 6 \| Phylogenetic generalized least squares regression results (Study 2). Estimated βPGLS (vertical line) and 95% confidence interval (horizontal line) per LM and per symbol (see Supplementary Table 9 for numerical results). (a) Results for NT (N = 504). (b) Results for OT (N = 138). Olive colour – words as information encoding units. Blue colour – byte-pair encoding. Figure 6 \| Phylogenetic generalized least squares regression results (Study 2). Estimated βPGLS (vertical line) and 95% confidence interval (horizontal line) per LM and per symbol (see Supplementary Table 9 for numerical results). (a) Results for NT (N = 504). (b) Results for OT (N = 138). Olive colour – words as information encoding units. Blue colour – byte-pair encoding. Figure 6 \| Phylogenetic generalized least squares regression results (Study 2). Estimated βPGLS (vertical line) and 95% confidence interval (horizontal line) per LM and per symbol (see Supplementary Table 9 for numerical results). Results (a) Results for NT (N = 504). (b) Results for OT (N = 138). Olive colour – words as information encoding units. Blue colour – byte-pair encoding. genetic generalized least squares regression results (Study 2). Estimated βPGLS (vertical line) While the PGLS analyses explicitly control for genealogical relatedness, spatial proximity can also generate non-independence in comparative language data.21,41,84 To control for both sources of influence simultaneously, we use two weighting matrices: While the PGLS analyses explicitly control for genealogical relatedness, spatial proximity can also generate non-independence in comparative language data.21,41,84 To control for both sources of influence simultaneously, we use two weighting matrices: (i) to control for spatial proximity, we generate a matrix containing the geographical distances between languages; (i) (ii) to control for genealogical relatedness, we used a phylogenetic dissimilarity matrix provided by Ref.85 that is based on word lists from the Automated Similarity Judgment Program (ASJP).86 (ii) 18 We conduct spatial autoregressive errors regression models (SAR)45 with learning difficulty as the outcome and speaker population size as a covariate. We add two spatially lagged error terms specified by the inverse of each weighting matrix using a generalized spatial two-stage least- squares estimator (GS2SLS).87 We conduct spatial autoregressive errors regression models (SAR)45 with learning difficulty as the outcome and speaker population size as a covariate. We add two spatially lagged error terms specified by the inverse of each weighting matrix using a generalized spatial two-stage least- squares estimator (GS2SLS).87 Figure 7 \| Spatial autoregressive error regression results (Study 2). Bars – estimated coefficients, βSAR, for the effect of speaker population size per LM and per symbol. (a) Results for NT (N = 414). (b) Results for OT (N = 126). Each model contains autoregressive error terms for phylogenetic relatedness and geographical proximity simultaneously estimated by two inverse-distance matrices. p < 0.05, * p < 0.01, *** p < 0.005. Statistical significance is determined based on non-parametric permutation tests (see Methods for details and Supplementary Table 10 for numerical results). Olive colour – words as information encoding units. Blue colour – byte-pair encoding. Figure 7 \| Spatial autoregressive error regression results (Study 2). Bars – estimated coefficients, βSAR, for the effect of speaker population size per LM and per symbol. (a) Results for NT (N = 414). (b) Results for OT (N = 126). Results Each model contains autoregressive error terms for phylogenetic relatedness and geographical proximity simultaneously estimated by two inverse-distance matrices. p < 0.05, * p < 0.01, *** p < 0.005. Statistical significance is determined based on non-parametric permutation tests (see Methods for details and Supplementary Table 10 for numerical results). Olive colour – words as information encoding units. Blue colour – byte-pair encoding. The SAR results are visualized in Figure 7. Again, all results are in close agreement with the other analyses presented in this section: 27 out of 28 estimated βSAR-coefficients are negative. With the exception of the small transformer model for the NT version, all coefficients pass a non- parametric permutation test (see Methods for details) with p < 0.05 in one case, p < 0.01 in four cases and p < 0.005 in the remaining 21 cases (goodness-of-fit: NT version, excluding NNCPsmall: 19 19 𝑅𝑅𝑚𝑚𝑚𝑚𝑚𝑚 2 = 8.64%, Q1 = 5.57%, Q3 = 10.44%; OT version: 𝑅𝑅𝑚𝑚𝑚𝑚𝑚𝑚 2 = 17.02%, Q1 = 14.32%, Q3 = 20.38%, see Supplementary Table 10 for numerical details). 8.64%, Q1 = 5.57%, Q3 = 10.44%; OT version: 𝑅𝑅𝑚𝑚𝑚𝑚𝑚𝑚 2 = 17.02%, Q1 = 14.32%, Q3 = 20.38%, see Supplementary Table 10 for numerical details). 20.38%, see Supplementary Table 10 for numerical details). Discussion In this article, we examined the assumption that all languages are equally difficult to learn by using LMs as computational working models for empirically studying various aspects of human language.26 In summary, we find that there is evidence for an effect of speaker population size on learning difficulty that questions the above assumption. This evidence turns out to be stable across different datasets and two different ways of operationalising learning difficulty. We have observed this relationship across a range of LMs, ranging from very basic n-gram models that use only the last few symbols for prediction to state-of-the-art deep neural network large language models that leverage complex computational architectures and mechanisms that allow them to capture long-range dependencies and contextual information in the text. Despite the differences in model complexity, the observed correlation held consistently, highlighting the robustness of this finding across the spectrum of LMs. To address the potential non-independence of observations resulting from the phylogenetic and geographic relationships between languages, we employed established methods from existing literature41 while also introducing novel approaches to analyse our data.43,45 In addition, we used both parametric and non-parametric tests to determine statistical significance, recognising that our data cannot be considered a random sample of all existing languages.61 By using a variety of methods and analyses, we have increased the reliability and generalisability of our primary finding, which challenges our initial expectations: contrary to what we expected, our research reveals a positive statistical association between population size and learning difficulty, suggesting that languages with more speakers tend to be harder to learn. 20 The expectation that there should be an inverse correlation between speaker population size and learning difficulty can be traced back to the linguistic niche hypothesis, which suggests that the social niche that a language occupies in a community affects its structural properties.2,18 The expectation that there should be an inverse correlation between speaker population size and learning difficulty can be traced back to the linguistic niche hypothesis, which suggests that the social niche that a language occupies in a community affects its structural properties.2,18 Specifically, the linguistic niche hypothesis suggests that languages with large numbers of speakers tend to simplify their grammar and have a reduced structural complexity. It assumes that languages that are spoken by more people over larger geographical areas are, on average, also learned by a larger proportion of adults. Discussion Since languages with complex structures appear to be difficult for adults to learn, the linguistic niche hypothesis conjectures that there should be a negative selection against complexity, i.e. languages tend to adapt and simplify when they are spoken by larger communities that include a significant number of adult learners.18,88,89 Subsequently, the linguistic niche hypothesis has been an important starting point that generated extensive research in the field.89–95 Only very recently, research has emerged that casts doubt on the validity of the specific assertions made by the linguistic niche hypothesis: it has been shown that the number of adult learners does not appear to impact language complexity21,23 and that languages with more speakers tend to be more complex, not less.21,25 In a similar vein, the results presented in this article suggest that languages with more speakers are not easier to learn but more difficult. It is important to point out in this context that LMs are only working models and that there are therefore important limitations.26 In particular, we neither claim that there is a one-to-one correspondence between human and machine language learning, nor that LMs understand language in a human-like sense.96 Future work could explore whether and to what extent our results also apply to human language learning. However, we agree with Refs.26,27 that, given their impressive performance in natural language processing71,97, LMs, especially so-called large 21 language models, are worthy of scientific investigation, because of their inherent “potential to inform future work in the cognitive science of language”.26 In our study we have tried to exploit this potential, and we hope that we have, at a very minimum, been able to show that the sociolinguistic structure of language and learnability by machines do not seem to be statistically independent of each other. Nevertheless, as language scientists, our primary objective is to comprehend the underlying reasons behind the specific characteristics of human language, and it is not clear whether the results regarding machine learnability presented in this study can be extrapolated to human language learning as well. A link to human language learning could be made through the field of artificial grammar learning, where participants are asked to judge the permissibility of an upcoming symbol in a sequence of symbols based on the rules of an underlying artificial grammar. Language models We use general-purpose data compression algorithms, taking advantage of the fact that there is a close connection between understanding, prediction and compression.100,101 All data compression algorithms consist of a model and a coder.78 Our focus is on the class of (lossless) compressors where the algorithm estimates a model, i.e. a conditional probability distribution, based on the training data, which can be used to generate predictions. To perform compression, the predicted probabilities are then used to encode symbols using a technique called arithmetic encoding.102 The language models that we use are summarized in Table 1. In what follows, further details are given for each language model. PPM is a dynamic and adaptive variable-order n-gram LM. The algorithm makes an assumption of the Markov property: to predict the next symbol, the algorithm uses the last o symbols that immediately precede the symbol of interest.47,103 For Study 2, we use two different values for o, 2 and 6, i.e. the last 2 resp. 6 symbols are used as context to generate predictions, whereas the optimal order in the range of [2, 32] is learned directly from the data in Study 1.25,46 In both studies, the level of compression is set to maximum and the size of used memory is set to 2000 megabytes. LZMA employs a compression strategy wherein repetitive segments within the data are identified and replaced by references pointing to a single instance of that segment occurring earlier in the uncompressed data stream. These matches are encoded using a length-distance pair, indicating that a specific number of symbols following the match are identical to the symbols located a certain distance back in the uncompressed stream.77,78 LZMA is only used in Study 2; the level of compression is set to maximum and the size of the compression dictionary is set to the maximum value of 1536 megabytes. PAQ can be described as a weighted combination of predictions from a large number of models, where the individual models are combined using a gated linear network.52,78–80 The network has a single layer with 552 input nodes and 3080 input weights. The model has a total of approximately 1.7 million weights, but due to the sparse updating scheme which leads to faster compression and decompression, the effective number of parameters used in training is significantly lower. Only 552∙7 = 3864 weights are updated for each bit of data. Discussion In this paradigm, it can be shown that the complexity of an artificial grammar is positively correlated with the error rate of participants who implicitly learn the underlying rules of an artificial grammar (i.e., by reading language material based on the artificial grammar).98,99 Our results suggest that rule complexity should also influence the speed of artificial grammar learning (measured, e.g., as number of experimental trials until the rules have been extracted from the linguistic material). Using advanced machine learning techniques, we found that speaker population size negatively affects learning speed after controlling for potential confounding from translation effects and several environmental variables such as geographic range size. To understand how speaker population size affects machine learning difficulty, future studies could build on this methodological approach and examine the impact of other potential covariates. Another promising avenue for future work would therefore be to investigate which types of grammatical 22 structures tend to be more difficult for LMs to learn, and whether those features covary with speaker population size. A recently published new cross-linguistic database of grammatical features of unprecedented size17 provides an ideal starting point for such endeavours. structures tend to be more difficult for LMs to learn, and whether those features covary with speaker population size. A recently published new cross-linguistic database of grammatical features of unprecedented size17 provides an ideal starting point for such endeavours. Language models In both studies, we use version PAQ8o and set the compression level to maximum, requiring 1712 megabytes of memory. As PAQ, lstm- 23 compress53, referred to as LSTMcomp throughout the manuscript, combines predictions from independent models. Predictions are combined using a long short-term memory deep neural network.54 The network is trained using backpropagation through time104 and Adam optimization is used to update network weights.105 The algorithm takes no options and we do not use any dictionary-based pre-processor in neither Study 1 nor Study 2. In total, the model has 508936 parameters (assuming the corresponding input file uses all 256 possible bytes. The model size may be smaller if the input file contains a smaller set of bytes). NNCP81 is a lossless data compressor that is based on the Transformer XL model defined in Ref.97. Modifications to the original Transformer XL model and algorithmic details are provided in Refs.106,107. As for lstm-compress, the Adam optimizer is used. For NNCPsmall, we use the default options with four layers and a resulting total number of parameters of ~2.24 million. For NNCPlarge, we change the default options to twelve layers. This results in a total of ~6.45 million parameters. For both versions, we use the Gaussian error linear unit activation function108, we do not use a text pre-processor or tokenizer and we use the faster “encode only” mode (the output cannot be decompressed, but the compression itself is still lossless). As for lstm-compress, the Adam optimizer is used. For NNCPsmall, we use the default options with four layers and a resulting total number of parameters of ~2.24 million. For NNCPlarge, we change the default options to twelve layers. This results in a total of ~6.45 million parameters. For both versions, we use the Gaussian error linear unit activation function108, we do not use a text pre-processor or tokenizer and we use the faster “encode only” mode (the output cannot be decompressed, but the compression itself is still lossless). Data For Study 1, we use part of a large-scale database of written multilingual texts compiled by Ref.25. In total, we use information on 3853 documents contained in 40 different multilingual corpora comprising a large variety of different text types. The documents range in length from a few tens to several hundreds of millions of words. 33 of these corpora consist of fully parallel texts. Parallel texts are texts in different languages that contain the same message, but differ in the language used (e.g. subtitles of a movie in different languages). The remaining seven corpora are comparable corpora, i.e. texts that are not parallel but come from comparable sources and are therefore similar in content (e.g. Wikipedia or newspaper articles). In-depth details on the database and each corpus are given in the Methods section and supplementary information of Ref.25 that is available at https://osf.io/f5mke/. For Study 2, we use data from the Parallel Bible Corpus made available by Ref.67, which contains 1568 unique translations of the Bible in 1166 different languages in a fine-grained parallel structure (in terms of book, chapter and verse). Each translation is already tokenized and Unicode normalized and spaces are inserted between words as well as punctuation marks and non-alphabetic symbols by Ref.67. In addition, all texts were manually checked and corrected by Ref.67 where necessary. In some texts without spaces or marks between words (e.g. for Khmer, Burmese, or Mandarin Chinese), we used a dictionary lookup method described in Ref.25 to detect word boundaries with detected word tokens then being space- separated. All uppercase characters are lowered based on the closest language-specific ISO-639-3 code. We then split each Bible translation into different books of the biblical canon, aggregating all books of the 24 New Testament (NT) and the Old Testament (OT). Beside the actual text, each element of each line of each Bible translation document contains information about the book, chapter and verse number.67 Per version, we dropped translations with no available verse. For languages with more than one available translation, we kept the translation with most available verses and broke ties at random. In total, we ended up with 1062 different languages for the NT version and 189 different languages for the OT version. On average, each NT translation consists of 7840 verses and each OT translation consists of 17086 verses. Data Per version, we then dropped partly incomplete translations and removed verses that are only available in some translations and selected verses that appeared in as many translations as possible. For the NT version, we selected 5000 verses that are available in 504 different languages. For the OT version, we selected 15000 parallel verses in 138 different languages. Note that the biblical canon consists of different books, for our selection 24 books for NT and 36 books for OT. Per version (NT/OT) we prepared stratified samples by randomly assigning each available verse from each available book to one of ten folds. In addition, we made sure that both (i) the verse order across translations and folds and (ii) the sequential training order is fully balanced and parallel; (i) means that each fold consists of the same verses that the LM is trained with, in the same order. From an information-theoretic perspective, this procedure ensures that – apart from random fluctuations – each fold contains text drawn from the same information source109 and thus induces stationarity.110 Regarding (ii), assume that fold 10 is the test fold, and the remaining folds are used to sequentially train the LM. We generated random training sequences, e.g. fold 3 – fold 2 – fold 8 – fold 5 – fold 4 – fold 9 – fold 6 – fold 7 – fold 1. This means that the LM is first trained on fold 3, we then compute H, i.e. the average number of bits per verse that are needed to encode each byte of fold 10, then the LM is trained on fold 3 and fold 2 and H is computed again, and so on. Training sequences are kept parallel across translations. Sociodemographic and linguistic variables Information on speaker population size, corpus, language family, language (identified by its ISO code), macro area, country, writing script, speaker population size, longitude and latitude are taken from Ref.25. EGIDS level information was initially sourced from Ref.113, which is reported in Glottolog114 (v4.2.1). Country is defined by Ethnologue as the primary country/country of origin of the language in question.15 To ensure completeness, we manually supplemented missing data from Ref.113 by cross-referencing with Glottolog and Ethnologue. The EGIDS level serves as a measure of a language's endangerment status.57 The purpose is to use the EGIDS level as a covariate to control for potential translation effects55,111, as languages with lower EGIDS levels could be more likely to be used as source languages, while languages with higher EGIDS levels could be more likely to be used as target languages. For example, an EGIDS level of 0 (labelled "International") pertains to the six official United Nations languages: Arabic, Chinese, English, French, Russian, and Spanish. On the other hand, languages with values of five and above pertain to languages that are not used in formal education, mass media or by the government, and they may consequently be more susceptible to (more) pronounced "translationese" influences.111 Additional information used in Study 2 regarding the classification of languages into family, subfamily and sub-branch are taken from Ref.67. We manually added information for five languages that was missing by using publicly available genealogical classifications (ISO codes gso, lbk, lsm, npi and yan, see https://osf.io/sa9x2/ for details). Classifications in Ref.67 are given as comma-separated values, we define the first value as the family, the second one as the subfamily and the third one as the sub-branch e.g. for the language “Ghotuo” the classification is “Niger-Congo, Atlantic-Congo, Volta-Congo, Benue-Congo, Edoid, North-Central, Ghotuo-Uneme-Yekhee”, so the family is “Niger-Congo”, the subfamily is “Atlantic-Congo” and the sub-branch is “Volta-Congo”. Additionally, we use a phylogenetic tree provided by Ref.82 for the PGLS regressions and a dissimilarity matrix provided by Ref.85 for the SAR regressions. We take information on language range size estimates from Ref.115 and information on distance to water resources, altitude and two variables on climatic information (Climate PC1 and Climate PC2) from Ref.116. Information on the number of countries in which each language is spoken was sourced from Glottolog (v4.2.1). Information encoding units For Study 1, we follow Ref.25 and compute the relevant quantities for both words and characters as information encoding units/symbols. For Study 2, we estimate on the level of words, but not on the level of characters, since there are idiosyncrasies/vagaries of the writing system that can lead to cross-linguistic differences in the mapping between phonemes and graphemes on the level of characters.111,112 Instead, we apply byte pair encoding (BPE)69,70 to split words into one or several units and the LM will be trained over the resulting sequences of sub-word units. BPE plays an important role in many state-of-the-art natural language modelling applications71,72 and provides strong baseline results on a multilingual corpus73. Note that the BPE is always extracted from the training data only and then applied to both the training and the test data. We follow Ref.69 and set the number of BPE merges to 0.4·C where C is the number of different word types observed in the training data. 25 On the level of words and sub-words, each unique symbol type is replaced by one unique 4-byte Unicode symbol. Each LM is then trained on the resulting sequence of Unicode symbols. On the level of characters, each LM is trained directly on the raw text. Estimating LM learning difficulty The link between understanding, prediction and compression mentioned above (“Language models”) directly implies that the better the compression, the better the language model.78 𝑟𝑙 In Study 1, we take advantage of this fact by measuring the compression rate 𝑟𝑟𝑙𝑙 for different sub- sequences of increasing length l where 𝑟𝑟𝑙𝑙 represents the number of bits per symbols that are needed to compress the first l symbols. Estimating the shape of the curve of the resulting series of compression lengths gives us a measure of how well language learning succeeds.32,118 For PPM as LM, we take the series of compression rates directly from Ref.25. Here, each document in each corpus is compressed every m symbols where m is some pre-defined corpus-specific chunk size, e.g. 1000 symbols. For the purpose of this study, we carried out a comprehensive retraining of all documents utilizing two additional language models, PAQ and LSTMcomp. Due to the significant increase in training time required (see Table 1), we did not use the corpus-specific chunk size pre-defined by Ref.25, but compressed each document of each multilingual corpus every 5% of all symbols, resulting in 20 data points, i.e. 𝑟𝑟𝑙𝑙-values per document. Note that consistency checks revealed that LSTMcomp repeatedly produced inconsistent results for the following documents that belong to the United Nations Parallel Corpus119, see Ref.25 for further details: ISO code “fra”, level: words, 𝑟𝑟𝑙𝑙 at 95% and 100%; ISO code “rus”, level: characters, all 𝑟𝑟𝑙𝑙-values from 65% to 100%; ISO code “rus”, level: words, 𝑟𝑟𝑙𝑙 at 100%. Since these inconsistencies could not be resolved, we exclude these 𝑟𝑟𝑙𝑙-values in what follows. We fit a variant of the ansatz suggested by Ref.65 to each series of compression rates: 𝑟𝐴𝑙 𝑟𝑟𝑙𝑙= ℎ+ 𝐴𝐴· log 𝑙𝑙 𝑙𝑙𝑏𝑏 (1) 𝑟𝑅𝑋𝑙𝑙 (1) where A > 0, b > 0 and ℎ> 0; 𝑟𝑟𝑙𝑙= 𝑅𝑅(𝑋𝑋1 𝑙𝑙)/𝑙𝑙 denotes the number of bits per symbol that are needed to compress the first l symbols of a document. h is the limiting entropy rate, A is a proportionality constant and b describes the shape of the curve and thus can be used to quantify learning difficulty as visualised in Figure 1 and Supplementary Figure 1. Sociodemographic and linguistic variables We manually supplemented missing data by cross-referencing with Ethnologue.83,117 The rationale behind considering this variable as a potential covariate is to account for the varying degrees of pluricentrism.56 For instance, languages such as Chinese or Spanish are spoken in several countries and may therefore have different codified standard forms. For further information and a discussion of potential 26 caveats and problems regarding the assignment of environmental variables to individual languages in order to reflect local grouping structure, see Refs.36,116. caveats and problems regarding the assignment of environmental variables to individual languages in order to reflect local grouping structure, see Refs.36,116. Estimating LM learning difficulty where f = 1, 2, …, 9 denotes the number of folds that are used to train the LM, 𝑁𝑁𝑣𝑣 denotes the number of verses and 𝑅𝑅(𝑋𝑋) denotes the compressed size of string X. 𝑇𝑇𝑓𝑓 denotes a string that consists of the concatenation of the first f training folds, while 𝑇𝑇𝑓𝑓𝑇𝑇𝑡𝑡𝑚𝑚𝑡𝑡𝑡𝑡 represents the concatenation of 𝑇𝑇𝑓𝑓 and the test fold 𝑇𝑇𝑡𝑡𝑚𝑚𝑡𝑡𝑡𝑡. Note that on both symbolic levels (words/BPE) we also compress the mapping of unique symbols to 4-byte Unicode symbol mentioned above (“Information encoding units”) and add the resulting compressed lengths to 𝑅𝑅𝑓𝑓൫𝑇𝑇𝑓𝑓𝑇𝑇𝑡𝑡𝑚𝑚𝑡𝑡𝑡𝑡൯ and 𝑅𝑅𝑓𝑓(𝑇𝑇𝑓𝑓). In general, there is a strong negative correlation between cross entropy and the number of folds, for both versions, both symbolic levels, all LMs and all languages (NT version, word level – PPM2: median Pearson correlation between the log of 𝐻𝐻𝑓𝑓 and the log of f, rmed = -0.90; PPM6: rmed = -0.97; LZMA: rmed = -0.97; PAQ: rmed = -0.97; LSTMcomp: rmed = -0.98; NNCPsmall: rmed = -0.97; NNCPlarge: rmed = -0.98; BPE l l PPM2 0 73 PPM6 0 94 LZMA 0 94 PAQ 0 94 LSTM In general, there is a strong negative correlation between cross entropy and the number of folds, for both versions, both symbolic levels, all LMs and all languages (NT version, word level – PPM2: median Pearson correlation between the log of 𝐻𝐻𝑓𝑓 and the log of f, rmed = -0.90; PPM6: rmed = -0.97; LZMA: rmed = -0.97; PAQ: rmed = -0.97; LSTMcomp: rmed = -0.98; NNCPsmall: rmed = -0.97; NNCPlarge: rmed = -0.98; BPE level – PPM2: rmed = -0.73; PPM6: rmed = -0.94; LZMA: rmed = -0.94; PAQ: rmed = -0.94; LSTMcomp: rmed = -0.98; rmed = NNCPsmall: rmed = -0.97; NNCPlarge: rmed = -0.98; OT version, word level – PPM2: rmed = -0.91; PPM6: rmed = -0.99; LZMA: rmed = -0.99; PAQ: rmed = -0.99; LSTMcomp: rmed = -0.99; NNCPsmall: rmed = - 0.98; NNCPlarge: rmed = -0.99; BPE level – PPM2: rmed = -0.77; PPM6: rmed = -0.98; LZMA: rmed = -0.99; PAQ: rmed = -0.99; LSTMcomp: rmed = -0.98; NNCPsmall: rmed = -0.98; NNCPlarge: rmed = -0.99). This demonstrates that all LMs – on average – improve with input. Estimating LM learning difficulty To estimate the three parameters of the ansatz function, we fit the following nonlinear function by log-least squares: 𝑟𝐴𝑙 𝑟𝑟𝑙𝑙= exp ቀℎ∗+ exp (𝐴𝐴′) · log 𝑙𝑙 𝑙𝑙exp (𝑏𝑏′)ቁ+ ό𝑙𝑙 (2) (2) where ό𝑙𝑙 is an independent and identically distributed (i.i.d.) error term and exp() denotes the exponential function. Since we want A and b to be positive, we set interval constraints that make sure that the 27 optimisation algorithm will not search in the negative subspace by fitting both parameters as exponentials, i.e. we estimate 𝐴𝐴′ = log (𝐴𝐴) and 𝑏𝑏′ = log(𝑏𝑏). The limiting entropy rate is recovered as ℎ= exp (ℎ∗). Since achieving convergence of the parameter estimates turned out to be difficult25, we approximate initial values in linear space, i.e., for each value of φ=.01, .02, …, 10, we calculate 𝛷𝛷= log 𝑙𝑙 𝑙𝑙𝜑𝜑 and fit the following linear regression by ordinary least squares: 𝑟𝑙𝛽𝛽𝐹𝛷𝑙 log(𝑟𝑟𝑙𝑙) = 𝛽𝛽ℎ+ 𝛽𝛽𝐹𝐹𝛷𝛷+ ό𝑙𝑙 (3) 𝛽 log(𝑟𝑟𝑙𝑙) = 𝛽𝛽ℎ+ 𝛽𝛽𝐹𝐹𝛷𝛷+ ό𝑙𝑙 (3) (3) where ό𝑙𝑙 is an i.i.d. error term. To provide initial values to fit equation (2), we pick the solution of equation (3) where the root mean squared error is smallest and where 𝛽𝛽𝐹𝐹> 0, then ℎ∗ is initialized as 𝛽𝛽ℎ, 𝐴𝐴′ is initialized as exp (𝛽𝛽𝐹𝐹) and 𝑏𝑏′ is initialized as exp (𝜑𝜑𝑚𝑚) where φm denotes the value of φ corresponding to the selected Φ. Further details are provided in Ref.25. In Study 2, we compute the cross entropy H, i.e. the number of bits needed on average to encode/predict a training verse for each document as a function of the number of training folds as follows: 𝐻𝐿𝑇𝑇𝐿𝑇 𝐻𝐻𝑓𝑓= 𝐿𝐿൫𝑇𝑇𝑓𝑓𝑇𝑇𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡൯−𝐿𝐿(𝑇𝑇𝑓𝑓) 𝑁𝑁𝑣𝑣 (4) 𝑁𝑣 (4) where f = 1, 2, …, 9 denotes the number of folds that are used to train the LM, 𝑁𝑁𝑣𝑣 denotes the number of verses and 𝑅𝑅(𝑋𝑋) denotes the compressed size of string X. 𝑇𝑇𝑓𝑓 denotes a string that consists of the concatenation of the first f training folds, while 𝑇𝑇𝑓𝑓𝑇𝑇𝑡𝑡𝑚𝑚𝑡𝑡𝑡𝑡 represents the concatenation of 𝑇𝑇𝑓𝑓 and the test fold 𝑇𝑇𝑡𝑡𝑚𝑚𝑡𝑡𝑡𝑡. Note that on both symbolic levels (words/BPE) we also compress the mapping of unique symbols to 4-byte Unicode symbol mentioned above (“Information encoding units”) and add the resulting compressed lengths to 𝑅𝑅𝑓𝑓൫𝑇𝑇𝑓𝑓𝑇𝑇𝑡𝑡𝑚𝑚𝑡𝑡𝑡𝑡൯ and 𝑅𝑅𝑓𝑓(𝑇𝑇𝑓𝑓). Estimating LM learning difficulty In general, there is a strong negative correlation between cross entropy and the number of folds, for both versions, both symbolic levels, all LMs and all languages (NT version, word level – PPM2: median Pearson correlation between the log of 𝐻𝐻𝑓𝑓 and the log of f, rmed = -0.90; PPM6: rmed = -0.97; LZMA: rmed = -0.97; PAQ: rmed = -0.97; LSTMcomp: rmed = -0.98; NNCPsmall: rmed = -0.97; NNCPlarge: rmed = -0.98; BPE level – PPM2: rmed = -0.73; PPM6: rmed = -0.94; LZMA: rmed = -0.94; PAQ: rmed = -0.94; LSTMcomp: rmed = -0.98; rmed = NNCPsmall: rmed = -0.97; NNCPlarge: rmed = -0.98; OT version, word level – PPM2: rmed = -0.91; PPM6: rmed = -0.99; LZMA: rmed = -0.99; PAQ: rmed = -0.99; LSTMcomp: rmed = -0.99; NNCPsmall: rmed = - 0.98; NNCPlarge: rmed = -0.99; BPE level – PPM2: rmed = -0.77; PPM6: rmed = -0.98; LZMA: rmed = -0.99; PAQ: rmed = -0.99; LSTMcomp: rmed = -0.98; NNCPsmall: rmed = -0.98; NNCPlarge: rmed = -0.99). This demonstrates that all LMs – on average – improve with input demonstrates that all LMs – on average – improve with input. demonstrates that all LMs – on average – improve with input. 28 To measure language-specific learning difficulty, we fit LMERs per LM, version (NT/OT) and level (words/BPE) with the log of 𝐻𝐻𝑓𝑓 as the outcome and a fixed effect for f. We include (crossed) random intercepts for language and for the test fold (1 – 10 for PPM2, PPM6, LZMA and PAQ and, due to the significant increase in training time, 1 – 5 for LSTMcomp, NNCPsmall and NCCPlarge). In addition, we include random slopes per language, i.e. we allow the relationship between 𝐻𝐻𝑓𝑓 and f to be different for each language. We model the covariance structure between the random effect and the random slope for language as either (i) independent, i.e. both the effect and slope have their own variance and the covariances between effect and slope are assumed to be independent of each other or (ii) unstructured, i.e. we allow the random effect and the random slope to be correlated. In all cases, an unstructured covariance structure turned out to be better as indicated by a lower AIC. Based on the LMERs, we obtained language- specific empirical Bayes predictions or best linear unbiased predictions (BLUPs)120 of the random slopes, represented by variable µ. Estimating LM learning difficulty These BLUPs capture the interactions between f and language, with higher values of µ indicating faster learning and lower values indicating slower learning as visualized in Figure 4. µ is assumed to be Gaussian with mean zero and variance 𝜎𝜎2. Models were fitted with gradient-based maximization and – since our main interest lies in the estimation of random slopes – via restricted maximum likelihood (REML) to avoid a downward-biased estimate of 𝜎𝜎2, for details, see Ref.120. Statistical analyses Multilevel mixed-effects linear regression (LMER) Multilevel mixed-effects linear regression (LMER) To enhance convergence for the LMERs conducted in Study 1 (Table 1), the outcome b and the fixed control variables h and L were standardized per corpus, i.e. the corpus-specific mean was subtracted from each observed value and the result was divided by the corpus-specific standard deviation. As described in the main body of the paper, our covariate candidate model set includes (i) random intercepts for corpus, language family, language, macro area, country and writing script and (ii) random slopes for corpus, language family, macro area, country and writing script. All effects are assumed to be crossed. Note, however, that – in the terminology of Ref.121 – countries are explicitly nested within macro areas, i.e. each country occurs in exactly one macro area. In the same sense, languages are explicitly nested within language families. 29 To compute differences in AIC, ΔAIC, we additionally fit LMERs without a fixed effect for speaker population size. Note that in models without a fixed effect for speaker population size, we also exclude potential random slopes. We then compute ΔAIC between the full model, which includes a fixed effect and potential random slopes for speaker population size, and a reduced model that does not include a fixed 29 effect or random slopes for speaker population size but otherwise has the same fixed and random effect structure. We counted a full model to be more apt if either its AIC value is lower than its reduced counterpart or if the fitting of the reduced model fails. In all other cases, we counted the reduced model to be better. We model all intercepts and slopes as i.i.d. and to be independently from each other. Models were fitted with gradient-based maximisation and – since our primary focus in this set of analyses is on estimating and comparing different fixed effects structures– via maximum likelihood (ML).122–124 We accepted any solution after a maximal number of 20 iterations. Full details on the fixed and random effect structure for each selected model are given in Supplementary Tables 1,2. As written above, we also exclude potential random slopes in models without a fixed effect for speaker population size, since excluding the fixed effect for speaker population size while including random slopes would constrain 𝛽𝛽LMER to be zero and thus force the random slopes to be evenly distributed around a slope of zero. Multilevel mixed-effects linear regression (LMER) 30 Frequentist model averaging (FMA) entist model averaging (FMA) Frequentist model averaging (FMA) In Study 1, the FMA estimator is computed per LM and symbol (words/characters) for all M = 2430 1, the FMA estimator is computed per LM and symbol (words/characters) for all M = 2430 In Study 1, the FMA estimator is computed per LM and symbol (words/characters) for all M = 2430 candidate models that include a fixed effect for the log of speaker population size as50,48,125: 𝛽𝐿𝐿𝐿𝐿𝐹𝐿𝐹𝜔𝑗𝐿𝑗𝛽𝑗 y , p p y ( ) candidate models that include a fixed effect for the log of speaker population size as50,48,125: 𝛽𝐿𝐿𝐿𝐿𝐹𝐿𝐹𝜔𝑗𝐿𝑗𝛽𝑗 candidate models that include a fixed effect for the log of speaker population size as50,48,125: 𝛽𝐿𝐿𝐿𝐿𝐹𝐿𝐹𝜔𝑗𝐿𝑗𝛽𝑗 ate models that include a fixed effect for the log of speaker population size as50,48,125: 𝛽𝐿𝐿𝐿𝐿𝐹𝐿𝐹𝜔𝑗𝐿𝑗𝛽𝑗 𝛽𝛽𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 𝐹𝐹𝐿𝐿𝐹𝐹= ∑ 𝜔𝜔𝑗𝑗 𝐿𝐿 𝑗𝑗=1 𝛽𝛽𝑗𝑗 (5) 𝛽𝛽𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 𝐹𝐹𝐿𝐿𝐹𝐹= ∑ 𝜔𝜔𝑗𝑗 𝐿𝐿 𝑗𝑗=1 𝛽𝛽𝑗𝑗 (5) 𝜔𝑗𝑐𝑗𝛺𝑗𝑐𝑗 (5) where 𝜔𝜔𝑗𝑗= 𝑐𝑐𝑗𝑗𝛺𝛺𝑗𝑗 ∑ 𝑐𝑐𝑖𝑖𝛺𝛺𝑖𝑖 𝑀𝑀 𝑖𝑖=1 ; 𝑐𝑐𝑗𝑗 is a binary indicator that is equal to 1 if j converged to a solution and 0 otherwise; 𝛺𝛺𝑗𝑗= 𝑒𝑒𝑒𝑒𝑒𝑒ቀ− 𝐹𝐹𝐴𝐴𝐴𝐴𝑗𝑗 2 ቁ where 𝐴𝐴𝐴𝐴𝐴𝐴𝑗𝑗 denotes the AIC value computed for j, likewise for i. Therefore, ∑ 𝜔𝜔𝑗𝑗 𝐿𝐿 𝑗𝑗=1 = 1. 𝛽𝐿𝐿𝐿𝐿𝐹𝐿𝐹 1. In Study 2, 𝛽𝛽𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 𝐹𝐹𝐿𝐿𝐹𝐹 is computed in an analogous way per LM, symbol (words/BPE) and version (NT/OT) for the set of models consisting of M = 728 candidates. In Study 2, 𝛽𝛽𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 𝐹𝐹𝐿𝐿𝐹𝐹 is computed in an analogous way per LM, symbol (words/BPE) and version (NT/OT) for the set of models consisting of M = 728 candidates. Double-selection lasso linear regression (DS) and permutation testing Double-selection lasso linear regression (DS) and permutation testing For the DS regressions, we use the log of b as the outcome. Our covariate of interest is the log of speaker population size. As potential control variables, we use the three different sets specified in the main part of the paper. We generate a set of variables that form third-order B-spline basis functions for both longitude and latitude each with three knots placed at the 25th, the 50th and the 75th percentiles. Multilevel mixed-effects linear regression (LMER) To make sure that this decision does not overly influence the results, we additionally ran constrained models where we allowed for reduced models that did not include a fixed effect for speaker population size but included potential random slopes for speaker population size. We then compared AIC values between full and reduced models with the same fixed effects, random effects and random slopes. Column 6 of Supplementary Tables 1 and 2 shows that our results are also valid if such constrained models are included. For the LMERs conducted as part of Study 2, our covariate candidate model set contains (crossed) random effects and slopes for macro area, country, language family, language subfamily and sub-branch. Again, countries are explicitly nested within macro areas. We also explicitly nest sub-branches within subfamilies and subfamilies within families by creating unique indicators for subfamilies/sub-branches that occur in more than one level, e.g. the sub-branch label “West” occurs in several subfamilies, e.g. “Germanic” and “Mande”. To create a unique sub-branch indicator, the corresponding sub-branches are replaced by “GermanicWest” and “MandeWest”. Further note that if there is no subfamily for a language, we also create a unique subfamily indicator within the corresponding family, e.g. for the Papuan language “Angor”, the only classification given in our data is “Senagi” for the language family. To fill in a unique group indicator for the subfamily, we use the language family. We proceed in the same way with missing sub-branches by filling in corresponding subfamilies. Analogous to Study 1, we compute ΔAIC-values. Again, we model all intercepts and slopes as i.i.d. and to be independent from each other. All models were fitted with gradient-based maximization and via ML. We accepted any solution after a maximal number of 100 iterations. Full details on the random intercept and slope structure for each selected model are given in Supplementary Table 7. Again, we tested if the inclusion of constrained models for model comparison changes the results. Columns 6 and 12 of Supplementary Table 7 show that this not the case. Multilevel mixed-effects linear regression (LMER) We regress the log of b against ϲ ෥ only to obtain fitted values and residuals. 2. We regress the log of b against ϲ ෥ only to obtain fitted values and residuals. 3. We randomly permute the residuals and generate a new variable b* that is computed as the fitted values from step 2 and the randomly permuted residuals. 4. We regress b* against speaker population size and ϲ ෥ and extract the t-statistic of the coefficient for speaker population size and call that quantity t. 5. Steps 3 and 4 are repeated 10000 times to build the distribution of t if the null hypothesis is true. 6. We count the number of times the absolute value of t* is at least as high as tobs and divide the result by the number of repetitions, i.e. 10000. The result is the permutation p-value. The idea of the permutation test is that if the null hypothesis is true, we do not lose “anything essential in the data”63 by permuting the residuals from the reduced model (step 2), because they should not be different from the full model (step 1) and can thus be used to generate the reference distribution of the test statistic. The idea of the permutation test is that if the null hypothesis is true, we do not lose “anything essential in the data”63 by permuting the residuals from the reduced model (step 2), because they should not be different from the full model (step 1) and can thus be used to generate the reference distribution of the test statistic. Phylogenetic Generalised Least Squares regression (PGLS) Phylogenetic Generalised Least Squares regression (PGLS) To account for historical relatedness among languages, we fit PGLS regressions129 per LM, version (NT/OT) and level (words/BPE) with learning difficulty µ as the outcome and speaker population size as a covariate. The PGLS approach incorporates a covariance matrix that captures the phylogenetic relatedness between languages.84 The covariance matrix is estimated using a Brownian motion model based on a tree that represents the evolutionary relationships between different languages and is used to model the degree of similarity or dissimilarity between languages. We use a phylogenetic tree provided by Ref.82 that was generated using language taxonomies from Ethnologue.83 This tree represents the evolutionary relationships among the languages in our sample and allows us to account for the non-independence of observations due to shared ancestry. Languages are identified by their ISO codes. Models are fitted by generalized least squares and estimates are derived by maximizing the log-likelihood. To compute ΔAIC, we additionally re-fit each model without including speaker population size. As a measure of fit, we compute the coefficient of determination, R2, as the squared Pearson correlation between the observed value of µ and the regression model-based prediction. Spatial autoregressive error regression (SAR) and permutation testing Multilevel mixed-effects linear regression (LMER) In addition to the log of h, the log of L, the basis functions for longitude and latitude and the set of indicator variables for the levels of corpus, language family, writing script, macro area and EGIDS, we include (log) language range, (log) distance to water resources, (log) altitude, Climate PC1, Climate PC2 and the (log) number of countries in which a language is spoken as potential controls. The DS approach works by (i) running a lasso of speaker population size on the potential covariates, (ii) running a lasso of the log of b on the potential covariates. Let ϲ ෥ denote the union of the covariates selected in (i) and (ii). As a third step, the log of b is regressed on the log of speaker population size and ϲ ෥. Further information on this approach is given in Refs.43,126. To select the optimal value for the penalty parameter The DS approach works by (i) running a lasso of speaker population size on the potential covariates, (ii) running a lasso of the log of b on the potential covariates. Let ϲ ෥ denote the union of the covariates selected in (i) and (ii). As a third step, the log of b is regressed on the log of speaker population size and ϲ ෥. Further information on this approach is given in Refs.43,126. To select the optimal value for the penalty parameter for each lasso, we use cross-validation. Standard errors are clustered at the level of individual languages, i.e. we allow for intra-language correlation. Since, as written above, the sample of languages for which we have available documents cannot be considered a random sample of the population of all languages61,62, we use the controls selected in step (i) and step (ii) as input for non-parametric Freedman-Lane permutation tests.63,127 Here, we wish to test the null hypothesis that speaker population size provides no information about the outcome b, i.e. that the corresponding estimate coefficient is equal to zero. The procedure is as follows128: 31 1. We regress b (logged) against speaker population size (logged) and ϲ ෥ and extract the observed t- statistic tobs of the coefficient for speaker population size. 1. We regress b (logged) against speaker population size (logged) and ϲ ෥ and extract the observed t- statistic tobs of the coefficient for speaker population size. 2. Spatial autoregressive error regression (SAR) and permutation testing As written above, the PGLS framework uses a single covariance matrix that represents the phylogenetic relatedness between languages.84 That means we do not take potential non-independence due to spatial proximity into account.41 We fit SAR regressions45 using a GS2SLS estimator87 where autocorrelated 32 errors are treated as heteroskedastic. Individual regressions are fitted per LM, version (NT/OT) and level (words/BPE) with learning difficulty µ as the outcome and speaker population size as a covariate. To control for both potential sources of non-independence simultaneously, we add two spatially lagged error terms to the regression equation that are specified by the inverse of two weighting matrices. To control for spatial proximity, we compute the Haversine distance130 between each pair of languages based on longitudinal and latitudinal information to generate a spatial distance matrix. To control for genealogical relatedness, we used a matrix provided by Ref.85 that is based on word lists from the Automated Similarity Judgment Program (ASJP).86 Again, languages are identified by their ISO codes. To select a specific language in case there are multiple languages with the same ISO code, we select either the language whose name begins with “STANDARD_”, e.g. “STANDARD_ARABIC” or the name with the shortest length, e.g. we select “JAPANESE” over “JAPANESE_2” or “TOKYO_JAPANESE”. 𝛽𝑂𝐹𝐿 To assess the significance of the estimated 𝛽𝛽𝑂𝑂𝐹𝐹𝐿𝐿-coefficients, we use the following permutation procedure: To assess the significance of the estimated 𝛽𝛽𝑂𝑂𝐹𝐹𝐿𝐿-coefficients, we use the following permutation procedure: 1. We fit a SAR regression of µ against speaker population size (logged) and extract the observed z- statistic zobs of the coefficient for speaker population size. 2. We randomly permute the speaker population size variable, re-fit the SAR model and extract the z-statistic of the coefficient for speaker population size and call that quantity z. 𝛽 3. Step 2 is repeated 10000 times to build the distribution of z if the null hypothesis is true (i.e. 𝛽𝛽𝑂𝑂𝐹𝐹𝐿𝐿 = 0). 4. We count the number of times the absolute value of z* is at least as high as zobs and divide the result by the number of repetitions, i.e. 10000. The result is the permutation p-value. R2-values are computed in the same way as for the PGLS regressions. Author contributions Conceptualisation: AK; Data curation: AK; Formal Analysis: AK, SW; Investigation: AK; Methodology: AK; Software: AK; Validation: AK; Visualisation: AK; Writing – original draft: AK; Writing – review & editing: AK, SW Data, code and materials availability All parallel text data, bibliographic information on languages and the compression algorithms were taken from the sources mentioned in the Methods section. Data preparation, management and statistical analyses were done in Stata/MP4 (version 18.0) on a Linux server (CentOS 7.9.2009) with 756GB of available RAM. Commented Stata code plus additional R (version 4.2.2) and Python code (version 3.6.8) are available at https://osf.io/sa9x2/. 33 Competing interests We declare no competing interests. Funding We received no external funding. References 1. Nettle, D. Social scale and structural complexity in human languages. Philosophical Transactions of the Royal Society B: Biological Sciences 367, 1829–1836 (2012). 1. Nettle, D. Social scale and structural complexity in human languages. Philosophical Transactions of the Royal Society B: Biological Sciences 367, 1829–1836 (2012). 2. Lupyan, G. & Dale, R. Why Are There Different Languages? The Role of Adaptation in Linguistic Diversity. TRENDS in Cognitive Science 20, 649–660 (2016). 3. Wells, R. Archiving and Language Typology. International Journal of American Linguistics 20, 101–107 (1954). 4. Hockett, C. F. A course in modern linguistics. (Collier-Macmillan, 1958). 5. Trudgill, P. Accent, Dialect and the School. (Edward Arnold, 1975). 6. Crystal, D. The Cambridge encyclopedia of language. (Cambridge University Press, 1987). 7. O’Grady, W., Dobrovolsky, M. & Aronoff, M. Contemporary linguistics: an introduction. (St. Martin’s Press, 1993). 8. Edwards, J. Multilingualism. (Penguin Books, 1995). 8. Edwards, J. Multilingualism. (Penguin Books, 1995). Bickerton, D. Language and human behavior. (Univ. of Washington Press, 1996). 34 10. Ridley, M. Genome: The Autobiography of a Species in 23 Chapters. (HarperCollins, 1999). 11. Fortson, B. W. Indo-European language and culture: An introduction. (Blackwell, 2004). 10. Ridley, M. Genome: The Autobiography of a Species in 23 Chapters. (HarperCollins, 1999). 11. Fortson, B. W. Indo-European language and culture: An introduction. (Blackwell, 2004). 12. Sweet, H. The Practical Study of Languages: A Guide for Teachers and Learners. (Oxford University Press 1899) 13. Gibson, E. et al. How Efficiency Shapes Human Language. TRENDS in Cognitive Science 23, 389–407 (2019). 14. Hammarström, H., Forkel, R. & Haspelmath, M. Glottolog 3.2. (2019). 15. Simons, G. F. & Fennig, C. D. Global Dataset Ethnologue: Languages of the World, Twentieth edition. (2017). 16. WALS Online. (Max Planck Institute for Evolutionary Anthropology, 2013). 17. Skirgård, H. et al. Grambank reveals the importance of genealogical constraints on linguistic diversity and highlights the impact of language loss. Sci. Adv. 9, eadg6175 (2023). 18. Lupyan, G. & Dale, R. Language Structure Is Partly Determined by Social Structure. PLoS ONE 5, e8559 (2010). 19. Greenhill, S. J. Overview: Debating the effect of environment on language. Journal of Language Evolution 1, 30–32 (2016). 20. Bentz, C., Dediu, D., Verkerk, A. & Jäger, G. The evolution of language families is shaped by the environment beyond neutral drift. Nature Human Behaviour 2, 816–821 (2018). 21. Shcherbakova, O. et al. Societies of strangers do not speak grammatically simpler languages. https://osf.io/svfdx (2023) doi:10.31235/osf.io/svfdx. 22. References Bromham, L., Hua, X., Fitzpatrick, T. G. & Greenhill, S. J. Rate of language evolution is affected by population size. Proceedings of the National Academy of Sciences 112, 2097– 2102 (2015). 35 35 23. Koplenig, A. Language structure is influenced by the number of speakers but seemingly not by the proportion of non-native speakers. Royal Society Open Science 6, 181274 (2019). 24. Sampson, G. A linguistic axiom challenged. in Language complexity as an evolving variable (eds. Sampson, G., Gil, D. & Trudgill, P.) 1–18 (Oxford University Press, 2009). 25. Koplenig, A., Wolfer, S. & Meyer, P. A large quantitative analysis of written language challenges the idea that all languages are equally complex. Sci Rep 13, 15351 (2023). 26. Contreras Kallens, P., Kristensen‐McLachlan, R. D. & Christiansen, M. H. Large Language Models Demonstrate the Potential of Statistical Learning in Language. Cognitive Science 47, e13256 (2023). 27. Piantadosi, S. Modern language models refute Chomsky’s approach to language. (2023). 28. Chater, N. & Vitányi, P. ‘Ideal learning’ of natural language: Positive results about learning from positive evidence. Journal of Mathematical Psychology 51, 135–163 (2007). 29. Yang, Y. & Piantadosi, S. T. One model for the learning of language. Proc Natl Acad Sci USA 119, e2021865119 (2022). 30. Webb, T., Holyoak, K. J. & Lu, H. Emergent analogical reasoning in large language models. Nat Hum Behav (2023) doi:10.1038/s41562-023-01659-w. 31. Gold, E. M. Language identification in the limit. Information and Control 10, 447–474 (1967). 32. Chater, N. & Vitányi, P. Simplicity: a unifying principle in cognitive science? TRENDS in Cognitive Science 7, 19–22 (2003). 33. Kolmogorov, A. N. Three approaches to the quantitative definition of information. 33. Kolmogorov, A. N. Three approaches to the quantitative definition of information. International Journal of Computer Mathematics 2, 157–168 (1968). International Journal of Computer Mathematics 2, 157–168 (1968). 34. Kontoyiannis, I. The Complexity and Entropy of Literary Styles. NSF Technical Report, Department of Statistics, Stanford University 97, (1996). ment of Statistics, Stanford University 97, (1996). 36 35. Cover, T. M. Kolmogorov Complexity, Data Compression, and Inference. in The Impact of Processing Techniques on Communications (ed. Skwirzynski, J. K.) 23–33 (Springer Netherlands, 1985). doi:10.1007/978-94-009-5113-6_2. 36. Jaeger, T. F., Graff, P., Croft, W. & Pontillo, D. Mixed effect models for genetic and areal dependencies in linguistic typology. Linguistic Typology 15, (2011). 37. Roberts, S. & Winters, J. Linguistic Diversity and Traffic Accidents: Lessons from Statistical Studies of Cultural Traits. References PLoS ONE 8, e70902 (2013). 38. Bromham, L., Hua, X., Cardillo, M., Schneemann, H. & Greenhill, S. J. Parasites and politics: why cross-cultural studies must control for relatedness, proximity and covariation. R. Soc. open sci. 5, 181100 (2018). 39. Hua, X., Greenhill, S. J., Cardillo, M., Schneemann, H. & Bromham, L. The ecological drivers of variation in global language diversity. Nat Commun 10, 2047 (2019). 40. Bromham, L., Skeels, A., Schneemann, H., Dinnage, R. & Hua, X. There is little evidence that spicy food in hot countries is an adaptation to reducing infection risk. Nat Hum Behav 5, 878–891 (2021). 41. Bromham, L. Solving Galton’s problem: practical solutions for analysing language diversity and evolution. https://osf.io/c8v9r (2022) doi:10.31234/osf.io/c8v9r. 42. Claessens, S. & Atkinson, Q. The non-independence of nations and why it matters. https://osf.io/m6bsn (2022) doi:10.31234/osf.io/m6bsn. https://osf.io/m6bsn (2022) doi:10.31234/osf.io/m6bsn. 43. Belloni, A., Chernozhukov, V. & Hansen, C. Inference on Treatment Effects after Selection among High-Dimensional Controls. The Review of Economic Studies 81, 608–650 (2014). 44. Chernozhukov, V. et al. Double/debiased machine learning for treatment and structural parameters. The Econometrics Journal 21, C1–C68 (2018). 37 37 45. Drukker, D. M., Egger, P. & Prucha, I. R. On Two-Step Estimation of a Spatial Autoregressive Model with Autoregressive Disturbances and Endogenous Regressors. Econometric Reviews 32, 686–733 (2013). 46. Takahira, R., Tanaka-Ishii, K. & Dębowski, Ł. Entropy Rate Estimates for Natural Language—A New Extrapolation of Compressed Large-Scale Corpora. Entropy 18, 364 (2016). 47. Cleary, J. & Witten, I. Data Compression Using Adaptive Coding and Partial String Matching. IEEE Transactions on Communications 32, 396–402 (1984). 48. Model Selection and Multimodel Inference. (Springer New York, 2004). doi:10.1007/b97636. 49. Akaike, H. A new look at the statistical model identification. IEEE Transactions on Automatic Control 19, 716–723 (1974). 50. Buckland, S. T., Burnham, K. P. & Augustin, N. H. Model Selection: An Integral Part of Inference. Biometrics 53, 603 (1997). 51. Mahoney, M. PAQ8. (2007). 52. Knoll, B. & Freitas, N. de. A Machine Learning Perspective on Predictive Coding with PAQ8. in 2012 Data Compression Conference 377–386 (IEEE, 2012). doi:10.1109/DCC.2012.44. 53. Knoll, B. lstm-compress. GitHub repository (2019). 54. Hochreiter, S. & Schmidhuber, J. Long short-term memory. Neural computation 9, 1735– 1780 (1997). 55. Baker, M. Corpus Linguistics and Translation Studies — Implications and Applications. in Text and Technology (eds. Baker, M., Francis, G. & Tognini-Bonelli, E.) 233 (John Benjamins Publishing Company, 1993). doi:10.1075/z.64.15bak. 38 56. Stewart, W. Α. References A Sociolinguistic Typology for Describing National Multilingualism. in Readings in the Sociology of Language (ed. Fishman, J. A.) 531–545 (DE GRUYTER, 1968). doi:10.1515/9783110805376.531. 57. Lewis, M. P. & Simons, G. F. Assessing Endangerment: Expanding Fishman’s GIDS. Revue Roumaine de Linguistique 55, 103–120 (2010). 58. Kelly, M. DP17429 Improved Causal Inference on Spatial Observations: A Smoothing Spline Approach. CEPR Discussion Paper (2022). 59. Tibshirani, R. Regression Shrinkage and Selection via the Lasso. Journal of the Royal Statistical Society. Series B (Methodological) 58, 267–288 (1996). 60. Hastie, T., Tibshirani, R. & Friedman, J. H. The elements of statistical learning: data mining, inference, and prediction. (Springer, 2009). 61. Koplenig, A. Quantifying the efficiency of written language. Linguistics Vanguard 7, 20190057 (2021). 62. Koplenig, A. Against statistical significance testing in corpus linguistics. Corpus Linguistics and Linguistic Theory 15, 321–346 (2019). 63. Freedman, D. A. & Lane, D. A Nonstochastic Interpretation of Reported Significance Levels. Journal of Business & Economic Statistics 1, 292 (1983). 64. Vaswani, A. et al. Attention is All You Need. in Proceedings of the 31st International Conference on Neural Information Processing Systems 6000–6010 (Curran Associates Inc., 2017). 65. Schürmann, T. & Grassberger, P. Entropy estimation of symbol sequences. Chaos: An Interdisciplinary Journal of Nonlinear Science 6, 414 (1996). 39 39 66. Scannell, K. P. The Crúbadán Project: Corpus building for under-resourced languages. in Proceedings of the 3rd Web as Corpus Workshop: Building and Exploring Web Corpora vol. 4 5–15 (2007). 67. Mayer, T. & Cysouw, M. Creating a Massively Parallel Bible Corpus. in Proceedings of the Ninth International Conference on Language Resources and Evaluation (LREC’14) (eds. Chair), N. C. (Conference et al.) (European Language Resources Association (ELRA), 2014). 68. Futrell, R. & Hahn, M. Information Theory as a Bridge Between Language Function and Language Form. Front. Commun. 7, 657725 (2022). 69. Mielke, S. J., Cotterell, R., Gorman, K., Roark, B. & Eisner, J. What Kind of Language Is Hard to Language-Model? in Proceedings of the 57th Annual Meeting of the Association for Computational Linguistics 4975–4989 (Association for Computational Linguistics, 2019). doi:10.18653/v1/P19-1491. 70. Sennrich, R., Haddow, B. & Birch, A. Neural Machine Translation of Rare Words with Subword Units. in Proceedings of the 54th Annual Meeting of the Association for Computational Linguistics (Volume 1: Long Papers) 1715–1725 (Association for Computational Linguistics, 2016). doi:10.18653/v1/P16-1162. 71. Brown, T. et al. Language Models are Few-Shot Learners. in Advances in Neural Information Processing Systems (eds. References Larochelle, H., Ranzato, M., Hadsell, R., Balcan, M. F. & Lin, H.) vol. 33 1877–1901 (Curran Associates, Inc., 2020). 72. Kudo, T. & Richardson, J. SentencePiece: A simple and language independent subword tokenizer and detokenizer for Neural Text Processing. in Proceedings of the 2018 Conference on Empirical Methods in Natural Language Processing, EMNLP 2018: System Demonstrations, Brussels, Belgium, October 31 - November 4, 2018 (eds. Blanco, E. & Lu, W.) 66–71 (Association for Computational Linguistics, 2018). doi:10.18653/v1/d18-2012. 40 73. Mielke, S. J. & Eisner, J. Spell Once, Summon Anywhere: A Two-Level Open-Vocabulary Language Model. in Proceedings of the Thirty-Third AAAI Conference on Artificial Intelligence and Thirty-First Innovative Applications of Artificial Intelligence Conference and Ninth AAAI Symposium on Educational Advances in Artificial Intelligence (AAAI Press, 2019). doi:10.1609/aaai.v33i01.33016843. 74. Jurafsky, D. & Martin, J. H. Speech and Language Processing. (2021). 75. Shkarin, D. PPM: one step to practicality. in Proceedings DCC 2002. Data Compression Conference 202–211 (IEEE Comput. Soc, 2002). doi:10.1109/DCC.2002.999958. ence 202–211 (IEEE Comput. Soc, 2002). doi:10.1109/DCC.2002.999958. 76. Pavlov, I. 7-zip. (2023). 77. Ziv, J. & Lempel, A. A universal algorithm for sequential data compression. IEEE Trans. Inform. Theory 23, 337–343 (1977). 78. Mahoney, M. Data Compression Explained. (Dell Inc., 2013). 79. Veness, J. et al. Gated Linear Networks. (2019) doi:10.48550/ARXIV.1910.01526. 80. Mahoney, M. Adaptive weighing of context models for lossless data compression. Preprint at http://hdl.handle.net/11141/154 (2005). 81. Bellard, F. NNCP v3.1: Lossless Data Compression with Transformer. in (2021). 82. Dediu, D. Making genealogical language classifications available for phylogenetic analysis: Newick trees, unified identifiers, and branch length. Lang. Dyn. Change 8, 1–21 (2018). Newick trees, unified identifiers, and branch length. Lang. Dyn. Change 8, 1–21 (2018). 83. Simons, Gary F & Fennig, C. D. Ethnologue: Languages of the World. (SIL International, 2017). 84. Roberts, S. G., Winters, J. & Chen, K. Future Tense and Economic Decisions: Controlling for Cultural Evolution. PLoS ONE 10, e0132145 (2015). 85. Jäger, G. Global-scale phylogenetic linguistic inference from lexical resources. Scientific Data 5, 180189 (2018). 41 86. Wichmann, S., Holman, E. W., Brown, C. H., Forkel, R. & Tresoldi, T. CLDF dataset derived from Wichmann et al.’s ‘ASJP Database’ v17 from 2016. (2016) doi:10.5281/ZENODO.3835942. 87. Kelejian, H. H. & Prucha, I. R. Specification and estimation of spatial autoregressive models with autoregressive and heteroskedastic disturbances. Journal of Econometrics 157, 53–67 (2010). (2010). 88. Wray, A. & Grace, G. W. The consequences of talking to strangers: Evolutionary corollaries of socio-cultural influences on linguistic form. Lingua 117, 543–578 (2007). 89. Raviv, L., De Heer Kloots, M. & Meyer, A. What makes a language easy to learn? A preregistered study on how systematic structure and community size affect language learnability. Cognition 210, 104620 (2021). 90. Bentz, C. & Winter, B. Languages with More Second Language Learners Tend to Lose Nominal Case. Language Dynamics and Change 3, 1–27 (2013). 91. Bentz, C., Verkerk, A., Kiela, D., Hill, F. & Buttery, P. Adaptive Communication: Languages with More Non-Native Speakers Tend to Have Fewer Word Forms. PLOS ONE 10, e0128254 (2015). 92. Bentz, C. Adaptive languages: an information-theoretic account of linguistic diversity. (De Gruyter Mouton, 2018). 93. Atkinson, M., Smith, K. & Kirby, S. Adult Learning and Language Simplification. Cogn Sci 42, 2818–2854 (2018). 94. Walkden, G. & Breitbarth, A. Complexity as L2-difficulty: Implications for syntactic change. Theoretical Linguistics 45, 183–209 (2019). 95. Berdicevskis, A. & Semenuks, A. Imperfect language learning reduces morphological overspecification: Experimental evidence. PLoS ONE 17, e0262876 (2022). 42 96. Mitchell, M. & Krakauer, D. C. The debate over understanding in AI’s large language models. Proc. Natl. Acad. Sci. U.S.A. 120, e2215907120 (2023). 97. Dai, Z. et al. Transformer-XL: Attentive Language Models Beyond a Fixed-Length Context. arXiv:1901.02860 [cs, stat] (2019). 98. Schiff, R. & Katan, P. Does complexity matter? Meta-analysis of learner performance in artificial grammar tasks. Front. Psychol. 5, (2014). 99. Van Den Bos, E. & Poletiek, F. H. Effects of grammar complexity on artificial grammar learning. Memory & Cognition 36, 1122–1131 (2008). 100. Shannon, C. E. Prediction and Entropy of Printed English. Bell System Technical Journal 30, 50–64 (1951). 101. Chaitin, G. J. On the intelligibility of the universe and the notions of simplicity, complexity and irreducibility. arXiv:math/0210035 (2002). 102. Rissanen, J. J. Generalized Kraft Inequality and Arithmetic Coding. IBM Journal of Research and Development 20, 198–203 (1976). 103. Chen, S. F. & Goodman, J. An Empirical Study of Smoothing Techniques for Language Modeling. in 34th Annual Meeting of the Association for Computational Linguistics 310–318 (Association for Computational Linguistics, 1996). doi:10.3115/981863.981904. 104. Rumelhart, D. E., Hinton, G. E. & Williams, R. J. Learning representations by back- propagating errors. Nature 323, 533–536 (1986). 105. Kingma, D. P. & Ba, J. Adam: A Method for Stochastic Optimization. (2014) doi:10.48550/ARXIV.1412.6980. 106. Bellard, F. (2010). Lossless Data Compression with Neural Networks. (2019). . Bellard, F. NNCP v2: Lossless Data Compression with Transformer. (2021). 107. Bellard, F. NNCP v2: Lossless Data Compression with Transformer. (2021). 43 108. Hendrycks, D. & Gimpel, K. Gaussian Error Linear Units (GELUs). (2016) doi:10.48550/ARXIV.1606.08415. 109. Cover, T. M. & Thomas, J. A. Elements of information theory. (Wiley-Interscience, 2006). 110. Moscoso del Prado Martín, F. The mirage of morphological complexity. in Proceedings of Quantitative Measures in Morphology and Morphological Development (2011). 111. Cotterell, R., Mielke, S. J., Eisner, J. & Roark, B. Are All Languages Equally Hard to Language-Model? in Proceedings of the 2018 Conference of the North American Chapter of the Association for Computational Linguistics: Human Language Technologies, Volume 2 (Short Papers) 536–541 (Association for Computational Linguistics, 2018). doi:10.18653/v1/N18-2085. 110. Moscoso del Prado Martín, F. The mirage of morphological complexity. in Proceedings of Quantitative Measures in Morphology and Morphological Development (2011). 111. Cotterell, R., Mielke, S. J., Eisner, J. & Roark, B. Are All Languages Equally Hard to Language-Model? in Proceedings of the 2018 Conference of the North American Chapter of the Association for Computational Linguistics: Human Language Technologies, Volume 2 (Short Papers) 536–541 (Association for Computational Linguistics, 2018). doi:10.18653/v1/N18-2085. 112. Moran, S. & Cysouw, M. The Unicode Cookbook For Linguists: Managing Writing Systems Using Orthography Profiles. (Language Science Press, 2018). doi:10.5281/zenodo.1296780. 113. Bromham, L. et al. Global predictors of language endangerment and the future of linguistic diversity. Nat Ecol Evol 6, 163–173 (2022). 114. Hammarström, H., Forkel, R., Haspelmath, M. & Bank, S. glottolog/glottolog: Glottolog database 4.8. (2023) doi:10.5281/ZENODO.8131084. 115. Amano, T. et al. Global distribution and drivers of language extinction risk. Proceedings of the Royal Society B: Biological Sciences 281, 20141574–20141574 (2014). 116. Bentz, C., Dediu, D., Verkerk, A. & Jäger, G. The evolution of language families is shaped by the environment beyond neutral drift. Nature Human Behaviour 2, 816–821 (2018). 44 118. Jamison, D. & Jamison, K. A note on the entropy of partially-known languages. Information and control 12, 164–167 (1968). 119. Ziemski, M., Junczys-Dowmunt, M. & Pouliquen, B. The United Nations Parallel Corpus v1.0. in Proceedings of the Tenth International Conference on Language Resources and Evaluation (LREC’16) 3530–3534 (European Language Resources Association (ELRA), 2016). 120. Rabe-Hesketh, S. & Skrondal, A. Multilevel and longitudinal modeling using Stata. (Stata Press Publication, 2012). 121. Bates, D. M. lme4: Mixed-effects modeling with R. (2010). 122. Verbeke, G. & Molenberghs, G. (2010). Linear mixed models for longitudinal data. (Springer, 2001). 123. Zuur, A. F., Ieno, E. N., Walker, N., Saveliev, A. A. & Smith, G. M. Mixed effects models and extensions in ecology with R. (Springer New York, 2009). doi:10.1007/978-0-387- 87458-6. 124. Faraway, J. J. Extending the Linear Model with R: Generalized Linear, Mixed Effects and Nonparametric Regression Models, Second Edition. (Chapman and Hall/CRC, 2016). doi:10.1201/9781315382722. 125. Steel, M. F. J. Model Averaging and Its Use in Economics. Journal of Economic Literature 58, 644–719 (2020). 126. Belloni, A., Chernozhukov, V. & Hansen, C. High-Dimensional Methods and Inference on Structural and Treatment Effects. Journal of Economic Perspectives 28, 29–50 (2014). 127. Freedman, D. A. & Lane, D. Significance testing in a nonstochastic setting. in A Festschrift for Erich L. Lehmann 185–208 (Wadsworth, 1983). 45 Winkler, A. M., Ridgway, G. R., Webster, M. A., Smith, S. M. & Nichols, T. E. 128. Winkler, A. M., Ridgway, G. R., Webster, M. A., Smith, S. M. & Nichols, T. E. Permutation inference for the general linear model. NeuroImage 92, 381–397 (2014). ermutation inference for the general linear model. NeuroImage 92, 381–397 (2014) 129. Harvey, P. H. & Pagel, M. D. The comparative method in evolutionary biology. (Oxford University Press, 1991). 129. Harvey, P. H. & Pagel, M. D. The comparative method in evolutionary biology. (Oxford University Press, 1991). 130. Sinnott, R. W. Virtues of the haversine. Sky and Telescope 68, 158–159 (1984). Acknowledgments We thank Byron Knoll for his response to our question regarding lstm-compress. We also thank Sarah Ahrens and Oliver Czulo for input and feedback. 46 SMREVISION.pdf Supplementary Files This is a list of supplementary ¦les associated with this preprint. Click to download. SMREVISION.pdf
https://openalex.org/W4206280721	https://pubs.rsc.org/en/content/articlepdf/2022/ra/d1ra08196a	English	null	Photocatalytic dye degradation and photoexcited anti-microbial activities of green zinc oxide nanoparticles synthesized<i>via Sargassum muticum</i>extracts	RSC advances	2,022	cc-by	9,171	Harinee Subramanian, Muthukumar Krishnan * and Ashok Mahalingam* ukumar Krishnan * and Ashok Mahalingam nee Subramanian, Muthukumar Krishnan Drug-resistant superbugs (DRS) were isolated from hospital sewage waste and conﬁrmed by a 16S rDNA molecular technique as B. ﬁlamentosus, B. ﬂexus, P. stutzeri, and A. baumannii. Green nanotechnologies provide a new promising alternative pathway that was found to be much safer, eco-friendly, and has economic beneﬁts over physical/chemical methods. Sargassum muticum (SM) mediated zinc oxide nanoparticles (ZnO-NPs) were proved to be photocatalytic and anti-microbial agents. Anti-microbial action was demonstrated by a maximal growth inhibition activity of 18 mm against A. baumannii and a minimal of 12 mm against B. ﬂexus at 80 mg mL1 concentrations. The anti-microbial mechanism of SMZnO-NPs employed a biphasic phenomenon persuaded by an osmotic shock that can attack the DRS bacterial cells directly and lead to death. In addition, photocatalytic activity was investigated by SMZnO- NPs for the degradation of methylene blue (MB) dye under diﬀerent light conditions. Natural sunlight irradiation shows eﬀective enhancement with the highest eﬃciencies of 96% being achieved within 60 min compared to UV-light and visible-light. The reusability of SMZnO-NPs provides up to 6 consecutive cycles towards MB decolorization for environmental water cleansing. Received 8th November 2021 Accepted 14th December 2021 DOI: 10.1039/d1ra08196a rsc.li/rsc-advances Centers for Disease Control and Prevention (CDC) estimated that around 90 000 deaths occurred over the last few years were due to pathogenic bacterial infections and more than half were caused by DRS in the United States of America (USA).8 Infected human or animal DRS pathogens typically require a type of hospital care that uses specic antibiotics that are less eﬀective, are more toxic, and costly.9 Partially metabolized antibiotics along with excreta are commonly discharged either to sewage treatment plants or released as untreated to environmental waters or soils.10 Of particular concern for public health are the eﬀects of antibiotics used for the treatment of infections or for farming purposes in a selection of DRS pathogens. 1. Department of Physics, National Institute of Technology (NIT), Tiruchirappalli – 620 015, Tamil Nadu, India. E-mail: marinekmk@gmail.com; ashokm@nitt.edu; Fax: +91-431-2500133; Tel: +91-431-2503610 PAPER Open Access Article. Published on 05 January 2022. Downloaded on 10/24/2024 5:36:13 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Photocatalytic dye degradation and photoexcited anti-microbial activities of green zinc oxide nanoparticles synthesized via Sargassum muticum extracts Cite this: RSC Adv., 2022, 12, 985 Harinee Subramanian, Muthukumar Krishnan * and Ashok Mahalingam* RSC Advances Cite this: RSC Adv., 2022, 12, 985 Received 8th November 2021 Accepted 14th December 2021 DOI: 10.1039/d1ra08196a rsc.li/rsc-advances PAPER e. Published on 05 January 2022. Downloaded on 10/24/2024 5:36:13 AM. rticle is licensed under a Creative Commons Attribution 3.0 Unported Licence. 1. Introduction Water pollution caused by pathogenic bacteria and industrial dye eﬄuents imposes several health risks to humans and to the aquatic environment. Diﬀerent sources of water-polluting pathogenic bacteria and harmful waste containing dyes increasingly deteriorate environmental water quality. The World Health Organization (WHO) has declared the contami- nation of water by various types of microorganisms as a great concern for human health.1 Since the last decade drug-resistant superbugs (DRS) provide an increasingly serious threat to global public health all over the world that needs action. There are severe threats to public health care due to a progressive rise in DRS for nosocomial and community infection. This also holds for developing DRS pathogens whose resistance proles provide a major task for public wellbeing.2,3 Presently, in the United States (USA) alone, over 70% of nosocomial infections are caused by DRS that are resistant to one or more traditionally used antibiotic drugs.4–7 The last few years have seen an enor- mous increase in DRS; much emphasis was allocated to safety aspects of foods and water owing to cross spoilage or contam- ination caused by pathogenic microorganisms. g p p p g On the other hand, water pollution caused by organic dyes has been considered a major threat to aquatic ecosystems because some of these dyes are extremely toxic even at very low concentrations. The discharge of tannery eﬄuents, textile industries, paper, and pulp mill industries, create large amounts of harmful organic waste containing methylene blue (MB) dyes.11 Dyes are chemical and non-degradable waste compounds that are generally diﬃcult to biodegrade and are providing major environmental problems.12,13 Particularly organic dyes such as methylene blue (MB), methyl orange (MO), azorubine/Acid Red 14 (AC-14), malachite green (MG), Remazol Brilliant Blue R/Reactive Blue 19, and Remazol Red (RR) provide the main source of environmental pollution.14–17 Conventional biological treatments of dye-containing industrial wastewaters RSC Adv., 2022, 12, 985–997 \| 985 © 2022 The Author(s). Published by the Royal Society of Chemistry 985 Paper View Article Online View Article Online RSC Advances Paper are unsuccessful and oen result in an intensively colored discharge from industrial plants. The extensive use of several non-biodegradable organic dye manufacturing industries has increasingly become a source of groundwater pollution.18 Organic dyes (e.g. 2.3. Identication of DRS strains A total of 152 pure isolated bacterial strain cultures were chal- lenged against diﬀerent standard antibiotics discs (10 mcg) representing several chemical structural groups such as ampi- cillin (AMP), chloramphenicol (C), penicillin-G (P), amoxycillin (AMX), methicillin (MET), erythromycin (E), ciprooxacin (CIP), gentamicin (GEN), tetracycline (TE), and vancomycin (VA). The anti-bacterial resistance index (ARI) of each location was calculated: The green synthesis of NPs represents a promising and environmentally favorable technology with some exciting properties to their wide-ranging applications. (i) We used aqueous extracts from the marine brown alga Sargassum muti- cum (SM) in order to obtain a bio-reducing agent for the ZnO- NPs synthesis as a natural product-inspired method.27 This is a quite novel approach of green viable and facile methodology which plays a major role in several applications.28,29 (ii) The green synthesis of SMZnO-NPs showed remarkable antibacte- rial activity against Gram-negative drug-resistant superbugs (DRS) such as Pseudomonas stutzeri (P. stutzeri; NCBI accession no.: MN045185); Acinetobacter baumannii (A. baumannii; NCBI accession no.: MN045188) than the Gram-positive such as Bacillus lamentosus (B. lamentosus; NCBI accession no.: MN045186); and Bacillus exus (B. exus; NCBI accession no.: MN045189). (iii) DRS pathogens and organic dyes provide a newly emerging issue in aquatic pollution with great concern ARI ¼ y/nx (1) (1) where ‘y’ represented the resistance determinants. ‘n’ and ‘x’ was the total number of anti-bacterial sensitivities tested.30 Among 152 isolates 4 strains were identied as drug-resistant superbug (DRS) pathogens identied by ARI. The identied DRS pathogens were subjected to conventional 16S rDNA gene sequencing approaches. 2.1. Materials In this study analytical grade, chemical reagents were used without further purication. The chemical reagents zinc nitrate hexahydrate [reagent grade, 98%] and potassium hydroxide pellets ACS reagent $85%, (KOH) were obtained from Sigma- Aldrich (Mumbai, India). All of the culture media such as nutrient agar (NA), nutrient broth (NB), and specic culture media (SCM) such as blood agar (BLA); xylose lysine deoxy- cholate agar (XLD); MacConkey agar (MCA); eosin methylene blue agar (EMB); Pseudomonas isolation agar (PIA); thiosulfate citrate bile salt agar (TCBS); and Mueller Hinton agar (MHA) were purchased from Hi-Media, Pvt. Limited (Mumbai, India). In recent years, the removal of the dyes containing organic eﬄuents through various processes of biological treatment, chemical oxidation, coagulation, occulation, ion exchange, electrochemical treatment, membrane processes, and photo- catalytic degradation process. Among these, the green nanoparticle-based photocatalytic technique is one of the best methods of removal/decolorization of eﬄuents. Nano-green technologies are recently emerging as a fast-growing eld in the technological applications of science with its contribution of eco-friendly nanoscale materials.20 There is a growing urgent requirement to develop environmental technologies without using toxic compounds and replace those with the synthesis of green nanoparticles (NPs).21 Nowadays, green synthetic meth- odologies employing green extracts drawn of metal nano- particles provide a sustainable solution due to a green, bio-safe, bio-compatible, viable, and facile methodology rather than toxic classical physical and chemical methods.22 Synthesis of green zinc oxide nanoparticles (ZnO-NPs) has several economic advantages, compared to physical and chemical methods, such as lower cost and white appearance.23,24 Among various NPs, ZnO-NPs are considered to be the most promising semi- conductors acting as green promising technology and providing alternative ways for anti-bacterial activity which are eﬀective in killing pathogenic and non-pathogenic bacteria. In particular, ZnO-NPs attracted attention owing to their large band gaps and excitation binding energy, high photosensitivity, and stability. ZnO-NPs were also found to be non-toxic, bio-safe, and bio- compatible and have been extensively used as drug carriers, in cosmetics, solar cells, automotive, and for llings in medical applications.25,26 2.2. Collection of drug-resistant superbugs (DRS) DRS pathogens were collected from sewage water of a govern- mental hospital (GH) at Tiruchirappalli (latitude 78 400 East, and longitude 10 480 North) and Srirangam (latitude 78 680 East, and longitude 10 870 North), Tamil Nadu, India. As per the manufacturer's guidelines SCM was used for the cultivation of DRS. 1. Introduction MB, MO, AC-14) that are produced annually with about 450 000 tons worldwide, produce more than 11% of environmental burdens as eﬄuents during manufacturing and application processes.19 Therefore, there is a crucial and pressing demand to develop new anti-microbial approaches combined with dye-degradation technologies. to public health. (iv) This result reveals that seaweed (S. muti- cum) extract containing a phytochemical compound provides reducing properties for the fabrication of NPs. Green SMZnO- NPs could be employed eﬀectively for environmental (medical and biological) applications to inhibit the transmission of DRS pathogens in the future. 2.5. Collection and extraction of seaweed Brown seaweed S. muticum (Sargassum muticum) were collected at a marine biodiversity hotspot area in the intertidal zone at low tide (Fig. 1a–d) along the Gulf of Mannar coastline (latitude 78 80 East and longitude 9 170 North) in the eastern coastal region of Tamil Nadu, India. The collected seaweed was cleaned thoroughly and aqueous extracts were prepared based on our previous experience.34 2.7. Characterization study of SMZnO-NPs Green synthesized SMZnO-NPs was conrmed by UV-visible spectroscopy (SHIMADZU-1700 spectroscopy, Japan) and pho- toluminescence spectrophotometry (PL, JASCO FP-8500). Four- ier transformed infrared spectroscopy 4000 cm1 was applied for analyses by the KBr pellet technique (FTIR, 500–4000 cm1; Made spectrum RX-1, Male PerkinElmer) were used to examine the functional groups. Raman spectra (RS) operated in a back- scattering mode an Enspectr Raman spectrophotometer was equipped with a laser source of 532 nm wavelength (l). X-ray diﬀraction (XRD) analysis of the synthesized SMZnO-NPs studied (Rigaku ULTIMA-III with a Cu-ka anode l ¼ 1.54056 ˚A, Japan), followed by scanning electron microscopy (SEM- HITACHI) for a structural study. The elemental composition and the proportion of elements were estimated through energy- dispersive X-ray spectroscopy (EDXS-HITACHI, S-3000H, UK) and X-ray uorescence (XRF-Olympus DELTA Element) spectra. Reduction of nanoparticle size distribution and stabilization were studied by using a dynamic light scattering (DLS; ZEN 3600) analyzer. Dispersion status was described by the poly- dispersity index (PDI), which reects the broadness of the size distribution. Measurements were taken in triplicate and recor- ded as the mean standard deviation. Particle sizes of the NPs were studied by eld-emission scanning electron microscopy (Fe-SEM) imaging (ZEISS; SPSS-IBM). Article. Published on 05 January 2022. Downloaded on 10/24/2024 5:36:13 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Finally, PCR obtained product sequences were compared to known 16S rRNA sequences using the CLUSTAL W program. Furthermore, the sequences were compared by the Basic Local Alignment Searching Tool (BLAST) with phylogenetically related taxa. The phylogenetic relationships were indicated by neighbour-joining (NJ) technique in a BLAST search and DRS pathogen gene sequences were submitted to Gen Bank (NCBI) to retrieve and save their accession numbers. 2.4. 16S ribosomal DNA (16S rDNA) extraction of DRS isolates Isolated DRS n ¼ 4 strains were extracted and amplied through the AccuPrep genomic DNA kit. DNA was amplied by using universal primers 16S: 27f (50-GTT TGA AGA TCC TGG CTC GGT- 30) and 1450r (50-CTT GGT TAC GTT ACG ACT CTT-30) which was followed by conventional 16S rDNA gene sequencing. Wizard © 2022 The Author(s). Published by the Royal Society of Chemistry 986 \| RSC Adv., 2022, 12, 985–997 986 RSC Advances View Article Online RSC Advances View Article Online Paper Paper PCR Preps were used to purify the PCR products, DNA puri- cation by Promega kit conrmed 1.5 kb size on agarose gel. ABI Prism Big Dye Terminator Cycle Sequencing Ready Reaction Kit, auto sequence analyzer model 377 systems (Applied Biosystems, Bagalur, India) was used to determine the 16S rDNA (1200 bases) partial sequences. The primer set used for sequencing consisted of 518r (50-TTA GTA CGG CTG CCG CTG-30) and 338f (50-CCT ACT ACG GGA AGC GGC-30) with an ABI 3130X-Genetic analyzer (Yaazh Xenomics, Bagalur, India) sequencing process was performed.31–33 prepared 5 mL of 0.5 M potassium hydroxide (KOH) solution was continuously added drop-wise to the above mixture and stirred for 2 h until a white precipitate was formed and centri- fuged at 10 000 rpm for 15 min at 4 C. Finally, the obtained pale white solid product was collected and washed twice with double deionized water (DDW) thoroughly followed by keeping it in a hot air oven for drying at 60 C for 5 h aer which a ne powder was obtained.35 2.8. In vitro anti-bacterial eﬃcacies The anti-bacterial action of green synthesized SMZnO-NPs was evaluated against various DRS pathogen strains which occur in sewage water of a governmental hospital (GH). Agar well diﬀu- sion technique was examined the bactericidal capability of the green synthesized SMZnO-NPs. 16S rDNA conrmed DRS pathogens were grown on nutrient agar (NA) slants at 37 2 C for 24 h and stored at 4 C until further use. All test cultures such as both Gram-positive and-negative DRS pathogen strains were sub-cultured into fresh nutrient broth (NB) medium and incubate at 37 2 C for 24 h as per the Hi-Media guidelines. The optical density of 0.5 McFarland standard (corresponding to 1.5 108 colony-forming units per milliliter [CFUs mL1]) turbidity DRS broth was spread on the surface of the prepared Muller–Hinton agar (MHA) plates using sterile cotton swabbed for homogenous growth. Aer swabbing, the plates were allowed to stand for 30 min. Each test plate, the cork borer was used to create 6 mm diameter wells made and the test solutions (SMZnO-NPs and aqueous S. muticum extract) were loaded into the wells at 20, 40, 60, and 80 mg mL1 concentrations. Then the plates were incubated at 37 2 C for 48 h. Aer incubation, the growth of zones (mm) was measured by using a Zone Scale-C (Hi-Antibiotic)/Nataraj measuring scale to evaluate the inhibi- tion eﬀect.36,37 The growth of the inoculums in the broth is indicated by turbidity/cloudiness of the broth at the lowest concentration of the SMZnO-NPs. Which inhibited the growth of the test organism, were taken as the MIC. Aer 24 h incubation, the culture medium was centrifuged at 8000 rpm for 15 min, and then the supernatants were analyzed by UV-visible spectroscopy (SHIMADZU 1700; 600 nm). All experiments were carried out in triplicate and the average results have plotted on a graph.38 The MIC bacterial survival was optically studied by Fe-SEM. MIC solutions (0.2 mL) were placed on a glass slide, xed by air-drying at gentle heating, and allowed to cool. A controlled study was obtained, followed by the above procedure without the addition of SMZnO-NPs. 2.10. Photocatalytic eﬃcacies of green SMZnO-NPs Photocatalytic eﬃcacies of methylene blue (MB) dye was per- formed under irradiation of diﬀerent light sources, i.e., UV- light, visible-light, and natural sunlight in the presence of green SMZnO-NPs as a catalyst. A desired amount of the catalyst was dispersed in 200 mL of 105 M (samples were prepared in molar concentrations) of MB dye solution. Before light irradia- tion, the suspension was magnetically stirred at 150 rpm constantly for 20 to 30 min at dark conditions to obtain an adsorption/desorption equilibrium in the presence of the green catalyst. Aer that, the suspensions were irradiated separately by UV-light (Philips, 150 W, l ¼ 365 nm), visible-light (150 W, Tungsten halogen lamp), and under natural sunlight from 12 noon to 3:30 p.m. in an open atmosphere. During the Open Access Article. Published on 05 January 2022. Downloaded on 10/24/2024 5:36:13 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Erlenmeyer ask. Then various concentrations of SMZnO-NPs (40, 80, 120, 160, and 200 mg mL1) were added into ask and incubate in a shaker (under agitation, 150 rpm) at 37 2 C for 24 h. The control experiments were performed on the normal growth of the microbial cells without NPs (ask containing media + 0.5 McFarland standards DRS cells). 2.6. Green synthesis of seaweed based ZnO-NPs To synthesize SMZnO-NPs; 100 mL of 5 mM Zn(NO3)2 were mixed with 10 mL of SM aqueous extract and kept at continuous stirring (150 rpm) for 30 min to assist the electrostatic interac- tion of Zn2+ ions. Aer, the solution mixture was kept in a boiling water bath at 70 C for 20 min and allowed at room temperature for continuous stirring (150 rpm). Then, freshly Fig. 1 Sample collection areas: (a) Mandapam coastal region in the Gulf of Mannar Marine National Park in the Bay of Bengal, southeast coast of India; (b) behind the Central Electrochemical Research Institute (CECRI) Mandapam coast; (c) and (d) collection of seaweed. Gulf of Mannar Marine National Park in the Bay of Bengal, southeast coast of ECRI) Mandapam coast; (c) and (d) collection of seaweed. Fig. 1 Sample collection areas: (a) Mandapam coastal region in the Gulf of Mannar Marine National Park in the Bay of Bengal, southeast coast of India; (b) behind the Central Electrochemical Research Institute (CECRI) Mandapam coast; (c) and (d) collection of seaweed. © 2022 The Author(s). Published by the Royal Society of Chemistry RSC Adv., 2022, 12, 985–997 \| 987 Paper View Article Online RSC Advances Table 1 Details of BLAST analysis, similarity percentage, and NCBI accession numbers of drug-resistant superbugs (DRS) Assigned code Sequence length (bp) Similarity (%) BLAST results NCBI's accession Gram-positive NIT-PW-II 1250 100 Bacillus lamentosus MN045186 NIT-PW-V 1267 100 Bacillus exus MN045189 Gram-negative NIT-PW-III 1233 100 Pseudomonas stutzeri MN045185 NIT-PW-IV 1272 100 Acinetobacter baumannii MN045188 milarity percentage, and NCBI accession numbers of drug-resistant superbugs (DRS) 988 \| RSC Adv., 2022, 12, 985–997 2.9. Minimum inhibitory concentration (MIC) The MIC activity was determined using green synthesized SMZnO-NPs by broth dilution technique. Test organisms DRS pathogens were sub-cultured in a 500 mL Erlenmeyer ask containing 300 mL of fresh nutrient broth (NB) medium and incubated (37 2 C for 24 h) overnight. On a subsequent day, each 50 mL of the overnight 0.5 McFarland standards DRS pathogens broth was evenly transferred into a 100 mL Table 2 Systematic position of drug-resistant superbugs (DRS) Assigned code Phylum Class Order Family Genus Species NIT-PW-II Firmicutes Bacilli Bacillales Bacillaceae Bacillus lamentosus NIT-PW-V Firmicutes Bacilli Bacillales Bacillaceae Bacillus exus NIT-PW-III Proteobacteria Gammaproteobacteria Pseudomonadales Pseudomonadacea Pseudomonase stutzeri NIT-PW-IV Proteobacteria Gammaproteobacteria Pseudomonadales Moraxellaceae Acinetobacter baumannii 988 \| RSC Adv., 2022, 12, 985–997 © 2022 The Author(s). Published by the Royal Society of Chemistry © 2022 The Author(s). Published by the Royal Society of Chemistry 988 \| RSC Adv., 2022, 12, 985–997 RSC Advances View Article Online View Article Online Paper RSC Advances Fig. 2 (a) UV-visible spectroscopy. (b) X-ray diﬀraction pattern mixed phase of face-centered cubic (fcc) shape. (c) Photoluminescence (PL) spectra of synthesized green SMZnO-NPs. (d) Raman spectra of synthesized green SMZnO-NPs. article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 05 January 2022. Downloaded on 10/24/2024 5:36:13 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 2 (a) UV-visible spectroscopy. (b) X-ray diﬀraction pattern mixed phase of face-centered cubic (fcc) sh spectra of synthesized green SMZnO-NPs. (d) Raman spectra of synthesized green SMZnO-NPs. Fig. 2 (a) UV-visible spectroscopy. (b) X-ray diﬀraction pattern mixed phase of face-centered cubic (fcc) shape. (c) Photoluminescence (PL) spectra of synthesized green SMZnO-NPs. (d) Raman spectra of synthesized green SMZnO-NPs. degradation. Aer the experiments, the catalyst was separated by centrifugation at 8000 rpm for 15 min and the supernatant was discarded. The obtained catalyst pellet was washed twice with DDW thoroughly and kept in a hot air oven and dried at 60 C for 5 h for further reuse. The recovered catalyst was washed with DDW several times and oven-dried at 70 C under vacuum for further reuse. Then the green SMZnO-NPs were analyzed by X-ray diﬀraction (XRD) patterns to determine their structure and stability. The same procedure was adopted for all repeated cycles. Dye degradation efﬁciency% ¼ (c0 ct/c0) 100 (2) where ‘C0’ was the initial concentration (mg L1) of MB dye, ‘Ct’ was the remaining MB dye concentration (mg L1) of the aqueous solution and ‘t’ was the given time. where ‘C0’ was the initial concentration (mg L1) of MB dye, ‘Ct’ was the remaining MB dye concentration (mg L1) of the aqueous solution and ‘t’ was the given time. 3. Results and discussion Kinetics study of MB removal was tending to follow pseudo- rst-order kinetics. The photodecomposition rate (k) constant was calculated by the following equation: 3.1. Drug-resistant superbugs (DRS) patterns The widespread use of antibiotics has made great contributions to rescuing human life against infectious diseases. However, pathogens exerting resistance against available antibiotics are challenging the present individual and public health care. To develop some intriguing novel drugs against drug-resistant strains and the use of antibiotics could reverse global health by posing a key threat to human health and ecosystem functioning.39 ln(C0/Ct) ¼ kt (3) (3) where ‘kt’ is the apparent pseudo-rst order rate constant (min1) 2.9. Minimum inhibitory concentration (MIC) experiment, 2 mL of the suspension was sampled at certain time intervals the catalyst was separated by centrifugation at 6000 rpm for 20 min. MB dye concentration was further analyzed by UV-visible spectroscopy (SHIMADZU 1700) through monitoring the absorption spectra. Degradation eﬃciency was calculated by the following equation: Dye degradation efﬁciency% ¼ (c0 ct/c0) 100 (2) (2) 3.3. Green synthesis of SMZnO-NPs determined from various bacterial counts. The isolated bacte- rial strains were randomly chosen and colonies of dissimilar morphology were inoculated into rapid microbial limit test kits for their characterization and identication. Among the 152 bacterial isolates, 4 strains belonging to DRS genera were identied by standard antibiotics. The antibacterial resistance index (ARI) studies signicantly revealed that they were highly resistant Gram-negative and positive strains being conrmed by 16S rDNA sequence analysis. Recently, an increasing concen- tration, as well as persistent display of antibiotic traces in the aquatic environment, became an issue of high concern.40 Green technology as a rapidly growing and emerging eld from nano and life science shows pathways towards the development of more sustainable eco-friendly greener products for biological applications. S. muticum (SM) seaweed extract ZnO-NPs were conrmed by visual assessment. For the synthesis of SMZnO- NPs, desired amounts of zinc nitrate hexahydrate and seaweed extract were added, mixed, and let react. Upon the initiation of the reaction, the mixture turned from dark brown coloration to pale white, indicating the formation of SMZnO- NPs. The changes in color formation result from the interac- tion of possible functional groups present in the seaweed extract with zinc nitrate being reduced to Zn0 ions and stabi- lizing SMZnO-NPs. 3.2. Identication based on 16S rDNA-sequences Isolated DRS pathogens were subjected to BLAST analysis with their barcoding gene, 16S rDNA, and their nucleotide sequences were submitted to Gen Bank with corresponding accession numbers provided in Table 1. From the BLAST analysis, it was inferred that the sequenced nucleotides belonged to Bacillus lamentosus (B. lamentosus; NCBI accession no.: MN045186) and Bacillus exus (B. exus; NCBI accession no.: MN045189) and -negative Acinetobacter baumannii (A. baumannii; NCBI accession no.: MN045188) and Pseudomonas stutzeri (P. stutzeri; NCBI accession no.: MN045185). All of the resulting 16S sequences showed 100% similarity standards from drug- resistant generators in Gen Bank (NCBI) through nucleotide BLAST showed in Table 2. (4) ZnðNO3Þ2$6H2O þ 2KOH ! D ZnðOHÞ2 þ 2KNO3 (4) ZnðOHÞ2 þ 2KNO3 ! S: muticum=ð70 CÞ D ZnO nanoparticles ðNPsÞ (5) ZnðOHÞ2 þ 2KNO3 ! S: muticum=ð70 CÞ D ZnO nanoparticles ðNPsÞ (5) (5) 2.11. Photocatalytic stability of green SMZnO-NPs In order to inspect the photostability and reusability of the green SMZnO-NPs catalyst, this was tested by several photo- catalytic studies under natural sunlight irradiation for MB dye In the present study, we rst isolated drug-resistant super- bugs (DRS) pathogens. A total of 152 colonies were isolated from hospital sewage water and the average of diluted replicates was RSC Adv., 2022, 12, 985–997 \| 989 © 2022 The Author(s). Published by the Royal Society of Chemistry 989 View Article Online RSC Advances Paper Fig. 3 (a) and (b) SEM and Fe-SEM observation of green synthesized SMZnO-NPs (c) EDXS spectrum image of green synthesized SMZnO-NPs. (d) X-ray ﬂuorescence (XRF) spectrum of particle size distribution and of synthesized zinc oxide nanoparticles. Open Access Article. Published on 05 January 2022. Downloaded on 10/24/2024 5:36:13 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 3 (a) and (b) SEM and Fe-SEM observation of green synthesized SMZnO-NPs (c) EDXS spectrum image of green synthesized SMZnO-NPs. (d) X-ray ﬂuorescence (XRF) spectrum of particle size distribution and of synthesized zinc oxide nanoparticles. 3.4. Characterization of green synthesized SMZnO-NPs ZnO-NPs were synthesized by using marine brown alga S. muticum (SM). SM broth was added into the Zn(NO3)2 solution until a color change occurred which conrmed the generation © 2022 The Author(s). Published by the Royal Society of Chemistry 990 \| RSC Adv., 2022, 12, 985–997 990 RSC Advances View Article Online RSC Advances View Article Online Paper Fig. 4 (a) Green synthesized SMZnO-NPs size and distribution studied by zeta potential analysis. (b) FTIR spectra of Sargassum muticum (SM) extract (alone) and green synthesized SMZnO-NPs. (c) In vitro anti-microbial activity of green synthesized SMZnO-NPs against 16S rDNA conﬁrmed drug-resistant superbugs (DRS). Article. Published on 05 January 2022. Downloaded on 10/24/2024 5:36:13 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 05 January 2022. Downloaded on 10/24/2024 5:36:13 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 4 (a) Green synthesized SMZnO-NPs size and distribution studied by zeta potential analysis. (b) FTIR spectra of Sargassum muticum (SM) extract (alone) and green synthesized SMZnO-NPs. (c) In vitro anti-microbial activity of green synthesized SMZnO-NPs against 16S rDNA conﬁrmed drug-resistant superbugs (DRS). D ¼ (kl/b cos q) (7) (7) D ¼ (kl/b cos q) D ¼ (kl/b cos q) of green SMZnO-NPs. The UV-visible spectrum of the green SMZnO-NPs and S. muticum is shown in Fig. 2a. Excitonic absorption peaks at the wavelength of 350 nm elucidated the characteristics of ZnO NPs. Band gap energy of SMZnO-NPs were found to be 3.28 eV, calculated by the extrapolation plot of [ahn]2 versus photon energy (hn), according to the altered Kubelka–Munk function41 which is given as: where ‘D’ is the size of the average nanoparticles; ‘k’ is the Scherrer's constant (k ¼ 0.9); ‘b’ is the full width at half maximum (FWHM) of the peak; ‘q’ is the diﬀraction angle and ‘l’ is the wavelength of X-ray (l ¼ 1.5406 ˚A). An average crystal size has been calculated from XRD analysis using the Debye– Scherrer equation, which in this study was approximately equal to 15–50 nm. F(R) ¼ (1 R)2/2R (6) (6) Photoluminescence (PL) spectra of as-prepared SMZnO-NPs provided at room temperature under 325 nm excitation the wavelengths documented in Fig. 2c. In these luminescence characteristics surface states play a predominant role for the prepared samples. 3.4. Characterization of green synthesized SMZnO-NPs (b) Schematic structure representation of green synthesized SMZnO-NPs by the osmotic shock that could attack the cell wall directly and lead to bacterial death. Eﬀect of SMZnO-NPs against DRS pathogen observation by SEM. Inset in control (c) and (d) is a high-resolution Fe-SEM image of bacterial cell death. Open Access Article. Published on 05 January 2022. Downloaded on 10/24/2024 5:36:13 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 5 (a) MIC growth eﬀects of diﬀerent concentrations of SMZnO-NPs on the growth of Gram-positive and negative DRS pathogens. (b) Schematic structure representation of green synthesized SMZnO-NPs by the osmotic shock that could attack the cell wall directly and lead to bacterial death. Eﬀect of SMZnO-NPs against DRS pathogen observation by SEM. Inset in control (c) and (d) is a high-resolution Fe-SEM image of bacterial cell death were SMZnO-NPs and 10% were other than carbon elements (Zn, Al, and Fe) present in Fig. 3d. The high content of Zn in the sample is a clear indication of the complete reduction of zinc nitrate to ZnO-NPs. oxygen vacancies of ZnO.45 In addition, two Raman peaks at 331.7 and 950 cm1 can be explained with the E2(H) E2(L) and A1(LO) + E2(H) multi-photon scattering phenomenon associated with oxygen vacancies of ZnO.46 The size distributions of as-prepared SMZnO-NPs were studied by DLS analyzer in a standard mono-dispersed medium at 25 C kept at constant pressure (0.892 mPa) using 90 Plus. The graph image displayed an almost equivalent size distribu- tion of less than 25–50 nm as shown in Fig. 4a. Dispersion status was described by the polydispersity index (PDI), which reects the broadness of the size distribution. The measure- ments were taken in triplicate and recorded as the mean standard deviation. Zeta potential analysis exhibit a sharp peak at 21.4 mV (negative value) aﬃrms the prepared nanoparticles are highly stable and ascertain repulsion amidst the particles. The analysis soware of DLS provided the mean size, size distribution, and PDI of the green synthesized SMZnO-NPs suspension are monodispersed showing a narrow particle size distribution (Fig. 4a). SEM analysis showed that particle size, structure, and morphological shape of the green SMZnO-NPs were agglomer- ating to spherical/circular structures. Fig. 3a showed well- dispersed spherical ZnO nanoparticles with a size ranging between 15 and 50 nm with agglomeration. 3.4. Characterization of green synthesized SMZnO-NPs A band observed at 390 nm in the UV region exhibited near-band edge emissions of wurtzite hexagonal ZnO- NPs due to free excitation recombination's as a result of a radioactive recombination process of photo-generated elec- trons and holes.42,43 Emission at 465 nm assigned to the blue emission band may be due to a singly ionized Zn+ vacancy.44 where ‘R’ is reectance, ‘F(R)’ indicates the equivalent absorp- tion coeﬃcient. No color change was observed for the cessation of S. muticum broth. The characteristic gradual change of visual color was used as evidence to conrm the green nanoparticles (SMZnO-NPs). Fig. 2b shows the X-ray diﬀraction analysis of the green SMZnO-NPs showing several intensity peaks at 31.73, 34.39, 36.20, 47.56, 56.60, 63.02, 66.48, 69.1, 72.6, and 76.86, corresponding to (100), (002), (101), (102), (110), (103), (112), (201) and (004), respectively. The whole recorded diﬀraction peak intensities exhibited crystallographic struc- tures of ZnO coinciding well with the Joint Committee on Powder Diﬀraction Standards (JCPDS, card no.: 36-1451). The average NPs size was calculated using the following the Debye– Scherrer formula: where ‘R’ is reectance, ‘F(R)’ indicates the equivalent absorp- tion coeﬃcient. No color change was observed for the cessation of S. muticum broth. The characteristic gradual change of visual color was used as evidence to conrm the green nanoparticles (SMZnO-NPs). Fig. 2b shows the X-ray diﬀraction analysis of the green SMZnO-NPs showing several intensity peaks at 31.73, 34.39, 36.20, 47.56, 56.60, 63.02, 66.48, 69.1, 72.6, and 76.86, corresponding to (100), (002), (101), (102), (110), (103), (112), (201) and (004), respectively. The whole recorded diﬀraction peak intensities exhibited crystallographic struc- tures of ZnO coinciding well with the Joint Committee on Powder Diﬀraction Standards (JCPDS, card no.: 36-1451). The average NPs size was calculated using the following the Debye– Scherrer formula: Raman spectra (Fig. 2d) of prepared ZnO nanoparticles were recorded in the 200–1200 cm1 range. A prominent, strong peak at 438 cm1 corresponds to the E2H (high) mode of the Raman active mode of the wurtzite hexagonal phase of ZnO. A mode at 300 cm1 is ascribed as (high) E2L (low) mode due to the © 2022 The Author(s). Published by the Royal Society of Chemistry RSC Adv., 2022, 12, 985–997 \| 991 View Article Online RSC Advances Fig. 5 (a) MIC growth eﬀects of diﬀerent concentrations of SMZnO-NPs on the growth of Gram-positive and negative DRS pathogens. 992 \| RSC Adv., 2022, 12, 985–997 © 2022 The Author(s). Published by the Royal Society of Chemistry 3.4. Characterization of green synthesized SMZnO-NPs A Fe-SEM study further conrmed spherical poly-dispersed morphology and the average particle size was 50 nm showed in Fig. 3b. The high- magnied Fe-SEM image of synthesized NPs shows self- assembled spherical/circular morphology with well-dispersed due to electrostatic attraction forces. This is clearly matching with the calculated crystal diameter obtained from XRD patterns. Then EDXS analysis in the direct presence of Zn+ agglom- eration shows higher percentages of 92.77% and other carbon substances and elements comprising a portion of 4.23% as shown in Fig. 3c. In order to investigate the composition and quantication of elements in the green synthesized SMZnO- NPs, we employed X-ray uorescence (XRF) measurements. The XRF spectrum measurements of single-molecule behavior observations with abrupt spectral changes showed that 90% The FTIR spectra of biosynthesized SMZnO-NPs and the control spectrum of seaweed extract were shown in Fig. 4b. FTIR absorption peaks at 3432 and 1609 cm1 were related to the presence of an O–H stretching mode.47 Two strong sharp peaks appearing around 1402 cm1 and 1609 cm1 corresponded to 992 \| RSC Adv., 2022, 12, 985–997 © 2022 The Author(s). Published by the Royal Society of Chemistry 992 RSC Advances View Article Online Paper RSC Advances Fig. 6 Photocatalytic degradation using green synthesized SMZnO-NPs of MB dyes under the irradiation of (a) visible-light (b) UV-light and (c) natural sunlight. d on 05 January 2022. Downloaded on 10/24/2024 5:36:13 AM. ensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 05 January 2022. Downloaded on 10/24/2024 5:36:13 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 6 Photocatalytic degradation using green synthesized SMZnO-NPs of MB dyes under the irradiation of (a) visible-light (b) UV-light and (c) natural sunlight. a C–H hydroxyl group and a C]O carbonyl group.48 An FTIR band observed at lower wavenumber, i.e., below 500 cm1 refers to a Zn–O stretching vibration mode.49 SMZnO-NPs kill these DRS pathogens based on mechanisms including, self-destructive of the cell wall membrane, ROS generation can damage DNA leading to substantial treatment eﬀects in cell walls of both Gram-positive and negative genera. Scientists and researchers have strained an attempt to explain the damage of cell walls which may be caused by the incorpo- ration of foreign substances.55,56 In addition, 80 mg mL1 higher concentrations of green SMZnO-NPs showed a higher sensitivity compared to lower concentrations. 3.4. Characterization of green synthesized SMZnO-NPs Results show that a concentration depending manner plays an important role in antimicrobial eﬀects. The research has demonstrated that ZnO- NPs exhibit antibacterial activity based on the generation of ROS by oxidase and peroxidase-like catalytic activities that could aﬀect the bacterial cell wall directly leading to death.57,58 © 2022 The Author(s). Published by the Royal Society of Chemistry 3.5. In vitro anti-microbial eﬃcacy of SMZnO-NPs Published on 05 January 2022. Downloaded on 10/24/2024 5:36:13 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 7 Photocatalytic degradation of MB dyes under diﬀerent regimes of sunlight irradiation: (a)–(c). Calibration plot of ln(C0/Ct) vs. time for the catalytic degradation of MB dye; (d) XRD patterns results of the photocatalytic stability/cycling test after repetition reaction. Fig. 5a. It is clear evidence that green NPs at increasing concentration decrease the DRS growth populations. Anti- bacterial potency of DRS against SMZnO-NPs the Gram- negative bacteria was less susceptible than the positive causing a thinner peptidoglycan layer to be formed.59 A sche- matic diagram shows the prevalence of the possible antimi- crobial interaction mechanisms of ZnO-NPs with the bacterial cells detected, the osmotic shock of photo-induced holes (H+), and the hydroxyl radicals (OH) attack to bacterial cell wall causing their death shown in Fig. 5b. ZnO-NP inhibition mechanism in Gram-negative and -positive NP sensitivity can be explained while preventing bacterial growth mainly by the generation of ROS on the surface.60 Furthermore, our Fe-SEM study clearly showed that the anti-microbial mechanism was a biphasic phenomenon of osmotic shock that attacks the cell wall leading to death shown in Fig. 5c and d. of MB decolorization eﬃciency reached 84% in 105 min and its highest eﬃciency of 96% was achieved within 60 min duration when there was no further degradation of dye. During this process, the catalyst remained stable. These results demon- strated that the green SMZnO-NPs catalyst exhibited signi- cantly superior photocatalytic activity under natural sunlight irradiation in comparison to UV-light and visible-light sources as shown in Fig. 6a–c. A controlled study without adding green catalyst involved the degradation of dye under UV-light, visible-light, and natural sunlight. No obvious spectral changes were observed without the presence of a catalyst and the eﬃciency was less than 5% for MB under visible-light, 10% for natural sunlight while it was 12% under UV-light. Green prepared ZnO nanoparticles were further investigated for degradation kinetics. These revealed a linear relationship with time. A decolorization test tended to exhibit apparent pseudo-rst-order kinetics according to eqn (2), the comparison plot of Ct/C0 & ln Ct/C0 vs. irradiation time (t). 3.5. In vitro anti-microbial eﬃcacy of SMZnO-NPs Recently, several reports such as Chandra et al.,61 Korosi et al.,62 Bai et al.,63 Sirajudheen and Meenakshi,64 Gulce et al.,65 Chang et al.,66 Xiang et al.,67 Ju et al.,68 and Narayana et al.,69 discussed chemically prepared NPs and NCs metal and metal oxide nanoparticles of the Fe3O4, TiO2, ZnO, Chitosan-La3+- graphite composite, SiO2, polyaniline (PANI)/CdO, BiVO4, 3.5. In vitro anti-microbial eﬃcacy of SMZnO-NPs The four (n ¼ 4) selected antibiotic-sensitive strains, B. la- mentosus, B. exus, P. stutzeri, and A. baumannii were evaluated against SMZnO-NPs. An anti-microbial eﬀect concluding species-specic characteristics, NPs-shape, conguration, size, and concentrations of nano compounds play a key role to pertain antagonistic activity.50 According to our nding, SMZnO-NPs revealed signicant growth inhibition activity against DRS pathogens. The results demonstrated a maximal growth inhibition activity of 18 mm against the pathogen A. baumannii (NCBI accession no.: MN045188) and minimal growth inhibition of 12 mm against B. exus (NCBI accession no.: MN045189) shown in Fig. 4c, can be explained by their extracellular polymeric substance (EPS) secretion.51 Gram- positive bacterial cell walls are surrounded by multilayer peptidoglycan, which is signicantly thicker than in Gram- negative bacteria having a complex cell wall structure consist- ing of a thin peptidoglycan layer between the outer plasma and the cytoplasmic membrane.52 Minimum inhibitory concentration (MIC) growths were observed by optical density (OD) measurements of bacterial cultures and the inhibition rate was plotted against time to time intervals. MIC growths as a function of SMZnO-NPs and time intervals were employed to examine DRS pathogenic bacteri- cidal eﬀects. Furthermore, in the control experiment, the DRS pathogenic bactericidal activities, as well as growth, continued to be unchanged aer 24 h. Compared to the control the OD value of DRS growth populations was decreased by adding green synthesized SMZnO-NPs. When the green NPs were increased to 100 mg mL1, the growth of the DRS was gradually decreased. Following 200 mg mL1 at 24 h incubation, the growth was completely inhibited. For the green SMZnO-NPs compound, therefore, 200 mg mL1 is considered to be a MIC value shown in The anti-microbial mechanism of SMZnO-NPs employ a biphasic phenomenon persuaded by the osmotic shock which damages the cell membranes, thereby internalizing the SMZnO- NPs inside the cell, eventually inducing reactive oxygen species (ROS) followed by oxidative stress and cell death.53,54 Green © 2022 The Author(s). Published by the Royal Society of Chemistry RSC Adv., 2022, 12, 985–997 \| 993 993 View Article Online RSC Advances Fig. 7 Photocatalytic degradation of MB dyes under diﬀerent regimes of sunlight irradiation: (a)–(c). Calibration plot of ln(C0/Ct) vs. time for the catalytic degradation of MB dye; (d) XRD patterns results of the photocatalytic stability/cycling test after repetition reaction. article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Author contributions S. Harinee: data collection, interpretation, experiments and writing manuscript. Dr K. Muthukumar & Prof. M. Ashok: supervision, experiment, writing, reviewing, preparation and editing the manuscript. Open Access Article. Published on 05 January 2022. Downloaded on 10/24/2024 5:36:13 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Data availability Cyclic stability is another most important factor for practical/industrial applications. To inspect the stability of green SMZnO-NPs tested for the degradation cycles under natural sunlight irradiation reused up to 6 consecutive cycles. The degradation eﬃciency was found to be gradually decreased from 95% to 86% aer 6 cycles. In order to ensure stability XRD pattern of the catalyst was recorded before and aer 6 cycles of photocatalysis where the results remain the same as before as shown in Fig. 7a–d. There was no observation of any extra peak other than ZnO suggesting that the prepared sample was eﬃ- cient, more stable with higher durability, and recyclable. The data used to support the ndings of this study are included in the article. References 1 World Health Organization, Guidelines for drinking-water quality, WHO, Geneva, 4th edn, 2011. 2 Y. Zheng, W. Liu, Z. Qin, Y. Chen, H. Jiang and X. Wang, Bioconjugate Chem., 2018, 29, 3094–3103. 3 V. Dogra, G. Kaur, S. Jindal, R. Kumar, S. Kumar and N. K. Singhal, Sci. Total Environ., 2019, 681, 350–364. 4 F. C. Cabello, Environ. Microbiol., 2006, 8, 1137–1144. 5 World Health Organization, Water safety plan manual: step- by-step risk management for drinking-water suppliers, WHO, Geneva, 2009. 3.6. Photocatalytic activity and stability of SMZnO-NPs The photocatalytic performance of as-prepared green SMZnO- NPs was evaluated for MB dye degradation exposed to UV- light, visible-light, and natural sunlight. The process of spec- tral change for MB dyes was monitored for diﬀerent time intervals, under visible-light irradiation exhibiting a low pho- tocatalytic activity of 40% within 240 min. In another case under UV-light and direct natural sunlight irradiation, the absorbance © 2022 The Author(s). Published by the Royal Society of Chemistry 994 \| RSC Adv., 2022, 12, 985–997 RSC Advances View Article Online RSC Advances View Article Online Paper Bi2WO6/BiVO4 have been widely used into the degradation of organic dye and controlling microorganisms. Acknowledgements K. Muthukumar wishes to thank the Department of Science and Technology, Science Engineering Research Board (DST-SERB), New Delhi, India for awarding a National Postdoctoral Fellow- ship (PDF/2017/002213). The authors were reported no potential conict of interest. The authors were reported no potential conict of interest. Open Access Article. Published on 05 January 2022. Downloaded on 10/24/2024 5 This article is licensed under a Creative Commons Attribution 3.0 U Here the S. muticum (SM) mediated ZnO-NPs show remark- ably improved photocatalytic activity under sunlight irradiation for MB degradation up to 96% that was achieved within a short time (60 min). Additionally, an anti-bacterial performance of drug-resistant superbugs (DRS) inhibition of 18 mm was observed against A. baumannii and minimal inhibition of 12 mm in B. exus. This work provides the rst report of S. muticum mediated green catalyst SMZnO-NPs that provide an enhanced degradation of MB dye under natural sunlight irra- diation, as well as higher anti-microbial activity, was demon- strated against harmful DRS pathogens. When compared to earlier reports, these green SMZnO-NPs were attained within a short period compared to previous studies. It can be concluded that SMZnO-NPs showed superior photocatalytic activity for the degradation of MB dyes and also exhibited antibacterial performance towards DRS pathogens. Sunlight- mediated photocatalytic activity will also be benecial for environmental applications for good recyclability, high stability, cheap availability, and other practical purposes. Strongly enhanced activities of SMZnO-NPs conrmed signi- cant applications during water purication by converting hazardous materials into non-hazardous ones. Open Access Article. Published on 05 January 2022. Downloaded on 10/24/2024 5: This article is licensed under a Creative Commons Attribution 3.0 Un 45 C. K. Z. Padilla and S. A. Gamboa, J. Alloys Compd., 2018, 767, 932–937. 21 L. S. R. Yadav, S. Pratibha, K. Manjunath, M. Shivanna, T. Ramakrishnappa, N. Dhananjaya and G. Nagaraju, J. Sci.: Adv. Mater. Devices, 2019, 4, 425–431. 46 V. A. Soares, M. J. S. Xavier, E. S. Rodrigues, C. A. D. Oliveira, P. M. A. Farias, A. Stingl, N. Ferreira and M. S. Silva, Mater. Lett., 2020, 259, 126853. 22 E. F. El-Belely, M. M. S. Farag, H. A. Said, A. S. Amin, E. Azab, A. A. Gobouri and A. Fouda, Nanomater, 2021, 11, 95. 47 R. Yuvakkumar, J. Suresh, A. J. Nathanael, M. Sundrarajan and S. I. Hong, Mater. Sci. Eng., C, 2014, 41, 17–27. 23 Z. Ye, C. Li, N. Skillen, Y. Xu, H. McCabe, J. Kelly, P. Robertson and S. E. J. Bell, Applied Materials Today, 2019, 15, 398–404. 48 A. Raja, S. Ashokkumar, R. P. Marthandam, J. Jayachandiran, C. P. Khatiwada, K. Kaviyarasu, R. G. Raman and M. Swaminatha, J. Photochem. Photobiol., B, 2018, 181, 53–58. 24 N. Vigneshwaran, R. P. Nachane, R. H. Balasubramanya and P. V. Varadarajan, Carbohydr. Res., 2006, 341, 2012–2018. 49 S. Jeyabharathi, K. Kalishwaralal, K. Sundar and A. Muthukumaran, Mater. Lett., 2017, 209, 295–298. 25 S. P. Chandran, M. Chaudhary, R. Pasricha, A. Ahmad and M. Sastry, Biotechnol. Prog., 2006, 22, 577–583. 50 N. Matinise, X. G. Fuku, K. Kaviyarasu, N. Mayedwa and M. Maaza, Appl. Surf. Sci., 2017, 406, 339–347. 26 K. Tahir, S. Nazir, B. Li, A. U. Khan, Z. U. H. Khan, A. Ahmad and F. U. Khan, Sep. Purif. Technol., 2015, 150, 316–324. 51 L. Wang, C. Hu and L. Shao, Int. J. Nanomed., 2017, 12, 1227– 1249. 27 S. Azizi, M. B. Ahmad, F. Namvar and R. Mohamad, Mater. Lett., 2014, 116, 275–277. 52 N. Shanmugam, P. Rajkamal, S. Cholan, N. Kannadasan, K. Sathishkumar, G. Viruthagiri and A. Sundaramanickam, Appl. Nanosci., 2014, 4, 881–888. 28 R. Saravanan, N. Karthikeyan, V. K. Gupta, E. Thirumal, P. Thangadurai, V. Narayanan and A. Stephen, Mater. Sci. Eng., C, 2013, 33, 2235–2244. 53 C. Roca, V. D. Alves, F. Freitas and M. A. M. Reis, Front. Microbiol., 2015, 6, 288–295. 29 M. Zare, K. Namratha, S. Alghamdi, Y. H. E. Mohammad, A. Hezam, M. Zare, Q. A. Drmosh, K. Byrappa, B. N. Chandrashekar, S. Ramakrishna and X. Zhang, Sci. Rep., 2019, 9, 8303–8318. 54 O. Y. 4. Conclusion 6 N. J. Ashbolt, Curr. Environ. Health Rep., 2015, 2, 95–106. 7 B. Li and T. J. Webster, J. Orthop. Res., 2018, 36, 22–32. We have successfully identied drug-resistant superbugs (DRS; n ¼ 4) Gram-positive and negative B. lamentosus, B. exus, P. stutzeri, and A. baumannii through 16S rDNA conrmation. Eco- friendly and low-cost green synthesis was developed by S. muticum (SM) mediated ZnO-NPs through a complete green synthetic method. In vitro anti-microbial activity of SMZnO-NPs exhibited a higher potency on selected Gram-negative than on Gram-positive DRS pathogens. Caused by the secretion of EPS from Gram-positive bacteria a minimal zone of 12 mm occurred in B. exus and higher inhibition of 18 mm was observed against A. baumannii. The results suggest a modication of SMZnO-NPs that can eﬃciently target and kill DRS pathogens. Enhanced photocatalytic activity of SMZnO-NPs under natural sunlight irradiation eﬀectively MB dye degradation 96% within 60 min. The novel strategy to develop green synthesized SM prepared SMZnO-NPs is expected to have particular applica- tions in clinical water cleansing and in the biomedical industries. 8 R. Rawashdeh and Y. Haik, Dyn. Biochem. Process Biotechnol. Mol. Biol., 2009, 3, 12–20. 9 H. Dolliver, S. Gupta and S. Noll, J. Environ. Qual., 2008, 37, 1245–1253. 10 A. C. Singer, H. Shaw, V. Rhodes and A. Hart, Front. Microbiol., 2016, 7, 1728–1750. 11 T. M. Dhameliya, H. A. Donga, P. V. Vaghela, B. G. Panchal, D. K. Sureja, K. B. Bodiwala and M. T. Chhabria, RSC Adv., 2020, 10, 32740. 12 J. B. Fathima, A. Pugazhendhi, M. Oves and R. Venis, J. Mol. Liq., 2018, 260, 1–8. 13 A. Ali, Y. Liang, S. Ahmed, B. Yang, B. Guo and Y. Yang, Applied Materials Today, 2020, 18, 100534. 14 Z. C. Wu, Y. Zhang, T. X. Tao, L. Zhang and H. Fong, Appl. Surf. Sci., 2010, 257, 1092–1097. 15 J. Liang, X. Ning, M. Kong, D. Liu, G. Wang, H. Cai, J. Sun, Y. Zhang, X. Lu and Y. Yuan, Environ. Pollut., 2017, 231, 115–122. © 2022 The Author(s). Published by the Royal Society of Chemistry RSC Adv., 2022, 12, 985–997 \| 995 View Article Online RSC Advances Paper Paper 39 K. Punjabi, S. Mehta, R. Chavan, V. Chitalia, D. Deogharkar and S. Deshpande, Front. Microbiol., 2018, 9, 2207–2218. 16 S. Harinee, K. Muthukumar, A. Abirami, K. Amrutha, K. Dhivyaprasath and M. Ashok, Advanced Materials Proceedings, 2019, 4(3), 115–118. 40 M. Open Access Article. Published on 05 January 2022. Downloaded on 10/24/2024 5: This article is licensed under a Creative Commons Attribution 3.0 Un A. Costa, J. M. Raaijmakers and E. E. Kuramae, Front. Microbiol., 2018, 9, 1636–1650. 55 K. R. Raghupathi, R. T. Koodali and A. C. Manna, Langmuir, 2011, 27, 4020–4028. 30 M. Hinton, A. J. Hedges and A. H. Linton, J. Appl. Bacteriol., 1985, 58, 27–35. 31 M. Krishnan, H. U. Dahms, P. Seeni, S. Gopalan, V. Sivanandham, K. J. Hyoung and R. A. James, Mater. Sci. Eng., C, 2017, 73, 743–755. 56 T. Xia, M. Kovochich, M. Liong, L. M. Adler, B. Gilbert, H. Shi, J. I. Yeh, J. I. Zink and A. E. Nel, ACS Nano, 2008, 2, 2121–2134. 57 H. Yang, C. Liu, D. Yang, H. Zhang and Z. Xi, J. Appl. Toxicol., 2009, 29, 69–78. 32 J. D. Thompson, D. G. Higgins and T. J. Gibson, Nucleic Acids Res., 1994, 22, 4673–4680. 58 L. Zhang, Y. Jiang, Y. Ding, M. Povey and D. J. York, Nanopart. Res., 2007, 9, 479–489. 33 M. Drancourt, C. Bollet, A. Carlioz, R. Martelin, J. P. Gayral and D. Raoult, J. Clin. Microbiol., 2000, 38, 3623–3630. 59 R. Brayner, R. Ferrari-Iliou, N. Brivois, S. Djediat, M. F. Benedetti and F. Fievet, Nano Lett., 2006, 6, 866–870. 34 M. Krishnan, V. Sivanandham, D. Hans-Uwe, S. G. Murugaiah, P. Seeni, S. Gopalan and A. J. Rathinama, Mar. Pollut. Bull., 2015, 101, 816–825. 60 A. Sirelkhatim, S. Mahmud, A. Seeni, N. H. M. Kaus, L. C. Ann, S. K. M. Bakhori, H. Hasan and D. Mohamad, Nano-Micro Lett., 2015, 7(3), 219–242. 35 S. Rajaboopathi and S. Thambidurai, Current Applied Physics, 2017, 17, 1622–1638. 36 M. Balouiri, M. Sadiki and S. K. Ibnsouda, J. Pharm. Anal., 2016, 6, 71–79. 61 V. Chandra, J. Park, Y. Chun, J. W. Lee, I. C. Hwang and K. S. Kim, ACS Nano, 2010, 4, 3979–3986. 37 M. Goudarzi, H. Goudarzi, A. M. S. Figueiredo, E. E. Udo, M. Fazeli, M. Asadzadeh and S. S. Seyedjavadi, PLoS One, 2017, 1–13. 62 L. Korosi, B. Bognar, M. Horvath, G. Schneider, J. Kovacs, A. Scarpellini, A. Castelli, M. Colombo and M Prato, Appl. Catal., B, 2018, 231, 115–122. 38 Z. H. Jaﬀari, S. M. Lam, J. C. Sin, H. Zeng and A. R. Mohamed, Sep. Purif. Technol., 2020, 236, 116195. 63 S. L. Bai, H. Fu, Y. Y. Zhao, K. Tian, R. X. Luo, D. Q. Li and A. F. Chen, Sens. Actuators, B, 2018, 266, 692–702. 4. Conclusion Bilal, S. S. Ashraf, D. Barcelo and H. M. N. Iqbal, Sci. Total Environ., 2019, 691, 1190–1211. 17 S. Harinee, K. Muthukumar, H. U. Dahms, M. Koperuncholan, S. Vignesh, R. J. Banu, M. Ashok and R. A. James, Int. Biodeterior. Biodegrad., 2019, 145, 104790. 17 S. Harinee, K. Muthukumar, H. U. Dahms, M. Koperuncholan, S. Vignesh, R. J. Banu, M. Ashok and 41 I. S. Saputra and Y. Yulizar, IOP Conf. Ser.: Mater. Sci. Eng., 2017, 188, 012004. n Access Article. Published on 05 January 2022. Downloaded on 10/24/2024 5:36:13 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. 42 S. M. Hosseini, I. A. Sarsari, P. Kameli and H. Salamati, J. Alloys Compd., 2015, 640, 408–415. 18 V. K. Sharma, R. A. Yngard and Y. Lin, Adv. Colloid Interface Sci., 2009, 145, 83–96. 19 S. Gao, X. Jia, S. Yang, Z. Li and K. Jiang, J. Solid State Chem., 2011, 184, 764–769. 43 M. M. Alves, S. M. Andrade, L. Grenho, M. H. Fernandes, C. Santos and M. F. Montemor, Mater. Sci. Eng., C, 2019, 101, 76–87. 20 M. Krishnan, H. Subramanian, H. U. Dahms, V. Sivanandham, P. Seeni, S. Gopalan, A. Mahalingam and A. J. Rathinam, Sci. Rep., 2018, 8, 2609. 44 M. Karthikeyan, A. J. Ahamed, C. Karthikeyan and P. V. Kumar, Appl. Sci., 2019, 1, 355–374. 64 P. Sirajudheen and S. Meenakshi, Int. J. Biol. Macromol., 2019, 133, 253–261. 67 Y. H. Xiang, P. Ju, Y. Wang, Y. Sun, D. Zhang and J. Q. Yu, Chem. Eng. J., 2016, 288, 264–275. 65 H. Gulce, V. Eskizeybek, B. Haspulat, F. Sarl, A. Gulce and A. Avcl, Ind. Eng. Chem. Res., 2013, 52, 10924–10934. 68 P. Ju, Y. Wang, Y. Sun and D. Zhang, Dalton Trans., 2016, 45, 4588–4602. RSC Adv., 2022, 12, 985–997 \| 997 66 X. Chang, Z. Li, X. Zhai, S. Sun, D. Gu, L. Dong, Y. Yin and Y. Zhu, Mater. Des., 2016, 98, 324–332. 69 A. Narayana, S. A. Bhat, A. Fathima, S. V. Lokesh, S. G. Suryad and C. V. Yelamaggad, RSC Adv., 2020, 10, 13532. © 2022 The Author(s). Published by the Royal Society of Chemistry Open Access Article. Published on 05 January 2022. Downloaded on 10/24/2024 5: This article is licensed under a Creative Commons Attribution 3.0 Un 996 \| RSC Adv., 2022, 12, 985–997 © 2022 The Author(s). Published by the Royal Society of Chemistry 996 View Article Online View Article Online Paper RSC Advances mol., 67 Y. H. Xiang, P. Ju, Y. Wang, Y. Sun, D. Zhang and J. Q. Yu, Chem. Eng. J., 2016, 288, 264–275. 64 P. Sirajudheen and S. Meenakshi, Int. J. Biol. Macromol., 2019, 133, 253–261. 67 Y. H. Xiang, P. Ju, Y. Wang, Y. Sun, D. Zhang and J. Q. Yu, Chem. Eng. J., 2016, 288, 264–275. 65 H. Gulce, V. Eskizeybek, B. Haspulat, F. Sarl, A. Gulce and A. Avcl, Ind. Eng. Chem. Res., 2013, 52, 10924–10934. 68 P. Ju, Y. Wang, Y. Sun and D. Zhang, Dalton Trans., 2016, 45, 4588–4602. nd 68 P. Ju, Y. Wang, Y. Sun and D. Zhang, Dalton Trans., 2016, 45, 4588–4602. 66 X. Chang, Z. Li, X. Zhai, S. Sun, D. Gu, L. Dong, Y. Yin and Y. Zhu, Mater. Des., 2016, 98, 324–332. 69 A. Narayana, S. A. Bhat, A. Fathima, S. V. Lokesh, S. G. Suryad and C. V. Yelamaggad, RSC Adv., 2020, 10, 13532. 9 A. Narayana, S. A. Bhat, A. Fathima, S. V. Lokesh, S. G. Suryad and C. V. Yelamaggad, RSC Adv., 2020, 10, 13532. RSC Adv., 2022, 12, 985–997 \| 997 © 2022 The Author(s). Published by the Royal Society of Chemistry
W4210718875.txt	https://www.sciendo.com/pdf/10.2478/picbe-2021-0037	en		Approaching globalization from the perspective of socio-economic effects	Proceedings of the ... International Conference on Business Excellence	2,021	cc-by	3,784	Approaching globalization from the perspective of socio-economic effects Catalin Razvan DOBREA Bucharest University of Economic Studies, Bucharest, Romania razvan.dobrea@man.ase.ro Ovidiu Andrei Cristian BUZOIANU Bucharest University of Economic Studies, Bucharest, Romania buzoianuovidiu@yahoo.com Cristina DIMA Bucharest University of Economic Studies, Bucharest, Romania cristina.dima@man.ase.ro Evelina Petronela BALU Bucharest University of Economic Studies, Bucharest, Romania evelina.balu@yahoo.com Abstract. The topicality of the topic lies in the importance of globalization from the perspective of sustainable development in the socio-economic and ecological sphere. Globalization cannot be considered as representing a completely new phenomenon in the history of the world, it has certain historical antecedents, many specialists arguing that it is nothing but a new name for an older phenomenon. Globalization is the global trend of the world's economies to become borderless and interconnected. Globalization has caused major damage to nature and the drying up of non-renewable resources, creating the possibility of global campaigns to develop. Civil societies are less efficient in the face of global factors, which cannot be held accountable, such as governments or companies anchored in the territory, visible and affordable. Keywords: management, globalisation, economy, demography. Introduction In the new era of globalization, borders have opened up, trade barriers have been removed, and information is circulating at an unprecedented rate (Radulescu et al., 2020). The turnover of transnational companies is exploding, as is that of cross-border criminal organizations. Colossal fortunes are often the result of drug and arms trafficking, smuggling, prostitution, money laundering, all in the shadow of corruption. Nothing that happens in our society today remains untouched by the complexity of this process. So globalization has become a symbol of the times we live in, which has both advantages and disadvantages for the countries already involved and for those to be swallowed by this wave (Bran et al., 2020). This paper seeks to provide an image of the modern world, of economic and social changes as a result of globalization. It is dedicated to researching issues in the global economy, the evolution of economic and social gaps, global demographic trends, and environmental protection. The choice of theme was influenced by the topicality of the phenomenon as well as by the problems involved in the process of globalization (Dobrescu, 2010). It has also created the DOI: 10.2478/picbe-2021-0037 © 2021 C. R. Dobrea, O. A. C. Buzoianu, C. Dima, E. P. Balu, published by Sciendo. This work is licensed under the Creative Commons Attribution 4.0 License. possibility of global campaigns to develop. Civil societies are less efficient in the face of global factors, which cannot be held accountable, such as governments or companies anchored in the territory, visible and affordable. The issue of globalization has economic effects not only on individuals, businessmen, governments, nation-states and state unions, with an impact on labor markets, financial markets, economic growth and convergence processes of national economies (Brecher, 2012). Like any political and social process, globalization cannot be considered an unlimited source of benefits for humanity, especially for areas where labor productivity and efficiency are very low, but neither can it be blamed for all the evils that some social structures are forced to overcome (Dinu, 2004). However, the most important factor of globalization is the economic one, which directly influences the political factor, more precisely the policy of expansion and domination of the developed countries over the world. The dimensions of globalization cannot be clearly delimited from each other. Global environmental issues cannot be explored in isolation from either the economic or the political dimension. Globalization encompasses more than the increased integration of the world economy, so it cannot be strictly limited to economic processes, which is often the case (Stiglitz, 2008). Literature review Globalization is an abstract concept, it does not materialize in a concrete object, not being highlighted by its natural dimensions, easily identifiable by established units of measurement (Radulescu, 2020). There is no indicator or statistical index obtained by mathematical calculations that can quantify the size of this phenomenon (Androniceanu et al., 2017). Due to this fact, the phenomenon is not easy to define (Dima et al., 2020). The topic of globalization first entered a dictionary in 1961. Since the mid-80s it has gained considerable circulation, joining as an adjective other terms such as: markets, institutions, ecology, finance, lifestyles, communications, migration, laws, factories, conferences, civil society, events and risks (Burlacu et al., 2020). An answer to the question “what is globalization” we find in Ioan Bari defines globalization as a phenomenon whereby geographical distance becomes an increasingly important factor in cross-border socio-economic, socio-cultural, political and development stability (Bari, 2005). Networks of relationships and dependencies are gaining increasing potential to become international and global (Negescu et al., 2020). Antony Giddens offers a definition containing predominantly sociological elements. "Globalization can be defined as the intensification of social relations throughout the world, which connects such distant localities to such an extent that events that take place locally are viewed through the prism of similar ones, spent many miles away and vice versa." The World Bank defines "globalization as the observable fact that in recent years a growing share of global economic activity has taken place between individuals and firms in different countries." Globalization involves many things: an international circulation of ideas. and information, common cultural experiences, a global civil society and a common environmental movement (Ioniţă et al., 2009). Globalization is a process of interaction and integration among people, companies and governments of different nations, a process driven by international trade and investment and aided by information technology (Held, 2016). Globalization can best be explained as a spatial phenomenon, with two extremes: at one end are local communities with their specific problems, and at the other end is the global society we aspire to, but we do not yet know if it will ever happen (Bodislav et al., 2020). Between these DOI: 10.2478/picbe-2021-0037, pp. 406-414, ISSN 2558-9652 \| Proceedings of the 15th International Conference on Business Excellence 2021 PICBE \| 407 points the whole human activity moves, in the sense that day by day it is more and more influenced by what is happening in other parts of the world, and it is influenced by the practices and decisions of local groups and communities. which may have global influences (Costache et al., 2015). Methodology This article aims to test the hypothesis that globalization is a decisive factor in global economic, environmental and social modeling. That said, certain statistical indicators are analyzed quantitatively, based on data provided by FAO and Worldbank. From a qualitative point of view, an analysis of some notoriety concepts from the specialized literature is used. The analysis is based on the comparative method between certain regions of the world, exposed over a wide area of time. Results and discussions The dimensions of the globalization phenomenon Globalization brings with it extraordinary opportunities that have allowed some countries to capitalize on the opening of markets and the opportunity to assimilate new technologies. This is the case in several Southeast Asian countries, especially as long as they have not opened financially prematurely. In the economically advanced world, new information technologies have greatly prolonged the U.S. boom. what made some believe in the incredible virtues of the new economy and thus spoke of a new paradigm, able to combine uninterrupted economic growth with low unemployment and low inflation. Globalization can have five dimensions: economic, political, social, cultural and ecological. The economic dimension of globalization is of great importance, it being one of the most important causes for globalization processes in other fields (huge growth of trade and direct investment, globalization of financial markets, transnational integrated production, transnational corporations, competition in local level between states and regions, end of national economies) (Ioniţă et al., 2009). Particularly significant are the transformations produced by globalization in terms of production. The national classical corporation has today been transformed into a network, incorporating into a finished product a suite of elements collected from the farthest sources (Bergh and Gustafsson, 2019). Markets tend to globalize due to the fact that domestic markets can no longer sustain the rising costs of research and development nor the shorter life cycles of technologically advanced products. A global production network allows firms to diversify their sources of supply and markets. to mitigate the risks assumed (Rodrik, 2018). The ecological dimension. Some global problems, such as global warming or deforestation, most impressively illustrate the phenomenon of globalization, because in this case it is certainly global issues that require a global approach. The social dimension. The world has become a "global village" (Bari, 2003), with innovative long-distance communication networks (chat, e-mail) adding to traditional communities such as family or neighborhood. However, they cannot replace these traditional spheres of communication. The political dimension. This dimension refers to the “internationalization and institutionalization of political structures as a result of changes in the decision-making process to the regional and international level. Politics faces major problems. Globalization and competition at the local level limit the scope of national policies, many problems can only be properly resolved DOI: 10.2478/picbe-2021-0037, pp. 406-414, ISSN 2558-9652 \| Proceedings of the 15th International Conference on Business Excellence 2021 PICBE \| 408 at the international, global level. In this sense, European integration is seen as a successful response to the challenges of globalization. Globalization has greatly stimulated international trade and the distribution of production activities based on a logic of comparative advantage applied on a global scale, hence the rapid increase in the number of companies with global activity (Pieterse, 2015). The effects of globalization The reaction to a rapid and comprehensive process is not long in coming. From total rejection, fierce resistance to the critical identification of especially social implications, contemporary writings have raised globalization to the rank of the main subject. Analyzed in terms of economic efficiency of resource allocation and use, globalization appears as a rational phenomenon, designed to provide increasing quantities of services with fewer and cheaper material, financial and human resources. Globalization is advantageous and very advantageous for countries with high economic competitiveness: advanced technologies, efficient labor, high labor productivity, low production costs. In these conditions it can be stated that the main source of competitiveness is represented by capital (Holton, 2005). The process of globalization offers multiple and rapid advantages to developed economies, which are overwhelmingly concentrated in world capital and where large transnational companies are headquartered. The process of globalization, in its essence, contains all the premises for widening the economic gaps existing at the end of the twentieth century between developed countries, developing countries, including former socialist states, because it globalizes only the creation of gross domestic products, without globalizing their distribution. Today, "being local in a globalized world is a behavior of social inadequacy and degradation." For globalization to mean progress for all the states of the world, it is necessary for the developed states, which establish the international rules, to avoid understanding the representation of globalization for some and localization for others. Markets tend to be globalized due to the fact that "internal markets can no longer sustain the rising costs of research and development, nor the shorter life cycles of technologically advanced products." (Bari, 2005). The globalization of markets determines the "homogenization of the tastes of consumers around the world" within a market, which acquires a global character. The globalization of markets is directly related to the formation and development of multinational companies and transnational companies. An objective analysis of the globalization process attests that economic benefits are more inclined towards developed countries and the great economic powers where transnational society finds its origin. Globalization, poverty and development inequalities The globalization of the economy can have beneficial effects by facilitating the access of underdeveloped countries to technologies and efficient management, ensuring on the whole an increased economic efficiency, lower costs, more products and services for consumers, new market opportunities (Bergh and Nilsson, 2014). Countries more strongly integrated into the global economy have recorded much higher income convergence than geographically isolated countries. The greatest benefits from DOI: 10.2478/picbe-2021-0037, pp. 406-414, ISSN 2558-9652 \| Proceedings of the 15th International Conference on Business Excellence 2021 PICBE \| 409 globalization have been recorded by developing countries that have integrated into the global economy, those that have chosen to actively participate in the process of globalization. The recent financial crisis reduced the pace of poverty reduction, estimates suggest that the economic crisis pushed 64 million people into extreme poverty by the end of 2010 (Nissanke and Thorbecke, 2010). In East Asia and the Pacific, about 14% of the population lives below $ 1.25 a day, down from 76% in 1990, when it was the region with the highest poverty rate in the world. Figure 1. Peoples living on less than $ 1.25 per day (million people) Source: WorldBank Data. In developing countries, the extreme poverty rate was 25% in 2008, down from 38% in 1990. In South America and the Caribbean, the poverty rate has reached its lowest level so far of 6.5% in 2019, in 1990 being 14%. Regions such as North Africa and The Middle East had 8.6 million people or 2.7% of the population living on less than $ 1.25 a day in 2008, down from 10.5 million in 2005 and 16, 5 million in 1990. The percentage of people living on less than $ 1.25 in 2019 was below 0.5% in Eastern Europe and Central Asia, after reaching 3.8 percent in 2000. In addition to the negative effects of civilization on natural resources and environmental factors, social disparities are the most serious challenges of the 21 st century for international stability and the sustainable development of human society. Global hunger Agriculture and food have proven to be central to both poverty generation and poverty reduction. Malnutrition is a specific feature of poverty and is a direct violation of universal human rights (Desai and Rudra, 2018). From here start all the problems related to the standard of living: health, infant mortality, training of children in the labor force, illiteracy, poor labor productivity. The food problem is not limited to developing countries, in one way or another it affects all countries of the world, including developed ones, hence its global character. DOI: 10.2478/picbe-2021-0037, pp. 406-414, ISSN 2558-9652 \| Proceedings of the 15th International Conference on Business Excellence 2021 PICBE \| 410 Hunger is a mass phenomenon, it is the most critical form of malnutrition, its consequences are catastrophic. They result in loss of life and lead to high health risks throughout life, especially children who usually do not survive. The risk of death for an underweight child, weighing twothirds of normal weight, is ten times higher than for a healthy child. Hungry babies, as well as older children or adults, are prone to infectious diseases because they do not have the energy to fight them. Hungry children are marked for life due to neurological disorders and delayed physical growth. The problem of hunger has worsened in recent years due to rising food prices, which affects the poor as they cannot adapt to rapid change and cannot adjust their budget, eventually resorting to lower quality food or giving up products. basic in daily nutrition. Figure 2. Number of malnourished people in the world 1996-2010 (millions of people) Source: FAO Data- 2010-2019. According to the FAO, there has been a certain decline in the number of undernourished people in recent decades. In 2019, 925 million people were registered in a state of chronic hunger, compared to 844 million people in the period 1990-1992. A total of 921 million people are malnourished in 2010, compared to 1.23 billion in 2009, although almost one in six people suffer from hunger, which is still higher than before the food and economic crises in 2008-2009 and above the level that existed when world leaders agreed to halve the number of people suffering from hunger. The explanation for this decline is explained by a favorable economic environment in 2010, especially in emerging countries, as well as the fall in food prices from 2008 to the present. Of the malnourished, 98% live in developing countries and two-thirds live in just seven countries (Bangladesh, China, the Democratic Republic of the Congo, Ethiopia, India, Indonesia and Pakistan), and over 40% live in China and India. DOI: 10.2478/picbe-2021-0037, pp. 406-414, ISSN 2558-9652 \| Proceedings of the 15th International Conference on Business Excellence 2021 PICBE \| 411 PICBE \| 412 Figure 3. Undernourished people in 2019 by region (millions) Source: FAO Data 2010-2019. The most malnourished region continues to be Asia and the Pacific, but down 12 percent from 658 million people in 2009 to 578 million, followed by South Saharan Africa (239 million), Latin America and the Caribbean-53 million), and the North Africa and Middle East (37 million). Conclusions Long discussed and analyzed, globalization has today become an objective phenomenon, unprecedented in universal history, through its magnitude and evolution. Criticized by some, glorified by others, globalization is following in its footsteps. This controversial process consists, in essence, in the stronger integration of countries and their populations as a result of the significant reduction of transport and communication costs and the elimination of barriers to the movement of goods, services, capital, knowledge and people between states. Globalization can be a force for good: it has brought hundreds of millions of people to a higher standard of living; the globalization of the economy has benefited the countries that have taken advantage of it by identifying new export markets and attracting foreign investment. The growth of international trade, massive international financial flows, and the activities of multinational corporations are increasingly closely linked to each other's world economies, making globalization a controversial feature of the world economy. Thanks to a complicated network of mechanisms, processes, such as the rapid unification of markets and capital and technological processes, accelerated by the combination of computers, telecommunications satellites and submarine and fiber optic cables, globalization has reached its highest stage today. We are currently witnessing a global reconfiguration in which US supremacy is gradually giving way to new powers rising at the beginning of this 21st century. The new foreshadowed global order does not lead to an anti-American world, the US still dominating the economy and geopolitics of tomorrow's world. We can speak, rather, of a post-American world, in which the G20 member DOI: 10.2478/picbe-2021-0037, pp. 406-414, ISSN 2558-9652 \| Proceedings of the 15th International Conference on Business Excellence 2021 countries (Group of 20) will consolidate the future multipolarity, and the G2 formed by the USA and China will mean progress or the new world division. China's growing role in global economics and geopolitics forces us to look at China from a new perspective: an emerging world power capable of influencing the effects and duration of the economic crisis and a world power that plays an important role in the world geopolitical scene with consequences that are difficult to predict and at the same time difficult to accept. References Androniceanu, A., Burlacu, S., Drăgulănescu, I. V., & Nicolae, E. E. (2017, May). New trends of businesses digitalization in Romania and the behaviour of young consumers. In BASIQ International Conference: New Trends in Sustainable Business and Consumption, Graz, Vol. 31, 27-35. Bari, I. (2003). Contemporary global issues, Editura Economica, Bucuresti. Bari, I. (2005). Globalization and global issues, Editura Economica, Bucuresti. Bergh, A., Gustafsson, A. (2019). Globalization and Populism in Europe, SSRN Electronic Journal. Bergh, A., Nilsson, T. (2014). Is globalization reducing absolute poverty? World Development 62, 42-61. Bodislav, D.A., Radulescu, C.V., Bran, F., and Burlacu, S. (2020). Public Policy in the Areas of Environment and Energy. 6th BASIQ International Conference on New Trends in Sustainable Business and Consumption. Messina, Italy, 4-6 June 2020. Bucharest: ASE, 228-235. Bran, F., Rădulescu, C. V., Bodislav, D. A., & Burlacu, S. (2020). Environmental risks in the context of globalization, Economic Convergence in European Union, 350. Brecher, J. (2012). De-centering globalization from below. Social Policy 42(2): 30-34. Burlacu, S., Vasilache, P. C., Velicu, E. R., Curea, Ș. C., & Margina, O. (2020, December). Management of Water Resources at Global Level, In Proceedings of the International Conference on Economics and Social Sciences, 998-1009. Sciendo. Costache, G., Marinas, C. V., Igret, R., & Burlacu, S. (2015). Internship in the Hr Department– Organizational and Individual Perspectives, In Proceedings of the International Management Conference, 9(1), 359-370, Faculty of Management, Academy of Economic Studies, Bucharest, Romania. Desai, R. M, Rudra, N. (2018). Trade, poverty, and social protection in developing countries, European Journal of Political Economy. Dima, C., Burlacu, S., & Buzoianu, O. A. C. (2020). Strategic Options for the Development of Ecotourism in the Danube Delta in the Context of Globalization, In SHS Web of Conferences, Vol. 74, 04005. EDP Sciences. Dinu, M. (2004). Globalizarea si aproximarile ei, Editura Economica, Bucuresti. Dobrescu, P. (2010). Viclenia globalizarii, Institutul European, Iasi. Held, D. (2016). Elements of a theory of global governance, Philosophy & Social Criticism, 42(9), 837-846. Holton, R. J. (2005). Making Globalisation, Basingstoke, Palgrave Macmilla. Ioniță, F., & Burlacu, S. (2009). Public Administration from Romania in the Knowledge Society and E-Learning, In Proceedings of the Fifth" Administration and Public Management" International Conference:" Public Institutions' Capacity to Implement the Administrative Reform Process", Bucharest, June 23-24, No. 25. DOI: 10.2478/picbe-2021-0037, pp. 406-414, ISSN 2558-9652 \| Proceedings of the 15th International Conference on Business Excellence 2021 PICBE \| 413 Ioniţă, F., Burlacu, S., & Gaidargi, A. (2009). Modern Approaches of the Management of Alternative Trade Systems, Revista de Management Comparat Internațional / Review of International Comparative Management, 51, 473-480. Negescu, M. D., Burlacu, S., Mitriţă, M., Buzoianu, O. C. A. (2020). Managerial Analysis of Factoring at the International Level Challenges of the Contemporary Society. Proceedings; Cluj-Napoca, 13(1), 99-102. Cluj-Napoca: Babes Bolyai University. Nissanke, M., Thorbecke, E. (2010). Globalization, poverty, and inequality in Latin America: Findings from case studies, World Development 38(6), 797-802 Pieterse, J. N. (2015). Globalization and Culture: Cultural Melange, 3rd edn. Lanham, MD, Rowman & Littlefield. 13. Radulescu D., (2007). Probleme globale contemporane, Editura Universitatii Petrol-Gaze. Rădulescu, C. V., Bran, F., Burlacu, S., Dobrea, C. R., & Diaconu, S. (2020, December). Challenges Regarding Food Resources in the Context of Globalization and Population Growth, In Proceedings of the International Conference on Economics and Social Sciences, 1041-1052, Sciendo. Rădulescu, C. V., Burlacu, S., Bodislav, D. A., & Bran, F. (2020). Entrepreneurial Education in the Context of the Imperative Development of Sustainable Business, European Journal of Sustainable Development, 9(4), 93-93. Radulescu, C.V., Ladaru, G.-R., Burlacu, S., Constantin, F., Ioanăș, C., Petre, I.L. (2021). Impact of the COVID-19 Pandemic on the Romanian Labor Market, Sustainability 2021, 13, 271. https://doi.org/10.3390/su13010271. Rodrik, D. (2018). Populism and the Economics of Globalization, Journal of International Business Policy, Vol. 1. Stiglitz, J. (2008). Mecanismele globalizarii, Editura Polirom, Iasi. DOI: 10.2478/picbe-2021-0037, pp. 406-414, ISSN 2558-9652 \| Proceedings of the 15th International Conference on Business Excellence 2021 PICBE \| 414
https://openalex.org/W4283079174	https://www.frontiersin.org/articles/10.3389/fncel.2022.876805/pdf	English	null	Vestibular Hair Cells Require CAMSAP3, a Microtubule Minus-End Regulator, for Formation of Normal Kinocilia	Frontiers in cellular neuroscience	2,022	cc-by	13,987	Vestibular Hair Cells Require CAMSAP3, a Microtubule Minus-End Regulator, for Formation of Normal Kinocilia Josephine O’Donnell1 and Jing Zheng1,2* 1 Department of Otolaryngology, Feinberg School of Medicine, Northwestern University, Chicago, IL, United States, 2 Knowles Hearing Center, Northwestern University, Evanston, IL, United States Josephine O’Donnell1 and Jing Zheng1,2* Josephine O’Donnell1 and Jing Zheng1,2* 1 Department of Otolaryngology, Feinberg School of Medicine, Northwestern University, Chicago, IL, United States, 2 Knowles Hearing Center, Northwestern University, Evanston, IL, United States Kinocilia are exceptionally long primary sensory cilia located on vestibular hair cells, which are essential for transmitting key signals that contribute to mammalian balance and overall vestibular system function. Kinocilia have a “9+2” microtubule (MT) conﬁguration with nine doublet MTs surrounding two central singlet MTs. This is uncommon as most mammalian primary sensory cilia have a “9+0” conﬁguration, in which the central MT pair is absent. It has yet to be determined what the function of the central MT pair is in kinocilia. Calmodulin-regulated spectrin-associated protein 3 (CAMSAP3) regulates the minus end of MTs and is essential for forming the central MT pair in motile cilia, which have the “9+2” conﬁguration. To explore the role of the central MT pair in kinocilia, we created a conditional knockout model (cKO), Camsap3-cKO, which intended to eliminate CAMSAP3 in limited organs including the inner ear, olfactory bulb, and kidneys. Immunoﬂuorescent staining of vestibular organs demonstrated that CAMSAP3 proteins were signiﬁcantly reduced in Camsap3-cKO mice and that aged Camsap3-cKO mice had signiﬁcantly shorter kinocilia than their wildtype littermates. Transmission electron microscopy showed that aged Camsap3-cKO mice were in fact missing that the central MT pair in kinocilia more often than their wildtype counterparts. In the examination of behavior, wildtype and Camsap3-cKO mice performed equally well on a swim assessment, right-reﬂex test, and evaluation of balance on a rotarod. However, Camsap3-cKO mice showed slightly altered gaits including reduced maximal rate of change of paw area and a smaller paw area in contact with the surface. Although Camsap3-cKO mice had no differences in olfaction from their wildtype counterparts, Camsap3-cKO mice did have kidney dysfunction that deteriorated their health. Thus, CAMSAP3 is important for establishing and/or maintaining the normal structure of kinocilia and kidney function but is not essential for normal olfaction. Our data supports our hypothesis that CAMSAP3 is critical for construction of the central MT pair in kinocilia, and that the central MT pair may be important for building long and stable axonemes in these kinocilia. ORIGINAL RESEARCH published: 17 June 2022 doi: 10.3389/fncel.2022.876805 ORIGINAL RESEARCH published: 17 June 2022 doi: 10.3389/fncel.2022.876805 Vestibular Hair Cells Require CAMSAP3, a Microtubule Minus-End Regulator, for Formation of Normal Kinocilia Whether shorter kinocilia might lead to abnormal vestibular function and altered gaits in older Camsap3-cKO mice requires further investigation. Keywords: CAMSAP3, kinocilia, “9+2” conﬁguration, gait, vestibular function, kidney dysfunction INTRODUCTION The actin-based stereocilia and MT-based kinocilia on vestibular hair cells are connected by lateral links and tip links (Figure 3B). Because of their length, the tips of vestibular kinocilia reach into the overlying otoconial layer. This structural arrangement allows vestibular kinocilia to transmit positions and movements of the head by change the otoconial membrane mass to the adjacent stereocilia, which generate electrical signals in sensory hair cells and ultimately controls mammalian balance and awareness of spatial orientation. As such, the mechanical properties and length of vestibular kinocilia play important roles in determining the operating range of the vestibular system (Spoon and Grant, 2011, 2013; Nam, 2018). Unlike most of primary sensory cilia, vestibular kinocilia have the “9+2” conﬁguration, a common conﬁguration found in motile cilia (Wersall et al., 1965; Rosenhall and Engstrom, 1974; Choksi et al., 2014). Unlike the outer 9 MT doublets, the central MT pair is not continuous with MT triplets in the basal body (Lechtreck et al., 2013). In motile cilia, the central MTs and their MT-associated proteins are required for the synchronized motion needed to remove debris (Loreng and Smith, 2017). The function of central MTs in primary cilia remains unknown. Primary cilia on olfactory sensory neurons and vestibular hair cells have the longest axoneme in mammals. Intriguingly, both cilia have the “9+2” conﬁguration, which leads us to suspect that the central MT pair may be essential for the formation of the long axoneme. q CAMSAP3 is expressed ubiquitously in mice. The global KD and global knockout (KO) CAMSAP3 in mice could potentially aﬀect all cells that express CAMSAP3. Indeed, removal of Camsap3 gene from a mouse genome leads to global Camsap3- KO mice dying prematurely (Muroyama et al., 2018). Even the Camsap3-KD mice, which reduce CAMSAP3 expression through RNA processing (Testa et al., 2004; White et al., 2013) without deleting any exons of Camsap3 gene, showed some degree of embryonic lethality, sub-infertility, hydrocephalus, anosmia or hyposmia, sinusitis, respiratory distress, otitis media, and hearing loss (Perez-Garcia et al., 2018; Ingham et al., 2019; Robinson et al., 2020). To investigate the function of CAMSAP3 in a reliable living mouse model, we created a conditional knockout (cKO) mouse model known as Camsap3tm1d/tm1d (Camsap3-cKO for short) for tissue speciﬁc cKO by using Cre- loxP system. Camsap3-cKO would allow us to examine CAMSAP3’s role in vestibular hair cells without inﬂuence from other health issues. INTRODUCTION roles for hearing and balance. The C isoform of CDH23 can directly bind to CAMSAP3 and modiﬁes CAMSAP3-associated MT networks (Takahashi et al., 2016). In addition, CAMSAP3 is involved in the formation of MTs from non-centrosomal MT organizing centers (MTOCs). CAMSAP3-coated MTs are stable and can be used to seed tubulin polymerization at non-centrosomal sites (Jiang et al., 2014, 2018; Zenker et al., 2017). We recently discovered that CAMSAP3 is necessary to form the central MT pairs in motile cilia (Robinson et al., 2020). In the Camsap3tm1a/tm1a mice, the global knockdown (KD) of CAMSAP3 expression is associated with impaired ciliary motion, leading to phenotypes of Primary Ciliary Dyskinesia (PCD), which includes hydrocephalus, subfertility, and impaired mucociliary clearance. Dysfunctional mucociliary clearance leads to hyposmia, anosmia, rhinosinusitis, and otitis media. We occasionally noticed Camsap3tm1a/tm1a mice (Camsap3-KD for short) with a head tilt to one side. Therefore, we suspect that CAMSAP3 is also needed to construct the central MT pair in kinocilia on vestibular hair cells and predict that the central MT pair in vestibular kinocilia is essential to establish the long and stable cilia required for vestibular function. Cilia, found on cell surfaces, are microtubule (MT)-based organelles that play essential roles for cell development, proliferation, diﬀerentiation, migration, signal transduction, etc. The structure, length, and function of cilia must be tightly regulated because their dysfunction is associated with numerous diseases collectively called ciliopathy disorders (Lee and Gleeson, 2011; Falk et al., 2015; Reiter and Leroux, 2017; Andreu-Cervera et al., 2021; Lee and Ostrowski, 2021). Based on their mobility, cilia are divided into two types: motile cilia and non-motile cilia like primary cilia. Motile cilia beat rhythmically to transport ﬂuids across epithelia, while non-motile, as known as primary cilia, serve as sensory organelles gathering information about their environment. The majority of motile cilia in mammals are composed of MTs in the “9+2” conﬁguration, i.e., nine doublet MTs surrounding two central singlet MTs. In contrast, most of the primary sensory cilia in mammals do not have central MT pairs, and instead, their axonemes have a “9+0” conﬁguration. p g Vestibular kinocilia, located on hair cells of the vestibular organ, are primary sensory cilia present throughout the murine lifespan. Compared to other primary sensory cilia, vestibular kinocilia are exceptionally long, some being 3–4 times longer than adjacent microvilli called stereocilia (Xue and Peterson, 2006). Edited by: Bernd Fritzsch, The University of Iowa, United States Reviewed by: Katie Kindt, National Institutes of Health (NIH), United States Kristen Rak, Comprehensive Hearing Center, Germany Correspondence: Jing Zheng jzh215@northwestern.edu Specialty section: This article was submitted to Non-Neuronal Cells, a section of the journal Frontiers in Cellular Neuroscience Received: 15 February 2022 Accepted: 30 May 2022 Published: 17 June 2022 Citation: O’Donnell J and Zheng J (2022) Vestibular Hair Cells Require CAMSAP3, a Microtubule Minus-End Regulator, for Formation of Normal Kinocilia. Front. Cell. Neurosci. 16:876805. doi: 10.3389/fncel.2022.876805 Specialty section: This article was submitted to Non-Neuronal Cells, a section of the journal Frontiers in Cellular Neuroscience Received: 15 February 2022 Accepted: 30 May 2022 Published: 17 June 2022 June 2022 \| Volume 16 \| Article 876805 1 Frontiers in Cellular Neuroscience \| www.frontiersin.org CAMSAP3 Is Important for the Vestibular Kinocilium Formation O’Donnell and Zheng Frontiers in Cellular Neuroscience \| www.frontiersin.org INTRODUCTION TgPax2-Cre mouse line is widely used to establish cKO mouse models for inner ear (Pan et al., 2011; Cox et al., 2012; Wiwatpanit et al., 2018). In this transgenic mouse line, IRES-Cre was inserted into Pax2 (paired box gene 2), a transcription factor that is crucial in the developmental and proliferation of multiple cells and organs. Cre expression was detected at E8.5 in the otic placode in the Pax2-Cre mouse line (Ohyama and Groves, 2004), which is earlier than when hair cell diﬀerentiation typically occurs (around E13.5) (Mbiene et al., 1984). Thus, we created Camsap3-cKO by crossing TgPax2- Cre mice with Camsap3tm1c/tm1c (ﬂ/ﬂ), aiming to eliminate CAMSAP3 expression in Cre-expressed tissues, which include most of the cells in the inner ear, midbrain, cerebellum, the olfactory bulb, and kidneys (Ohyama and Groves, 2004). If CAMSAP3 is important for establishing normal vestibular kinocilia as we predict, we expect Camsap3-cKO mice to show behaviors associated with vestibular dysfunction. Diﬀerent assessments, including Rotarod balance tests, DigiGait analyses, 3 Calmodulin-regulated spectrin-associated protein 3 (CAMSAP3), also called Marshalin (Zheng et al., 2013), is a MT minus-end regulator. Because CAMSAP3 can interact with other proteins through its protein-protein interaction domains, it has been reported to play several regulatory roles through interactions with multiple proteins (Meng et al., 2008; Goodwin and Vale, 2010; Toya et al., 2016; Gibieža et al., 2021). For example, CDH23 is an adhesive protein that plays crucial June 2022 \| Volume 16 \| Article 876805 Frontiers in Cellular Neuroscience \| www.frontiersin.org 2 CAMSAP3 Is Important for the Vestibular Kinocilium Formation O’Donnell and Zheng along with anatomical examination, were used to characterize the vestibular functions of Camsap3-cKO mice. We also evaluated olfactory function and monitored kidney health in order to determine whether removing CAMSAP3 causes detrimental eﬀects outside of our expected outcomes in the vestibular system. Our data suggests that CAMSAP3 has an important role in establishing or maintaining normal vestibular kinocilia and kidney function, but is not essential for the task of olfactory bulbs. step 2–95◦C, 30 s; step 3–55◦C, 30 s, step 4–72◦C, 90 s; repeat steps 2–4, 35 × ; step 5–72◦C, 10 min. Expected sizes were 1,307 base pairs (bp) for WT, and 199 bp for Camsap3-cKO mice. Both male and female mice were used in all experiments. Evaluation of Vestibular Function Swimming Test WT mice and their Camsap3-cKO littermates were placed in a 5-gallon bucket ﬁlled with room temperature water and were required to swim for 3 min. Behavior was observed, and any abnormal behavior was recorded. The swim test protocol was a modiﬁed version of a protocol described previously (Hardisty-Hughes et al., 2010). Right Reﬂex Test We modiﬁed a protocol described previously (Pau et al., 2004) to assess right reﬂexes. WT mice and their Camsap3-cKO littermates were placed in a clear plastic box (1′′ × 2′′ × 5′′). The box was quickly ﬂipped upside down and the time it took each mouse to ﬂip themselves back upright was recorded. p The Camsap3tm1a(EUCOMM) Wtsi (referred to as Camsap3- KD) mouse model was obtained from the Wellcome Trust Sanger Institute. The original Camsap3-KD line on the C57B6N background was re-derived on FVB murine backgrounds to increase their viability (Robinson et al., 2020). As shown in Figure 1A, a Camsap3-KD mouse was created using the “knockout ﬁrst, conditional-ready” strategy (Skarnes et al., 2011). A targeted trap allele was inserted into the intron between the exon 6 and 7 of the Camsap3 gene to knock out Camsap3 expression through RNA processing (Testa et al., 2004; White et al., 2013) without deleting any exons. To create a cKO mouse model, Camsap3-KD mice were ﬁrst crossed with B6-Tg(CAG-FLPe)36 (Kanki et al., 2006) to delete the neo and LacZ cassettes ﬂanked by FRT sites and create Camsap3tm1c (ﬂoxed allele), a strain of pseudo- wildtype (WT) mice. This WT Camsap3tm1c ﬂoxed line was further crossed with the TgPax2Cre mouse line, Tg(Pax2- Cre)1Akg/Mmnc (MMRRC stock number 10569) (Ohyama and Groves, 2004) to create a conditional knockout mouse model: TgPax2Cre/+; Camsap3tm1c/tm1c and their WT littermates: TgPax2Cre+/+;Camsap3tm1c/tm1c. We call this mouse model Camsap3tm1d/tm1d (null allele) Camsap3-cKO for short. All animals were maintained by heterotypic breeding, and the genotypes were determined by detecting the inserted cassettes in mouse tail samples performed by Transnetyx (Cordova, TN, United States). Creation of Camsap3-cKO Mice All experiments utilizing animals were approved by the Institutional Animal Care and Use Committees of Northwestern University and in accordance with the National Institutes of Health guidelines. Animals were housed in Northwestern’s Center for Comparative Medicine facilities. Rotarod Test The rotarod test was created by Dunham and Miya (1957) to assess neuromuscular function in rodents and is commonly used to assess balance (Jones et al., 2018). The Rotarod apparatus (TSE Systems, Chesterﬁeld, MO, United States) was used to assess balance in Camsap3-cKO and their WT littermates. Following the Jones et al. (2018) protocol, mice were placed on a rotating beam and their time to fall (TTF) was recorded. Mice were tested 3 at a time with their cage mates. The rotarod was set to start rotating at a speed of 5 rpm and accelerate to a top speed of 44 rpm over the course of 300 s (the maximum duration of the trial). The mice were given 10-min trial intervals and tested again. Ten Camsap3-cKO mice and 10 WT littermates completed eight trials, with the ﬁrst three trials functioning as an acclimation and familiarization period. The ﬁnal ﬁve trials were recorded for analysis. Gait Analysis Th TM The DigiGaitTM Imaging System apparatus (Mouse Speciﬁcs Inc., Framingham, MA, United States) consists of a motorized transparent treadmill belt sitting above a digital camera which records the ventral view (Supplementary Movie 1). WT mice and their Camsap3-cKO littermates were placed on the treadmill one at a time and were required to run. The mice were then evaluated at treadmill speeds of 10, 17, and 24 cm/s. The DigiGait assay was run by Northwestern Behavioral Phenotyping Core. The DigiGait Imaging System software allowed us to examine 52 diﬀerent aspects of each mouse’s gait (Rostosky and Milosevic, 2018). The analysis software can detect slight diﬀerences in angles, lengths, and speeds of the paws and strides of the mice. The key behaviors we were interested in, shown in Supplementary Figures 1, 2, included: swing to stance ratio, paw angle, gait symmetry, stance width, step angle, breaking duration, stride length, the ataxia Since Cre expression was limited in a few tissues including the inner ear, olfactory bulb, and kidneys of TgPax2Cre mice, the exon 7 of camsap3 was expected to be fully removed from these tissues of Camsap3-cKO mice. To verify the exon 7 exclusion, genomic DNA was puriﬁed from the inner ear of WT and Camsap3-cKO mice using the QIAamp DNA FFPE Tissue Kit (Qiagen). PCR was used to verify the exon 7 exclusion using the PCR Master Mix (Thermo Fisher Scientiﬁc, A44647100) with forward primer L1L2-BactP-MD-F (GCTGGCGCCG GAACC) and reverse primer C3Floxed5-R (TTGGCCTGGGGAACATGAC). Conditions for the thermal cycler were: step 1–95◦C, 2 min; June 2022 \| Volume 16 \| Article 876805 Frontiers in Cellular Neuroscience \| www.frontiersin.org 3 CAMSAP3 Is Important for the Vestibular Kinocilium Formation O’Donnell and Zheng FIGURE 1 \| Creation and validation of the Camsap3-cKO mouse. (A) A schematic diagram of the Camsap3 knockout ﬁrst targeting strategy and conversion of tm1a (KO ﬁrst) to tm1c (ﬂoxed), and tm1d (cKO) alleles, respectively. TgPax2Cre/+; Camsap3tm1c/tm1c mice, known as Camsap3-cKO, are a cKO model for the vestibular organ. (B) The exon 7 of Camsap3 execution was validated by PCR genotyping. The inner ear genomic DNA of offspring derived from crossing TgPax2Cre and Camsap3tm1c/tm1c were isolated and used for PCR (n = 9 from two separate litters). No DNA and ﬂoxed allele Camsap3tm1c/tm1c were used as the negative and positive controls. The bands were expected to be 1307 bp for WT (TgPax2Cre+/+; Camsap3tm1c/tm1c) and 199 bp for Camsap3-cKO mice. Gait Analysis Th TM (C,D) Representative immunoﬂuorescent images of a crista from WT (C) and Camsap3-cKO (D) mouse at P75 were shown. The dashed line outlines the edge of the crista of a Camsap3-cKO mouse. Scale Bars = 50 µm. (E) Vestibular cells from Camsap3-cKO mice had signiﬁcantly less CAMSAP3 staining than their WT counterparts. The bars represent mean ± SD. Statistically signiﬁcant difference (p = 0.03). WT and Camsap3-cKO mice littermate (P75) replicates (n) were as indicated. (F,G) Representative confocal maximum projection of z-stack images taken from whole-mounts utricle samples of a WT (F) and a Camsap3-cKO mouse (G). More CAMSAP3 staining dots were found on the apical surface of WT utricle compared to those on Camsap3-cKO mouse. Scale Bars = 10 µm. Antibodies: anti-Camsap3 (Green), phalloidin (Red). coeﬃcient, paw area, and the maximum change in paw area (maximum dA/dt). were cryosectioned before staining. These samples were ﬁrst decalciﬁed in 0.125M EDTA for 2 days. Decalciﬁed samples were then placed in a series of sucrose solutions in 1X PBS (10–30%) then two changes of Tissue-Tek Optimal Cutting Temperature (OCT) Embedding Medium (Sakura Finetek, 4583), and embedded in fresh OCT. The inner ear samples were then sectioned into 12 micrometer thick slices, and post-ﬁxed in 2% formaldehyde for 10 min and blocked at room temperature for 1 h in blocking solution: 5% goat serum, 2% Triton X-100 in Tris-buﬀered saline (TBS). The following primary antibodies were used for immunostaining: anti-Camsap3 (Robinson et al., 2020) at 1:2500, anti-acetylated tubulin at 1:500 (Thermo Fisher Gait Analysis Th TM (C–G) CAMSAP3 protein expression in the vestibular system as examined by immunoﬂuorescence. (C,D) Representative immunoﬂuorescent images of a crista from WT (C) and Camsap3-cKO (D) mouse at P75 were shown. The dashed line outlines the edge of the crista of a Camsap3-cKO mouse. Scale Bars = 50 µm. (E) Vestibular cells from Camsap3-cKO mice had signiﬁcantly less CAMSAP3 staining than their WT counterparts. The bars represent mean ± SD. Statistically signiﬁcant difference (p = 0.03). WT and Camsap3-cKO mice littermate (P75) replicates (n) were as indicated. (F,G) Representative confocal maximum projection of z-stack images taken from whole-mounts utricle samples of a WT (F) and a Camsap3-cKO mouse (G). More CAMSAP3 staining dots were found on the apical surface of WT utricle compared to those on Camsap3-cKO mouse. Scale Bars = 10 µm. Antibodies: anti-Camsap3 (Green), phalloidin (Red). FIGURE 1 \| Creation and validation of the Camsap3-cKO mouse. (A) A schematic diagram of the Camsap3 knockout ﬁrst targeting strategy and conversion of tm1a (KO ﬁrst) to tm1c (ﬂoxed), and tm1d (cKO) alleles, respectively. TgPax2Cre/+; Camsap3tm1c/tm1c mice, known as Camsap3-cKO, are a cKO model for the vestibular organ. (B) The exon 7 of Camsap3 execution was validated by PCR genotyping. The inner ear genomic DNA of offspring derived from crossing TgPax2Cre and Camsap3tm1c/tm1c were isolated and used for PCR (n = 9 from two separate litters). No DNA and ﬂoxed allele Camsap3tm1c/tm1c were used as the negative and positive controls. The bands were expected to be 1307 bp for WT (TgPax2Cre+/+; Camsap3tm1c/tm1c) and 199 bp for Camsap3-cKO mice. (C–G) CAMSAP3 protein expression in the vestibular system as examined by immunoﬂuorescence. (C,D) Representative immunoﬂuorescent images of a crista from WT (C) and Camsap3-cKO (D) mouse at P75 were shown. The dashed line outlines the edge of the crista of a Camsap3-cKO mouse. Scale Bars = 50 µm. (E) Vestibular cells from Camsap3-cKO mice had signiﬁcantly less CAMSAP3 staining than their WT counterparts. The bars represent mean ± SD. Statistically signiﬁcant difference (p = 0.03). WT and Camsap3-cKO mice littermate (P75) replicates (n) were as indicated. (F,G) Representative confocal maximum projection of z-stack images taken from whole-mounts utricle samples of a WT (F) and a Camsap3-cKO mouse (G). More CAMSAP3 staining dots were found on the apical surface of WT utricle compared to those on Camsap3-cKO mouse. Scale Bars = 10 µm. Antibodies: anti-Camsap3 (Green), phalloidin (Red). Gait Analysis Th TM FIGURE 1 \| Creation and validation of the Camsap3-cKO mouse. (A) A schematic diagram of the Camsap3 knockout ﬁrst targeting strategy and conversion of tm1a (KO ﬁrst) to tm1c (ﬂoxed), and tm1d (cKO) alleles, respectively. TgPax2Cre/+; Camsap3tm1c/tm1c mice, known as Camsap3-cKO, are a cKO model for the vestibular organ. (B) The exon 7 of Camsap3 execution was validated by PCR genotyping. The inner ear genomic DNA of offspring derived from crossing TgPax2Cre and Camsap3tm1c/tm1c were isolated and used for PCR (n = 9 from two separate litters). No DNA and ﬂoxed allele Camsap3tm1c/tm1c were used as the negative and positive controls. The bands were expected to be 1307 bp for WT (TgPax2Cre+/+; Camsap3tm1c/tm1c) and 199 bp for Camsap3-cKO mice. (C–G) CAMSAP3 protein expression in the vestibular system as examined by immunoﬂuorescence. (C,D) Representative immunoﬂuorescent images of a crista from WT (C) and Camsap3-cKO (D) mouse at P75 were shown. The dashed line outlines the edge of the crista of a Camsap3-cKO mouse. Scale Bars = 50 µm. (E) Vestibular cells from Camsap3-cKO mice had signiﬁcantly less CAMSAP3 staining than their WT counterparts. The bars represent mean ± SD. Statistically signiﬁcant difference (p = 0.03). WT and Camsap3-cKO mice littermate (P75) replicates (n) were as indicated. (F,G) Representative confocal maximum projection of z-stack images taken from whole-mounts utricle samples of a WT (F) and a Camsap3-cKO mouse (G). More CAMSAP3 staining dots were found on the apical surface of WT utricle compared to those on Camsap3-cKO mouse. Scale Bars = 10 µm. Antibodies: anti-Camsap3 (Green), phalloidin (Red). FIGURE 1 \| Creation and validation of the Camsap3-cKO mouse. (A) A schematic diagram of the Camsap3 knockout ﬁrst targeting strategy and conversion of tm1a (KO ﬁrst) to tm1c (ﬂoxed), and tm1d (cKO) alleles, respectively. TgPax2Cre/+; Camsap3tm1c/tm1c mice, known as Camsap3-cKO, are a cKO model for the vestibular organ. (B) The exon 7 of Camsap3 execution was validated by PCR genotyping. The inner ear genomic DNA of offspring derived from crossing TgPax2Cre and Camsap3tm1c/tm1c were isolated and used for PCR (n = 9 from two separate litters). No DNA and ﬂoxed allele Camsap3tm1c/tm1c were used as the negative and positive controls. The bands were expected to be 1307 bp for WT (TgPax2Cre+/+; Camsap3tm1c/tm1c) and 199 bp for Camsap3-cKO mice. (C–G) CAMSAP3 protein expression in the vestibular system as examined by immunoﬂuorescence. Immunoﬂuorescence and Microscopy The inner ears, olfactory bulbs, and olfactory mucosa in nasal cavities were collected from WT mice and their Camsap3- cKO littermates for immunoﬂuorescence as previously described (Sekerka et al., 2011; Robinson et al., 2020). Brieﬂy, samples were immersion ﬁxed in 4% formaldehyde in phosphate buﬀered solution (PBS). Some samples underwent whole mount preparation, in which the utricles and cristae were further dissected out and used for immunostaining. Other samples June 2022 \| Volume 16 \| Article 876805 Frontiers in Cellular Neuroscience \| www.frontiersin.org 4 CAMSAP3 Is Important for the Vestibular Kinocilium Formation O’Donnell and Zheng Scientiﬁc 32-2700, AB_2533073). Samples were incubated with primary antibodies at 4◦C overnight. The next day samples were then washed in PBS and incubated with appropriate ﬂuorophore-conjugated secondary antibodies, including goat anti-rabbit Alexa 488 at 1:500 (Thermo Fisher Scientiﬁc 32- 2700, AB_143165), goat anti-mouse Alexa Fluor 488 (RRID: AB_2556548), goat anti-mouse IgG2b Alexa 647 at 1:500 (Thermo Fisher Scientiﬁc AB_143165), Hoechst at 1:1000 (Thermo Fisher Scientiﬁc, H3570), and Phalloidin-Alexa 568 at 1:2000 (Thermo Fisher Scientiﬁc 21838, AB_2532159), or Phalloidin-Alexa 647 at 1:400 (Thermo Fisher Scientiﬁc A22287) for 2.5 h at room temperature. The stained samples were subsequently mounted in Fluoromount Aqueous Mounting Medium (Sigma, F4680). When mounting the whole mount utricle samples, a 0.12 mm deep Secure-Seal spacer (Thermo Fisher Scientiﬁc, S24735) was placed between the slide and the cover glass in order to avoid destruction of the vestibular kinocilia on hair cells. Immunostained samples were imaged using a Nikon C2 or A1R+ confocal microscope using objects of a plan Apo VC 20×/0.75 DIC N2 (Nikon) and a plan Apo 60×/1.4 oil (Nikon). All images were taken as 12-bit, which allows for 0–4,095 range. For vestibular kinocilia length measurement, Z-stacks images were captured from the apical surface of utricle hair cells to the tip of vestibular kinocilia (from the position “A” extended to “C-D” shown in Figure 3B) using the optical section (0.5 µm). These Z-stack images were then reconstructed into 3D images, which were analyzed using Imaris 8 (Bitplane) and Nikon NIS Element software. Mean gray value, the sum of the gray values of all the pixels in the selection divided by the number of pixels, was measured using FIJI. at 9 AM. The base of a Sterilite translucent storage box (cage) [47 cm (L) × 37.8 cm (W) × 28.3 cm (H)] was ﬁlled with bedding to a depth of 3 cm. Immunoﬂuorescence and Microscopy Each mouse was placed in an acclimation bin which was identical to the testing bin in every way, with the exception of no food being present, and were given 5 min to adjust to the enclosure. Mice were then transferred individually to the testing cage, which had ∼0.5 g of Nutter Butter cookie hidden in the center of the cage under the bedding. As soon as the mouse entered the testing bin a stopwatch was started and the latency to ﬁnd (LTF) the cookie was recorded. Following the experiment, mice were returned to their home cages and given their full allotment of food. Transmission Electron Microscopy py Utricles from WT mice and their Camsap3-cKO littermates were dissected out and immersion ﬁxed in 0.1 M sodium cacodylate buﬀer pH 7.3, containing 2% paraformaldehyde and 2.5% glutaraldehyde for at least 24 h and post-ﬁxed with 2% osmium tetroxide followed by 3% uranyl acetate. Tissues were then dehydrated in ascending grades of ethanol, transitioned with propylene oxide and embedded in resin mixture from the Embed 812 kit, cured in a 60◦C oven. Samples were sectioned on a UCT ultramicrotome (Leica Microsystems). To identify regions of interest 1 µm thick sections were collected, stained with Toluidine Blue O and examined by light microscopy. 70 nm tissue sections were collected on 200 mesh copper grids and stained with uranyl acetate and Reynolds lead citrate, and examined using a FEI Tecnai G2 Spirit Transmission Electron Microscope. The number of MTs in kinocilium was counted and sorted by structure: “9+2,” “9+0,” “8+2,” or “8+0.” The ﬁrst number indicates the numbers of MT doublets outlying, and the second number implies the amount of MTs in the center. RESULTS Camsap3-cKO Is a Camsap3 Conditional Knockout Model for the Vestibular Organ As previously reported, the global KD mice, Camsap3-KD, were born with smaller bodies along with sub-infertility, hydrocephalus, anosmia or hyposmia, sinusitis, respiratory distress, otitis media, and hearing loss (Perez-Garcia et al., 2018; Ingham et al., 2019; Robinson et al., 2020). In contrast to Camsap3-KD, Camsap3-cKO had normal fertility and a normal appearance comparable to their WT littermates regardless of their sex prior to 4 months of age, The normal appearance of Camsap3-cKO was not surprising as Camsap3 was expected to be eliminated only in the inner ear, midbrain, cerebellum, olfactory bulb, and the kidneys (Ohyama and Groves, 2004). To test whether the exon 7 of Camsap3 was indeed removed from the genome of aﬀected cells, we performed PCR genotyping using genomic DNA isolated from the inner ear, where Cre was expressed to remove the ﬂoxed exon 7 of Camsap3. As shown in Figure 1B, in all Camsap3-cKO mice (Pax2- Cre positive), the expected short fragments (199 bps) were detected, but not in mice without the Pax2-Cre allele (Pax2- Cre negative) or original homozygous Camsap3tm1c mice. We noted that the long PCR band, 1307 bp, was also detected in Camsap3-cKO mice with low intensity. As we did not perform cardiac perfusion before extracting genomic DNA from the inner ear, we suspected that cells from the circular bloodstream in the inner ear could contribute to the long PCR bands as the genomic DNA from these cells were not aﬀected by Pax2-Cre expression. It is also possible that the exon 7 of Camsap3 was not completely removed from all cells in the inner ear. To verify whether Camsap3 was expressed in the vestibular hair cells, we performed immunoﬂuorescence using vestibular organs collected from Camsap3-cKO and their WT littermates, with the ages of postnatally day (P) 23 to 260. In WT, CAMSAP3 expression was found in the vestibular organ including the hair cells in cristae (Figure 1C), utricle (Figure 1F), and saccule (data not shown). CAMSAP3 expression in Camsap3-cKO was absent or reduced in vestibular cells from Camsap3-cKO mice as shown in Figures 1D,G. CAMSAP3 staining was expressed in both hair cells and their surrounding supporting cells in vestibular organs from WT samples. CAMSAP3 signals were more concentrated in the apical cortical area of the WT vestibular organ (Figure 1C). Evaluation of Olfaction The Buried Food Test was used to evaluate olfaction in mice (Yang and Crawley, 2009; Robinson et al., 2020). WT mice and their Camsap3-cKO littermates were food restricted but provided water ad libitum for 24 h prior to testing. Mice were given a piece of a Nutter Butter cookie during this day to familiarize the mice with its scent. Testing was performed during daylight, starting Frontiers in Cellular Neuroscience \| www.frontiersin.org June 2022 \| Volume 16 \| Article 876805 5 CAMSAP3 Is Important for the Vestibular Kinocilium Formation O’Donnell and Zheng Our previous work demonstrated that CAMSAP3 was needed for de novo formation of the central MT pair in a “9+2” conﬁguration observed in the axons of motile cilia (Robinson et al., 2020). To test whether CAMSAP3 was also needed for formation of the central MT pair in the kinocilia on vestibular hair cells, we ﬁrst examined the connection between CAMSAP3 and basal bodies by immunoﬂuorescence. The basal bodies that extend to 9 peripheral MT doublets were labeled by anti-γ-tubulin, and phalloidin was used to label the actin-based stereocilia that were adjacent to the kinocilium. As shown in Figure 3A, a group of Z-stack images taken from the apical surface of utricle hair cells (the position “A” shown in Figure 3B) showed two vestibular kinocilia labeled by γ-tubulin (arrows). A stereocilia bundle was next to one of kinocilia, indicating these cells were hair cells from an utricle. Similar to other epithelial cells, CAMSAP3 punctuates (green) were found on the apical surface of hair cells. Two CAMSAP3-stained green dots (arrows) were colocalized with two basal bodies (red dots). As the focus pointing moved step by step (0.5 µm/per section) from the apical surface toward the nucleus, green CAMSAP3 dots (arrows in Figure 3A) in both vestibular kinocilia disappeared before the red basal bodies punctuates, suggesting that CAMSAP3 was at the base of vestibular kinocilia just above the basal bodes, as illustrated in Figure 3B. Such a close connection between CAMSAP3 and basal bodies was of particular interest because CAMSAP3 is not a centrosome- or basal bodies-bound protein. In fact, CAMSAP3 was not observed in the basal bodies of primary cilia with “9+0” MT conﬁguration (Robinson et al., 2020). Evaluation of Olfaction More importantly, the close association between γ-tubulin and CAMSAP3 was comparable to that observed in motile cilia with “9+2” MT conﬁguration (Robinson et al., 2020; Saito et al., 2021), suggesting that CAMSAP3 was also involved in de novo formation of the central MT pair in the kinocilia on the vestibular hair cells. Such distribution patterns are unsurprising as CAMSAP3 is needed at apical cortical area of epithelial cells to establish the intercellular connection and orient the apical-to-basal polarity of microtubule arrays (Meng et al., 2008; Toya et al., 2016). We further quantiﬁed CAMSAP3 staining intensities in the apical cortical area that included both vestibular hair cells and supporting cells. The mean gray value (intensity) of CAMSAP3 staining in these areas were measured using FIJI software. As shown in Figure 1E, CAMSAP3 expression was signiﬁcantly decreased in the hair cell area and supporting cells from Camsap3-cKO mice, with CAMSAP3 expression in Camsap3- cKO mice equaling 48.74% of expression in WT mice at P75 (p = 0.031, unpaired t-test, n = 4). In sum, we created a cKO model that signiﬁcantly reduced CAMSAP3 proteins in the vestibular system. Camsap3-cKO Mice Have Abnormal Kinocilia on Vestibular Hair Cells To investigate CAMSAP3’s role in establishing kinocilia on the vestibular hair cells, utricles collected from Camsap3-cKO mice (3 male) and their WT littermates (3 male) around 1- year old (P296-P384) were examined using immunoﬂuorescent staining of anti-acetylated tubulin and phalloidin, which labeled vestibular kinocilia (green) and stereocilia (red, dash lines), respectively (Figure 2A). We measured the length of kinocilia on vestibular hair cells of utricles from Camsap3-cKO mice and their WT littermates at the most anterior portion for consistency (Figure 2B). From each mouse sample, the length of kinocilium was measured from base to tip in micrometers. Great care was taken to exclude vestibular kinocilia whose full length was not captured in the conﬁnes of the reconstructed 3D image. The average length of the vestibular kinocilia, in mean ± SD, were 20.39 µm ± 3.21 for WT and 17.16 µm ± 3.03 for Camsap3- cKO (Figure 2C). An unpaired t-test revealed a signiﬁcant reduction in vestibular kinocilia length in Camsap3-cKO mice when compared with their WT littermates (p < 0.0001). To further verify the role of CAMSAP3 in formation of the central MT pair in vestibular kinocilia, the MT structure FIGURE 2 \| Kinocilia on utricles hair cells from Camsap3-cKO are shorter than their WT littermates. (A) A representative 3D-reconstruction from z-stacks images of bundles of stereocilia (red) and kinocilia (green) located on the apical surface of hair cells from an utricle of a WT mouse (P386). Antibodies: anti-acetylated tubulin (Green), phalloidin (Red). The dashed line outlines the edge of actin-based stereocilia bundles. Scale Bar = 10 µm. (B) A schematic diagram showing the region over which Z-stack images of utricle hair cells were collected. (C) Quantiﬁcation of kinocilia length collected from male WT (n = 3) and male Camsap3-cKO littermates (n = 3) with ages ranging from 10- to 13-month. Each dot represented one kinocilium. Bars represent mean ± SD. Kinocilia on utricles hair cells from Camsap3-cKO were statistically signiﬁcant shorter than WT (p < 0.0001). Statistically signiﬁcant difference. FIGURE 2 \| Kinocilia on utricles hair cells from Camsap3-cKO are shorter than their WT littermates. (A) A representative 3D-reconstruction from z-stacks images of bundles of stereocilia (red) and kinocilia (green) located on the apical surface of hair cells from an utricle of a WT mouse (P386). Antibodies: anti-acetylated tubulin (Green), phalloidin (Red). The dashed line outlines the edge of actin-based stereocilia bundles. Scale Bar = 10 µm. Camsap3-cKO Mice Have Abnormal Kinocilia on Vestibular Hair Cells (B) A schematic diagram showing the region over which Z-stack images of utricle hair cells were collected. (C) Quantiﬁcation of kinocilia length collected from male WT (n = 3) and male Camsap3-cKO littermates (n = 3) with ages ranging from 10- to 13-month. Each dot represented one kinocilium. Bars represent mean ± SD. Kinocilia on utricles hair cells from Camsap3-cKO were statistically signiﬁcant shorter than WT (p < 0.0001). Statistically signiﬁcant difference. FIGURE 2 \| Kinocilia on utricles hair cells from Camsap3-cKO are shorter than their WT littermates. (A) A representative 3D-reconstruction from z-stacks images of bundles of stereocilia (red) and kinocilia (green) located on the apical surface of hair cells from an utricle of a WT mouse (P386). Antibodies: anti-acetylated tubulin (Green), phalloidin (Red). The dashed line outlines the edge of actin-based stereocilia bundles. Scale Bar = 10 µm. (B) A schematic diagram showing the region over which Z-stack images of utricle hair cells were collected. (C) Quantiﬁcation of kinocilia length collected from male WT (n = 3) and male Camsap3-cKO littermates (n = 3) with ages ranging from 10- to 13-month. Each dot represented one kinocilium. Bars represent mean ± SD. Kinocilia on utricles hair cells from Camsap3-cKO were statistically signiﬁcant shorter than WT (p < 0.0001). Statistically signiﬁcant difference. June 2022 \| Volume 16 \| Article 876805 Frontiers in Cellular Neuroscience \| www.frontiersin.org 6 CAMSAP3 Is Important for the Vestibular Kinocilium Formation O’Donnell and Zheng IGURE 3 \| CAMSAP3 contributes to the formation of central MT pairs in axonemes of kinocilia on utricle hair cells. (A) The overlaps between CAMSAP3 (green) and asal bodies (red) were displayed by a group of consecutive Z-stack kinocilia images taken from a whole mount utricle sample of WT (P42). Z-stack images were aptured using the optical section (0.5 µm) starting from the apical surface of utricle hair cells toward its nucleus. Antibodies: anti-CAMSAP3 (Green), anti-γ-tubulin ed), phalloidin (white). Scale Bars = 5 µm. Two basal bodies of kinocilia were indicated by arrows. (B) A schematic diagram illustrates the region over which Z-stack mages of kinocilia immunostaining A, and TEM images C-D were collected. (C,D) Representative TEM images show transverse sections of kinocilia on utricle hair ells from 8-month-old WT with a “9+2” conﬁguration (black arrow) (C), and a “9+0” conﬁguration (black arrow) for their Camsap3-cKO littermates (D). Scale ars = 1 µm. Camsap3-cKO Mice Have Abnormal Kinocilia on Vestibular Hair Cells Our data suggests that vestibular kinocilia of Camsap3-cKO were more likely missing the central MT pair than WT vestibular kinocilia. Kinocilia are known to be essential for establishing the orientation of stereocilia bundles on hair cells. Data collected from P23 to 13-month showed that vestibular kinocilia were present on the vestibular hair cells of Camsap3-cKO mice just as their WT littermates. In addition, the shape and orientation of stereocilia on vestibular hair cells from Camsap3- cKO were quantitatively similar to those on WT mice despite the vestibular kinocilia from Camsap3-cKO mice were shorter and more likely to miss the central MT pair (Figures 2, 3). We also investigated the impact of CAMSAP3 on the orientation of stereocilia bundles by comparing auditory hair cells from WT and Camsap3-cKO mice with matched ages and sexes. As shown in Supplementary Figure 3, the orientation of stereocilia bundles on auditory hair cells from Camsap3-cKO diﬀerences between WT and Camsap3-cKO vestibular kinocilia were then evaluated using TEM (Figures 3C–E). We examined 42 axoneme structures (the position “C-D” shown in Figure 3B) from 8-month-old WT mice and 33 axonemes from their Camsap3-cKO littermates. The “9+2” conﬁguration here includes 9 MT doublets peripherally and two MT singlets or amorphous material with higher electron density (darker) at the center of axonemes. This is demonstrated in a transverse section of WT vestibular kinocilia (Figure 3C, indicated by an arrow). The “9+0” conﬁguration lacks the central MTs structure and electron-dense materials as seen in the Camsap3-cKO image in Figure 3D. More than 71% of vestibular kinocilia from WT mice have “9+2” conﬁguration, while only 24% vestibular kinocilia from Camsap3-cKO mice showed similar structure. The majority of Camsap3-cKO vestibular kinocilia (55%) have the “9+0” conﬁguration. In addition, irregular conﬁgurations, “8+2” and “8+0,” were found in both WT (12%) and Camsap3-cKO (21%). Distribution of the MT conﬁgurations between WT and KO was statistically signiﬁcant diﬀerent as analyzed using a Kolmogorov- Smirnov distribution comparison test (p = 0.0003). Our data suggests that vestibular kinocilia of Camsap3-cKO were more likely missing the central MT pair than WT vestibular kinocilia. Kinocilia are known to be essential for establishing the orientation of stereocilia bundles on hair cells. Data collected from P23 to 13-month showed that vestibular kinocilia were present on the vestibular hair cells of Camsap3-cKO mice just as their WT littermates. Camsap3-cKO Mice Have Abnormal Kinocilia on Vestibular Hair Cells (E) The distribution of MT arrangements for both WT and Camsap3-cKO mice was compared. Distribution of the MT conﬁgurations between WT and O was signiﬁcantly different as analyzed by a Kolmogorov–Smirnov test (p = 0.0003). FIGURE 3 \| CAMSAP3 contributes to the formation of central MT pairs in axonemes of kinocilia on utricle hair cells. (A) The overlaps between CAMSAP3 (green) and basal bodies (red) were displayed by a group of consecutive Z-stack kinocilia images taken from a whole mount utricle sample of WT (P42). Z-stack images were captured using the optical section (0.5 µm) starting from the apical surface of utricle hair cells toward its nucleus. Antibodies: anti-CAMSAP3 (Green), anti-γ-tubulin (red), phalloidin (white). Scale Bars = 5 µm. Two basal bodies of kinocilia were indicated by arrows. (B) A schematic diagram illustrates the region over which Z-stack images of kinocilia immunostaining A, and TEM images C-D were collected. (C,D) Representative TEM images show transverse sections of kinocilia on utricle hair cells from 8-month-old WT with a “9+2” conﬁguration (black arrow) (C), and a “9+0” conﬁguration (black arrow) for their Camsap3-cKO littermates (D). Scale Bars = 1 µm. (E) The distribution of MT arrangements for both WT and Camsap3-cKO mice was compared. Distribution of the MT conﬁgurations between WT and KO was signiﬁcantly different as analyzed by a Kolmogorov–Smirnov test (p = 0.0003). diﬀerences between WT and Camsap3-cKO vestibular kinocilia were then evaluated using TEM (Figures 3C–E). We examined 42 axoneme structures (the position “C-D” shown in Figure 3B) from 8-month-old WT mice and 33 axonemes from their Camsap3-cKO littermates. The “9+2” conﬁguration here includes 9 MT doublets peripherally and two MT singlets or amorphous material with higher electron density (darker) at the center of axonemes. This is demonstrated in a transverse section of WT vestibular kinocilia (Figure 3C, indicated by an arrow). The “9+0” conﬁguration lacks the central MTs structure and electron-dense materials as seen in the Camsap3-cKO image in Figure 3D. More than 71% of vestibular kinocilia from WT mice have “9+2” conﬁguration, while only 24% vestibular kinocilia from Camsap3-cKO mice showed similar structure. The majority of Camsap3-cKO vestibular kinocilia (55%) have the “9+0” conﬁguration. In addition, irregular conﬁgurations, “8+2” and “8+0,” were found in both WT (12%) and Camsap3-cKO (21%). Distribution of the MT conﬁgurations between WT and KO was statistically signiﬁcant diﬀerent as analyzed using a Kolmogorov- Smirnov distribution comparison test (p = 0.0003). Camsap3-cKO Mice Do Not Have Notable Vestibular Dysfunction y Since CAMSAP3 was essential to build or maintain normal structure of vestibular kinocilia (Figures 2, 3), we further investigated whether shorter kinocilia on vestibular hair cells caused vestibular dysfunction. Vestibular dysfunction can manifest as issues with balance or gait abnormalities. In their home cages, Camsap3-cKO mice did not show obvious head tilt, walked normally, and were indistinguishable from their WT littermates. To assess the vestibular function of Camsap3- cKO mice, we performed multiple methods aiming to detect more subtle abnormalities. The simplest methods of testing vestibular functions include a swim test and a right-reﬂex test (Hardisty-Hughes et al., 2010). The swim test aims to determine if the mice could properly swim in a straight line and keep themselves upright. After assessing 26 WT (14 male and 12 female), and 27 Camsap3-cKO (14 male and 13 female) mice, ages ranging from P22 to P388, we found that there were no notable diﬀerences between the Camsap3-cKO and WT mice regarding their ability to swim. The right-reﬂex test assesses whether the mice can re-right themselves when ﬂipped upside down. The same group of 26 WT and 27 Camsap3-cKO mice completed the right-reﬂex test. We found no diﬀerence in performance between the WT and Camsap3-cKO mice as each of the mice was able to ﬂip themselves back to upright immediately (within 1 s). Subsequently, we analyzed the balance ability of Camsap3- cKO mice by rotarod apparatus. On the Rotarod, we assessed Camsap3-cKO Mice Have Abnormal Kinocilia on Vestibular Hair Cells In addition, the shape and orientation of stereocilia on vestibular hair cells from Camsap3- cKO were quantitatively similar to those on WT mice despite the vestibular kinocilia from Camsap3-cKO mice were shorter and more likely to miss the central MT pair (Figures 2, 3). We also investigated the impact of CAMSAP3 on the orientation of stereocilia bundles by comparing auditory hair cells from WT and Camsap3-cKO mice with matched ages and sexes. As shown in Supplementary Figure 3, the orientation of stereocilia bundles on auditory hair cells from Camsap3-cKO June 2022 \| Volume 16 \| Article 876805 Frontiers in Cellular Neuroscience \| www.frontiersin.org Frontiers in Cellular Neuroscience \| www.frontiersin.org 7 CAMSAP3 Is Important for the Vestibular Kinocilium Formation O’Donnell and Zheng signiﬁcantly larger than the Camsap3-cKO mice regardless their sex (Figure 4). mice were comparable to those on WT auditory hair cells. This data is not surprising because most of kinocilia on auditory hair cells are with “9+0” conﬁguration (Sobkowicz et al., 1995), and CAMSAP3 signals do not co-localize with the basal bodies where “9+0” axonemes are originated (Robinson et al., 2020). In summary, CAMSAP3 is unlikely to have a signiﬁcant impact on cochlear kinocilia and the orientation of stereocilia. Body Weight Differences Between Wildtype and Camsap3-cKO Mice Arise as Mice Age June 2022 \| Volume 16 \| Article 876805 Frontiers in Cellular Neuroscience \| www.frontiersin.org 8 CAMSAP3 Is Important for the Vestibular Kinocilium Formation O’Donnell and Zheng FIGURE 5 \| Gait differences between WT and Camsap3-cKO mice. Two litters of mice, ages P130–P133, were tested using the DigiGait Imaging System. Multiple t-tests revealed that Paw Area (A,B) and Max dA/dt (C,D) were signiﬁcantly different between WT and Camsap3-cKO mice (p-values for each speed were as indicated). Bars represent mean ± SD. (E) Unpaired t-tests showed that there was no body length difference between male WT and Camsap3-cKO mice (ns, p = 0.1572). (F) Unpaired t-tests showed that there was also no body width difference between male WT and Camsap3-cKO mice (ns, p = 0.0539). N = 3 for both WT and Camsap3-cKO mice. Statistically signiﬁcant difference. FIGURE 5 \| Gait differences between WT and Camsap3-cKO mice. Two litters of mice, ages P130–P133, were tested using the DigiGait Imaging System. Multiple t-tests revealed that Paw Area (A,B) and Max dA/dt (C,D) were signiﬁcantly different between WT and Camsap3-cKO mice (p-values for each speed were as indicated). Bars represent mean ± SD. (E) Unpaired t-tests showed that there was no body length difference between male WT and Camsap3-cKO mice (ns, p = 0.1572). (F) Unpaired t-tests showed that there was also no body width difference between male WT and Camsap3-cKO mice (ns, p = 0.0539). N = 3 for both WT and Camsap3-cKO mice. Statistically signiﬁcant difference. 12 WT (6 male and 6 female) and 8 Camsap3-cKO (5 male and 3 female) mice, ages P37–P153, and found that there was no signiﬁcant diﬀerence in TTF between the two groups (data not shown). treadmill for both fore and hind limbs regardless of running speeds (Figures 5A,B). For example, at a speed of 17 cm/s, the paw area that Camsap3-cKO mice used to touch the treadmill was 62.1% (fore limbs) and 58.9% (hind limbs) of the paw area WT mice used. Maximum dA/dt (cm2/s) is the maximal rate of change of paw area in contact with the treadmill belt during the braking phase. At a low treadmill speed (10 cm/s), there was no substantial diﬀerence between the WT and Camsap3- cKO mice in their dA/dt for both fore and hind lambs. However, as the treadmill speeds increased, the diﬀerence between the WT and Camsap3-cKO mice became more apparent. Body Weight Differences Between Wildtype and Camsap3-cKO Mice Arise as Mice Age In our original measurements of young mice, ranging from 1 to 4 months old, there was no weight diﬀerence between Camsap3- cKO mice and their WT littermates (Figure 4A). This phenotype diﬀers from KD mice, Camsap3-KD, which were born smaller than their WT littermates (Robinson et al., 2020). Since we did not notice obvious body weight diﬀerences like those observed in the global Camsap3-KD mice, we assumed that their weights remained comparable. Though there were no obvious weight diﬀerences between WT and Camsap3-cKO mice when mice were less than 4-month old, weight diﬀerences emerged in 8- month old mice (Figure 4A). For mice 8 months and older (P237–P388), the average male WT mouse weighed (mean ± SD) 39.61 g ± 8.703 (n = 21), and the average male Camsap3-cKO mouse weighed 26.49 g ± 4.904 (n = 13). For female mice older than 8 months, the average WT mouse weighed 30.56 g ± 4.687 (n = 8), and the average female Camsap3-cKO mouse weighed 22.38 g ± 1.727 (n = 7). WT mice older than 8-month were FIGURE 4 \| Weight differences between WT and Camsap3-cKO mice. (A) Male Camsap3-cKO mice weigh less than their WT counterparts after the age of 8-month, while no body weight difference was observed in mice younger than 4-month old. Mice were weighed prior to euthanasia at ages P36–P388. ns, not statistically signiﬁcant difference. (B) Female Camsap3-cKO mice aged 8–13 months weigh less than their WT counterparts. For both male and female mice, the weights of old WT and Camsap3-cKO mice were signiﬁcantly different. p-values for each age group of mice were as indicated. Statistically signiﬁcant difference. FIGURE 4 \| Weight differences between WT and Camsap3-cKO mice. (A) Male Camsap3-cKO mice weigh less than their WT counterparts after the age of 8-month, while no body weight difference was observed in mice younger than 4-month old. Mice were weighed prior to euthanasia at ages P36–P388. ns, not statistically signiﬁcant difference. (B) Female Camsap3-cKO mice aged 8–13 months weigh less than their WT counterparts. For both male and female mice, the weights of old WT and Camsap3-cKO mice were signiﬁcantly different. p-values for each age group of mice were as indicated. Statistically signiﬁcant difference. Body Weight Differences Between Wildtype and Camsap3-cKO Mice Arise as Mice Age At a high treadmill speed (24 cm/s), the maximum dA/dt decreased in Camsap3-cKO mice’s hind limbs (−10.5%), while WT mice had increased maximum dA/dt (+10.0%). Similar to paw area, maximum dA/dt in Camsap3-cKO mice were always slower than their WT littermates independent of walking speed. These diﬀerences exist despite there being no diﬀerence in the body length, width (Figures 5E,F), and weight (Figure 4A) between the WT and Camsap3-cKO mice. Camsap3-cKO Mice Have Slightly Altered Gaits The vestibular function of Camsap3-cKO mice was also examined using the DigiGait Imaging System (Berryman et al., 2009). Since mice older than 8-month Camsap3-cKO mice were lighter than their WT littermates (Figure 4A), mice at 4 months old were selected for gait measurement. At this age, gait was fully developed (Akula et al., 2020; Rahn et al., 2021), and Camsap3- cKO mice were not signiﬁcantly lighter than their WT littermates (Figure 4A). The assessed litter consisted of 6 male mice: 3 WT and 3 Camsap3-cKO. We collected data from these mice using the DigiGaitTM Imaging System. Of all the aspects of gait analyzed by the DigiGait analysis software, most showed no signiﬁcant diﬀerence between the WT and Camsap3-cKO mice. Some of the results were shown in the Supplementary Figures 1, 2). Two aspects of gait with signiﬁcantly diﬀerences were paw area at peak stance and maximum dA/dt (Figure 5). Paw area (cm2) is the maximal paw area captured by the camera and corresponds with the time of “peak stance” (Supplementary Figure 1). When compared to their WT littermates, Camsap3- cKO mice had smaller areas of their paws in contact with the Older Camsap3-cKO Mice Have Renal Abnormalities In TgPax2Cre mouse line, Cre was detected in the embryonic kidney since the 12-somite stage to remove the ﬂoxed allele (Ohyama and Groves, 2004). Since CAMSAP3 is essential to orienting the apical-to-basal polarity of MT arrays in epithelial June 2022 \| Volume 16 \| Article 876805 Frontiers in Cellular Neuroscience \| www.frontiersin.org Frontiers in Cellular Neuroscience \| www.frontiersin.org 9 CAMSAP3 Is Important for the Vestibular Kinocilium Formation O’Donnell and Zheng et al., 2021), we weighed 19 WT mice and 7 Camsap3-cKO mice and their kidneys. Kidney weight as a proportion of body weight was recorded. Female mice that had previously given birth were excluded. Interestingly, we found that Camsap3-cKO mice, especially the males, had statistically signiﬁcant heavier cells (Toya et al., 2016), we suspected that kidney function might be aﬀected in Camsap3-cKO mice. Therefore, both kidneys were prepared out of euthanized WT and cKO mice with ages of P22– P339. Since the kidney-to-body weight ratio has been commonly used to predict kidney function (Hughes et al., 1999; Mitsuhata FIGURE 6 \| Renal abnormalities in Camsap3-cKO mice. (A,B) The kidney-to-body weight ratio in male (A) and female (B). Unpaired t-tests revealed that Camsap3-cKO mice (P22-P339) showed a statistically signiﬁcant increase in their kidney-to-body weight ratio relative to their WT counterparts. p-values for each age group of mice were as indicated. Bars represent mean ± SD. (C,D) Representative kidney images taken from male, 1-year-old WT (C) and Camsap3-cKO littermate (D). Kidneys from the Camsap3-cKO mouse were hypertrophic and discolored. Statistically signiﬁcant difference. FIGURE 7 \| WT and Camsap3-cKO mice show no differences in olfaction. (A) No differences in the reaction time of the sense of smell between WT and Camsap3-cKO mice. Bars represent mean ± SD. Unpaired t-tests revealed no signiﬁcant (ns) differences between WT and Camsap3-cKO mice in male or female mice. (B,C) CAMSAP3 had little expression in olfactory bulbs regardless of genotype, WT (B) or Camsap3-cKO (C). The representative images were taken from female WT and their Camsap3-cKO littermates at P180. (B’,C’) were the cells shown in (B,C) stained with anti-Camsap3 (green). (D–G) CAMSAP3 was expressed in olfactory sensory neurons (OSN) in both WT and Camsap3-cKO mice. Olfactory mucosa in the nasal cavity from WT (D,F) and Camsap3-cKO (E,G) mice were stained with anti-Camsap3 (green), anti-acetylated tubulin (red, olfactory cilia, and nerve ﬁbers), and Hoechst 33342 (blue, nuclei). Older Camsap3-cKO Mice Have Renal Abnormalities Scale Bars = 100 µm (B,C), 50 µm (D,E), 10 µm (F,G). FIGURE 7 \| WT and Camsap3-cKO mice show no differences in olfaction. (A) No differences in the reaction time of the sense of smell between WT and Camsap3-cKO mice. Bars represent mean ± SD. Unpaired t-tests revealed no signiﬁcant (ns) differences between WT and Camsap3-cKO mice in male or female mice. (B,C) CAMSAP3 had little expression in olfactory bulbs regardless of genotype, WT (B) or Camsap3-cKO (C). The representative images were taken from female WT and their Camsap3-cKO littermates at P180. (B’,C’) were the cells shown in (B,C) stained with anti-Camsap3 (green). (D–G) CAMSAP3 was expressed in olfactory sensory neurons (OSN) in both WT and Camsap3-cKO mice. Olfactory mucosa in the nasal cavity from WT (D,F) and Camsap3-cKO (E,G) mice were stained with anti-Camsap3 (green), anti-acetylated tubulin (red, olfactory cilia, and nerve ﬁbers), and Hoechst 33342 (blue, nuclei). (F,G) were images with higher magniﬁcation showing olfactory cilia and dendritic knobs layers on OSNs. (D’,E’,F”,G”) Showed the anti-CAMSAP3 (green) channel in (D–G). (F’,G’) Showed anti-acetylated tubulin (red) channel in (F,G). “[” Marks the OSN layer in the olfactory epithelium. Scale Bars = 100 µm (B,C), 50 µm (D,E), 10 µm (F,G). FIGURE 7 \| WT and Camsap3-cKO mice show no differences in olfaction. (A) No differences in the reaction time of the sense of smell between WT and Camsap3-cKO mice. Bars represent mean ± SD. Unpaired t-tests revealed no signiﬁcant (ns) differences between WT and Camsap3-cKO mice in male or female mice. (B,C) CAMSAP3 had little expression in olfactory bulbs regardless of genotype, WT (B) or Camsap3-cKO (C). The representative images were taken from female WT and their Camsap3-cKO littermates at P180. (B’,C’) were the cells shown in (B,C) stained with anti-Camsap3 (green). (D–G) CAMSAP3 was expressed in olfactory sensory neurons (OSN) in both WT and Camsap3-cKO mice. Olfactory mucosa in the nasal cavity from WT (D,F) and Camsap3-cKO (E,G) mice were stained with anti-Camsap3 (green), anti-acetylated tubulin (red, olfactory cilia, and nerve ﬁbers), and Hoechst 33342 (blue, nuclei). (F,G) were images with higher magniﬁcation showing olfactory cilia and dendritic knobs layers on OSNs. (D’,E’,F”,G”) Showed the anti-CAMSAP3 (green) channel in (D–G). (F’,G’) Showed anti-acetylated tubulin (red) channel in (F,G). “[” Marks the OSN layer in the olfactory epithelium. Scale Bars = 100 µm (B,C), 50 µm (D,E), 10 µm (F,G). Older Camsap3-cKO Mice Have Renal Abnormalities (F,G) were images with higher magniﬁcation showing olfactory cilia and dendritic knobs layers on OSNs. (D’,E’,F”,G”) Showed the anti-CAMSAP3 (green) channel in (D–G). (F’,G’) Showed anti-acetylated tubulin (red) channel in (F,G). “[” Marks the OSN layer in the olfactory epithelium. Scale Bars = 100 µm (B,C), 50 µm (D,E), 10 µm (F,G). FIGURE 6 \| Renal abnormalities in Camsap3-cKO mice. (A,B) The kidney-to-body weight ratio in male (A) and female (B). Unpaired t-tests revealed that Camsap3-cKO mice (P22-P339) showed a statistically signiﬁcant increase in their kidney-to-body weight ratio relative to their WT counterparts. p-values for each age group of mice were as indicated. Bars represent mean ± SD. (C,D) Representative kidney images taken from male, 1-year-old WT (C) and Camsap3-cKO littermate (D). Kidneys from the Camsap3-cKO mouse were hypertrophic and discolored. Statistically signiﬁcant difference. FIGURE 6 \| Renal abnormalities in Camsap3-cKO mice. (A,B) The kidney-to-body weight ratio in male (A) and female (B). Unpaired t-tests revealed that Camsap3-cKO mice (P22-P339) showed a statistically signiﬁcant increase in their kidney-to-body weight ratio relative to their WT counterparts. p-values for each age group of mice were as indicated. Bars represent mean ± SD. (C,D) Representative kidney images taken from male, 1-year-old WT (C) and Camsap3-cKO littermate (D). Kidneys from the Camsap3-cKO mouse were hypertrophic and discolored. *Statistically signiﬁcant difference. FIGURE 7 \| WT and Camsap3-cKO mice show no differences in olfaction. (A) No differences in the reaction time of the sense of smell between WT and Camsap3-cKO mice. Bars represent mean ± SD. Unpaired t-tests revealed no signiﬁcant (ns) differences between WT and Camsap3-cKO mice in male or female mice. (B,C) CAMSAP3 had little expression in olfactory bulbs regardless of genotype, WT (B) or Camsap3-cKO (C). The representative images were taken from female WT and their Camsap3-cKO littermates at P180. (B’,C’) were the cells shown in (B,C) stained with anti-Camsap3 (green). (D–G) CAMSAP3 was expressed in olfactory sensory neurons (OSN) in both WT and Camsap3-cKO mice. Olfactory mucosa in the nasal cavity from WT (D,F) and Camsap3-cKO (E,G) mice were stained with anti-Camsap3 (green), anti-acetylated tubulin (red, olfactory cilia, and nerve ﬁbers), and Hoechst 33342 (blue, nuclei). (F,G) were images with higher magniﬁcation showing olfactory cilia and dendritic knobs layers on OSNs. (D’,E’,F”,G”) Showed the anti-CAMSAP3 (green) channel in (D–G). (F’,G’) Showed anti-acetylated tubulin (red) channel in (F,G). “[” Marks the OSN layer in the olfactory epithelium. Camsap3-cKO Mice Maintain Normal Olfaction Since Cre was expressed in the olfactory bulb in TgPax2Cre mouse line to remove the ﬂoxed Camsap3 exon 7, we investigated whether Camsap3-cKO had normal olfaction. Olfaction evaluation was performed for WT (11 male and 10 female) and Camsap3-cKO mice (12 male and 12 female) from 8 litters of mice, ages P55–P313. Mice were placed in a large container ﬁlled with bedding, and their latency to ﬁnd (LTF) a buried Nutter Butter cookie was recorded. As shown in Figure 7A, WT and textitCamsap3-cKO mice took equal amounts of time to ﬁnd the buried cookie. There was no signiﬁcant diﬀerence between WT and Camsap3-cKO mice regardless their sex. We then examined CAMSAP3 expression in the olfactory bulb. Olfactory bulbs from P180 WT and Camsap3- cKO littermates were collected and stained with anti-Camsap3. As shown in Figures 7B,C, there was little CAMSAP3 expression in cells located in olfactory bulbs from both the WT and Camsap3-cKO mice, suggesting that CAMSAP3 has a minimal role in olfactory bulbs. We also compared CAMSAP3 expression in olfactory sensory neurons, where CAMSAP3 was abundantly expressed (Robinson et al., 2020). As shown in Figures 7D– G, CAMSAP3 was abundantly expressed in olfactory sensory neurons located in the nasal olfactory epithelia in both the WT and Camsap3-cKO mice. CAMSAP3 expression in olfactory sensory neurons is not aﬀected in Camsap3-cKO mice and remained concentrated at dendritic knobs under the olfactory cilia (Figures 7F,G). In other words, CAMSAP3 expression in Despite their abnormal vestibular kinocilia formation, Camsap3-cKO mice do not have notable vestibular dysfunction such as slower right-reﬂexes, inferior balance, and gait asymmetries (Hardisty-Hughes et al., 2010; Lu et al., 2011; Kopecky et al., 2012; Mathur et al., 2015). Such incoherent results between morphological data and behavioral assays are not uncommon, as many other mutant mice with damaged hair cells or neurons in the vestibular organ showed no detectable vestibular dysfunction (Jones and Jones, 2014). Since balance is achieved and maintained by multiple systems including the vestibular system, vision, proprioception, and central nervous system, it is possible that the Camsap3-cKO mice are able to retain normal vestibular function because of compensation or adaptation by the central nervous system and other peripheral systems. In addition, the central MT pairs are not completely eliminated from all kinocilia on vestibular hair cells from Camsap3-cKO mice as shown in Figure 3E. DISCUSSION Kinocilia on vestibular hair cells have MTs arranged in a “9+2” conﬁguration that diﬀers from most primary sensory cilia. Unlike the central MT pair in motile cilia, the function of the central MT pair in primary sensory cilia, including vestibular kinocilia, remains unknown. Our data shows that CAMSAP3 is located at the base of vestibular kinocilia (Figure 3). Removing or reducing CAMSAP3 in vestibular hair cells leads to abnormal kinocilia, which have shorter axonemes (Figure 2) and are more often missing the central MT pair (Figure 3). Since CAMSAP3 is required for de novo formation of the central MT pair for the “9+2” conﬁguration in motile cilia (Robinson et al., 2020; Saito et al., 2021), it is likely that lacking CAMSAP3 and CAMSAP3-stabilized MTs in vestibular hair cells also prevents the central MT pair formation in vestibular kinocilia. This likely leads to the shortened vestibular kinocilia observed in Camsap3-cKO mice (Figure 2). In addition, Camsap3-cKO mice have more irregular conﬁgurations (8+2, 8+0) in vestibular kinocilia than their WT littermates. Although a very small portion of irregular MT conﬁguration is commonly found in WT cilia, an increased proportion of irregular MT conﬁgurations in cilia was often associated with damage or degeneration of cilia (Piorunek et al., 2008). With increased irregular MT conﬁgurations and decreased “9+2” conﬁguration in vestibular kinocilia from Camsap3-cKO, our data suggests that the central MT pair in vestibular kinocilia is important in establishing and/or maintaining the long axonemes observed in vestibular kinocilia. Older Camsap3-cKO Mice Have Renal Abnormalities June 2022 \| Volume 16 \| Article 876805 Frontiers in Cellular Neuroscience \| www.frontiersin.org 10 CAMSAP3 Is Important for the Vestibular Kinocilium Formation O’Donnell and Zheng kidneys (as a function of body weight) than their WT littermates (Figure 6). In male WT mice, their kidneys made up (mean ± SD) 1.47 ± 0.13% of their body weight, and in male Camsap3- cKO mice, their kidneys equaled 3.90 ± 1.5% of their body weight (Figure 6A). In female WT mice, their kidneys made up 1.51 ± 0.34% of their body weight, while in female Camsap3- cKO mice, their kidneys equaled 2.23 ± 0.30% of their body weight (Figure 6B). Kidneys from the Camsap3-cKO mice were hypertrophic and discolored (Figure 6C). Such a phenotype was similar to those observed in CAMSAP3-mutant knocking in mice model Camsap3dc/dc, in which exons 14–17 of Camsap3 were removed from the genome of Camsap3dc/dc mice (Toya et al., 2016). Exons 14–17 encode the key MT binding domain of CAMSAP3, CKK domain. Because the mutant CAMSAP3 without the CKK domain is incapable of binding to MTs to form proper MT network in the epithelial cells in kidney, Camsap3dc/dc mice developed malfunction of kidneys with cyst at the proximal convoluted tubules starting at E17.5 even though kidney had normal appearance at P21 (Mitsuhata et al., 2021). Since renal dysfunction often caused body weight loss (Hickman and Swan, 2010), the deterioration of kidneys observed in Camsap3-cKO may contribute to the small body size of Camsap3- cKO mice older than 8-month (Figure 4). In consequence, health issues arose, and these mice became thin, hunched, and moved slowly. These health issues prevented further investigation of CAMSAP3’s impact on vestibular function in Camsap3-cKO mice that were older than 13 months of age. cells involved in olfaction were not changed in Camsap3-cKO mice compared to their WT littermates. Given the equivalent CAMSAP3 expression in cells involved in olfaction (Figures 7B– G), it was unsurprising that the Camsap3-cKO mice performed equally as well as WT mice on the open-ﬁeld olfaction assessment. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fncel. 2022.876805/full#supplementary-material Camsap3-cKO Mice Maintain Normal Olfaction We suspect that Camsap3 might not be completely removed from all cells in the vestibular organ. It is also possible that CAMSAP3’s role is compensated for by other members of CAMSAP family, such as CAMSAP2, which compensates for CAMSAP3 to maintain June 2022 \| Volume 16 \| Article 876805 Frontiers in Cellular Neuroscience \| www.frontiersin.org 11 O’Donnell and Zheng CAMSAP3 Is Important for the Vestibular Kinocilium Formation FUNDING This work was supported by the American Hearing Research Foundation and a Hugh Knowles Leadership Fund Award to JZ. Gait Analysis was performed by the Northwestern University Behavioral Phenotyping Core. Imaging was conducted at the Northwestern University Center for Advanced Microscopy, which was supported by the NCI CCSG P30 CA060553. The re- derived Camsap3-KD line on the FVB murine background was conducted at Transgenic and Targeted Mutagenesis Laboratory of Northwestern University. Balance disequilibrium is a signiﬁcant contributor to falls in the elderly. The most common cause of balance dysfunction is due to abnormal hair cells from the vestibular sensory epithelia of the vestibular organ. Because of malfunction of kidneys in Camsap3-cKO mice, we were not able to investigate the impact of CAMSAP3 on the vestibular function in older mice due to their health issues. Diﬀerent Camsap3 transgenic mouse models without damaged kidneys may be needed to investigate impact of CAMSAP3 and the central MT pair of kinocilia on vestibular function. In addition, it might be worthwhile to examine the vestibulo-ocular reﬂex (VOR) of Camsap3 transgenic mice using video-oculography. Video-oculography quantiﬁes eye movement data to evaluate vestibular function, and could highlight vestibular issues that we were not able to capture in this study. Akula, S. K., McCullough, K. B., Weichselbaum, C., Dougherty, J. D., and Maloney, S. E. (2020). The trajectory of gait development in mice. Brain Behav. 10:e01636. doi: 10.1002/brb3.1636 DATA AVAILABILITY STATEMENT MT networks (Tanaka et al., 2012). Nevertheless, Camsap3- cKO mice did show slightly altered gaits in their paw area at peak stance and maximum dA/dt. Assessment of paw placement during walking is often used to evaluate pain (Griﬃoen et al., 2015; Deuis et al., 2017), pain-killer medicines (Brings et al., 2021), diseases (Bernardes and Oliveira, 2017), and recovery of injuries including both peripheral and central nerve injury (Neumann et al., 2009; Heinzel et al., 2020). Mice with numbers of disorders decrease their paw area to form “tip-toe walking” patterns (Furusawa et al., 1996; Moskovitz et al., 2001). Such phenotypes are also found in humans, particularly in children. An underdeveloped or poor vestibular system is believed to contribute to some of the idiopathic toe walking cases observed in children (Montgomery and Gauger, 1978; Chu and Anderson, 2020). Camsap3-cKO mice also decreased their paw area in both hind paws that come into contact with the surface (Figure 5). It is, therefore, tempting to speculate that altered gaits observed in Camsap3-cKO mice is the consequence of shorter vestibular kinocilia due to a lack of CAMSAP3 and central MTs in their axoneme. However, vascular calciﬁcation can also cause tiptoe walking in mice (Okawa et al., 1998), and kidney disease is one etiology for vascular calciﬁcation (Palit and Kendrick, 2014). Although there is no evidence suggesting that Camsap3 expression is changed in the somatosensory system of Camsap3-cKO mice, but the midbrain and cerebellum may reduce CAMSAP3 expression due to Cre expression in these issues. Changed CAMSAP3 expression in the midbrain and cerebellum may interfere with the somatosensory information process that might lead to alternated gaits in Camsap3-cKO. The original contributions presented in this study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author. ETHICS STATEMENT The animal study was reviewed and approved by the Institutional Animal Care and Use Committees of Northwestern University. AUTHOR CONTRIBUTIONS JZ designed and performed some of the experiments and analyzed the data. JO’D performed some of the experiments and analyzed some of the data. JO’D and JZ wrote the manuscript. Both authors contributed to the article and approved the submitted version. ACKNOWLEDGMENTS We thank Alan M. Robinson, Satoe Takahashi, Yingjie Zhou, and Craig Weiss (Northwestern University) for their guidance and advice on several experiments, as well as MaryAnn Cheatham (Northwestern University) for creating Camsap3-cKO mice, and Jaime Garcia-Anoveros and Donna Whitlon (Northwestern University) for providing reagents. In summary, our data suggest that CAMSAP3 is critical for construction of the central MT pair in vestibular kinocilia, which may be essential for building long and stable axonemes in these kinocilia on vestibular hair cells. Whether abnormal vestibular kinocilia of Camsap3-cKO mice could directly lead to vestibular function requires further investigation. Andreu-Cervera, A., Catala, M., and Schneider-Maunoury, S. (2021). Cilia, ciliopathies and hedgehog-related forebrain developmental disorders. Neurobiol. Disease 150:105236. doi: 10.1016/j.nbd.2020.105236 Bernardes, D., and Oliveira, A. L. R. (2017). Comprehensive catwalk gait analysis in a chronic model of multiple sclerosis subjected to Andreu-Cervera, A., Catala, M., and Schneider-Maunoury, S. (2021). Cilia, ciliopathies and hedgehog-related forebrain developmental disorders. Neurobiol. Disease 150:105236. doi: 10.1016/j.nbd.2020.105236 Bernardes, D., and Oliveira, A. L. R. (2017). Comprehensive catwalk gait analysis in a chronic model of multiple sclerosis subjected to REFERENCES Neurosci. 10:284. doi: 10.3389/fnmol.2017. 00284 Lu, Y.-C., Wu, C.-C., Shen, W.-S., Yang, T.-H., Yeh, T.-H., Chen, P.-J. I, et al. (2011). Establishment of a knock-in mouse model with the SLC26A4 c.919- 2A>G mutation and characterization of its pathology. PloS one. 6, e22150– e22150. doi: 10.1371/journal.pone.0022150 Dunham, N. W., and Miya, T. S. (1957). A note on a simple apparatus for detecting neurological deﬁcit in rats and mice. J. Am. Pharm. Assoc. Am. Pharm. Assoc. 46, 208–209. doi: 10.1002/jps.3030460322 Falk, N., Losl, M., Schroder, N., and Giessl, A. (2015). Specialized Cilia in Mammalian Sensory Systems. Cells 4, 500–519. doi: 10.3390/cells4030500 Mathur, P. D., Vijayakumar, S., Vashist, D., Jones, S. M., Jones, T. A., and Yang, J. (2015). A study of whirlin isoforms in the mouse vestibular system suggests potential vestibular dysfunction in DFNB31-deﬁcient patients. Hum. Mole. Gene. 24, 7017–7030. doi: 10.1093/hmg/ddv403 Furusawa, N., Baba, H., Imura, S., and Fukuda, M. (1996). Characteristics and mechanism of the ossiﬁcation of posterior longitudinal ligament in the tip-toe walking Yoshimura (twy) mouse. Eur. J. Histochem. 40, 199–210. Mbiene, J. P., Favre, D., and Sans, A. (1984). The pattern of ciliary development in fetal mouse vestibular receptors. A qualitative and quantitative SEM study. Anat Embryol. 170, 229–238. doi: 10.1007/BF00318726 Gibieža, P., Peterman, E., Hoﬀman, H. K., Engeleburg, S. V., Skeberdis, V. A., and Prekeris, R. (2021). Rab14/MACF2 complex regulates endosomal targeting during cytokinesis. Mole. Biol.Cell 32, 554–566. doi: 10.1091/mbc.E20-09-0607 Goodwin, S. S., and Vale, R. D. (2010). Patronin regulates the microtubule network by protecting microtubule minus ends. Cell 143, 263–274. doi: 10.1016/j.cell. 2010 09 022 Gibieža, P., Peterman, E., Hoﬀman, H. K., Engeleburg, S. V., Skeberdis, V. A., and Prekeris, R. (2021). Rab14/MACF2 complex regulates endosomal targeting during cytokinesis. Mole. Biol.Cell 32, 554–566. doi: 10.1091/mbc.E20-09-0607 and Prekeris, R. (2021). Rab14/MACF2 complex regulates endosomal targeting during cytokinesis. Mole. Biol.Cell 32, 554–566. doi: 10.1091/mbc.E20-09-0607 Goodwin, S. S., and Vale, R. D. (2010). Patronin regulates the microtubule network by protecting microtubule minus ends. Cell 143, 263–274. doi: 10.1016/j.cell. 2010.09.022 Meng, W., Mushika, Y., Ichii, T., and Takeichi, M. (2008). Anchorage of microtubule minus ends to adherens junctions regulates epithelial cell-cell contacts. Cell 135, 948–959. doi: 10.1016/j.cell.2008.09.040 Goodwin, S. S., and Vale, R. D. (2010). Patronin regulates the microtubule network by protecting microtubule minus ends. Cell 143, 263–274. doi: 10.1016/j.cell. 2010.09.022 g by protecting microtubule minus ends. Cell 143, 263–274. doi: 10.1016/j.cell. REFERENCES Bernardes, D., and Oliveira, A. L. R. (2017). Comprehensive catwalk gait analysis in a chronic model of multiple sclerosis subjected to June 2022 \| Volume 16 \| Article 876805 Frontiers in Cellular Neuroscience \| www.frontiersin.org 12 CAMSAP3 Is Important for the Vestibular Kinocilium Formation O’Donnell and Zheng treadmill exercise training. BMC Neurol. 17:160. doi: 10.1186/s12883-017- 0941-z Activation Timing Dynamics and Impairs Balance. Front. Mole. Neurosci. 11:289. doi: 10.3389/fnmol.2018.00289 treadmill exercise training. BMC Neurol. 17:160. doi: 10.1186/s12883-017- 0941-z Berryman, E. R., Harris, R. L., Moalli, M., and Bagi, C. M. (2009). Digigait quantitation of gait dynamics in rat rheumatoid arthritis model. J. Musculoskelet Neuronal. Interact. 9, 89–98. Kanki, H., Suzuki, H., and Itohara, S. (2006). High-eﬃciency CAG-FLPe deleter mice in C57BL/6J background. Exp. Anim. 55, 137–141. doi: 10.1538/expanim. 55.137 Kopecky, B., Decook, R., and Fritzsch, B. (2012). Mutational ataxia resulting from abnormal vestibular acquisition and processing is partially compensated for. Behav. Neurosci. 126, 301–313. doi: 10.1037/a0026896 Brings, V. E., Payne, M. A., and Gereau, R. W. (2021). Paw placement during walking is altered by analgesic doses of opioids and post-surgical injury in mice. bioRxiv doi: 10.1101/2021.12.15.4728182012 Lechtreck, K. F., Gould, T. J., and Witman, G. B. (2013). Flagellar central pair assembly in Chlamydomonas reinhardtii. Cilia 2:15. doi: 10.1186/2046-2530- 2-15 Choksi, S. P., Lauter, G., Swoboda, P., and Roy, S. (2014). Switching on cilia: transcriptional networks regulating ciliogenesis. Development 141, 1427–1441. doi: 10.1242/dev.074666 Chu, V., and Anderson, L. (2020). Sensory-Processing Diﬀerences in ChildrenWith Idiopathic ToeWalking (ITW). Am. J. Occup. Therapy 74:74115051301. doi: 10.5014/ajot.2020.74S1-PO3205 Lee, J. E., and Gleeson, J. G. (2011). A systems-biology approach to understanding the ciliopathy disorders. Genome Med. 3:59. doi: 10.1186/gm275 Lee, L., and Ostrowski, L. E. (2021). Motile cilia genetics and cell biology: big results from little mice. Cell. Mole. Life Sci. 78, 769–797. doi: 10.1007/s00018-020- 03633-5 Lee, L., and Ostrowski, L. E. (2021). Motile cilia genetics and cell biology: big results Cox, B. C., Liu, Z., Lagarde, M. M., and Zuo, J. (2012). Conditional gene expression in the mouse inner ear using Cre-loxP. J.Assoc. Res. Otolaryngol. 13, 295–322. doi: 10.1007/s10162-012-0324-5 from little mice. Cell. Mole. Life Sci. 78, 769–797. doi: 10.1007/s00018-020- 03633-5 Loreng, T. D., and Smith, E. F. (2017). The Central Apparatus of Cilia and Eukaryotic Flagella. Cold Spring Harb. Perspect Biol. 9:a028118. doi: 10.1101/ cshperspect.a028118 Deuis, J. R., Dvorakova, L. S., and Vetter, I. (2017). Methods Used to Evaluate Pain Behaviors in Rodents. Front. Mole. REFERENCES 2010.09.022 Mitsuhata, Y., Abe, T., Misaki, K., Nakajima, Y., Kiriya, K., Kawasaki, M., et al. (2021). Cyst formation in proximal renal tubules caused by dysfunction of the microtubule minus-end regulator CAMSAP3. Sci. Rep. 11:5857. doi: 10.1038/ s41598-021-85416-x Griﬃoen, M. A., Dernetz, V. H., Yang, G. S., Griﬃth, K. A., Dorsey, S. G., and Renn, C. L. (2015). Evaluation of Dynamic Weight Bearing for Measuring Nonevoked Inﬂammatory Hyperalgesia in Mice. Nurs. Res. 64, 81–87. doi: 10.1097/NNR. 0000000000000082 Montgomery, P., and Gauger, J. (1978). Sensory Dysfunction in Children Who Toe Walk. Physical. Therapy. 58, 1195–1204. doi: 10.1093/ptj/58.10.1195 Hardisty-Hughes, R. E., Parker, A., and Brown, S. D. (2010). A hearing and vestibular phenotyping pipeline to identify mouse mutants with hearing impairment. Nat. Protoc. 5, 177–190. doi: 10.1038/nprot.2009.204 Moskovitz, J., Bar-Noy, S., Williams, W. M., Requena, J., Berlett, B. S., and Stadtman, E. R. (2001). Methionine sulfoxide reductase (MsrA) is a regulator of antioxidant defense and lifespan in mammals. Proc.Nat. Acad. Sci.U. S. A. 98, 12920–12925. doi: 10.1073/pnas.231472998 Heinzel, J., Swiadek, N., Ashmwe, M., Rührnößl, A., Oberhauser, V., Kolbenschlag, J., and Hercher, D. (2020). Automated gait analysis to assess functional recovery in rodents with peripheral nerve or spinal cord contusion injury. J. Vis. Exp. 164:e61852. doi: 10.3791/61852 Muroyama, A., Terwilliger, M., Dong, B., Suh, H., and Lechler, T. (2018). Genetically induced microtubule disruption in the mouse intestine impairs intracellular organization and transport. Mole. Biol. Cell 29, 1533–1541. doi: 10.1091/mbc.E18-01-0057 Hickman, D. L., and Swan, M. (2010). Use of a body condition score technique to assess health status in a rat model of polycystic kidney disease. J. Am. Assoc. Lab. Anim. Sci. 49, 155–159. Nam, J. H. (2018). An operating principle of the turtle utricle to detect wide dynamic range. Hearing Res. 360, 31–39. doi: 10.1016/j.heares.2017.09.015 Hughes, C. B., Bergstralh, E. J., Larson, T. S., Stegall, M. D., and Velosa, J. A. (1999). Kidney Weight (Kw) and Body Weight (Bw) Ratios Predict Glomerular Filtration Rate (GFR) in Transplant Recipients. Transplantation 67:S156. doi: 10.1097/00007890-199904150-00626 Neumann, M., Wang, Y., Kim, S., Hong, S. M., Jeng, L., Bilgen, M., et al. (2009). Assessing gait impairment following experimental traumatic brain injury in mice. J. Neurosci. Met. 176, 34–44. doi: 10.1016/j.jneumeth.2008.08.026 Ohyama, T., and Groves, A. K. (2004). Generation of Pax2-Cre mice by modiﬁcation of a Pax2 bacterial artiﬁcial chromosome. Genesis 38, 195–199. doi: 10.1002/gene.20017 Ingham, N. J., Pearson, S. A., Vancollie, V. E., Rook, V., Lewis, M. REFERENCES A reliable lacZ expression reporter cassette for multipurpose, knockout-ﬁrst alleles. Genesis. 38, 151–158. doi: 10.1002/gene.20012 Piorunek, T., Marszalek, A., Biczysko, W., Gozdzik, J., Cofta, S., and Seget, M. (2008). Correlation between the stage of cystic ﬁbrosis and the level of morphological changes in adult patients. J. Physiol. Pharmacol. 59, 565–572. Toya, M., Kobayashi, S., Kawasaki, M., Shioi, G., Kaneko, M., Ishiuchi, T., et al. (2016). CAMSAP3 orients the apical-to-basal polarity of microtubule arrays in epithelial cells. Proc.Nat. Acad. Sci.U. S. A. 113, 332–337. doi: 10.1073/pnas. 1520638113 Rahn, R. M., Weichselbaum, C. T., Gutmann, D. H., Dougherty, J. D., and Maloney, S. E. (2021). Shared developmental gait disruptions across two mouse models of neurodevelopmental disorders. J. Neurodev. Disorders. 13:10. doi: 10.1186/ s11689-021-09359-0 Wersall, J., Flock, A., and Lundquist, P. G. (1965). Structural basis for directional sensitivity in cochlear and vestibular sensory receptors. Cold Spring Harb. Symp. Quant. Biol. 30, 115–132. doi: 10.1101/SQB.1965.030.01.015 Reiter, J. F., and Leroux, M. R. (2017). Genes and molecular pathways underpinning ciliopathies. Nat. Rev. Mole. Cell Biol. 18, 533–547. doi: 10.1038/ nrm.2017.60 White, J. K., Gerdin, A. K., Karp, N. A., Ryder, E., Buljan, M., Bussell, J. N., et al. (2013). Genome-wide generation and systematic phenotyping of knockout mice reveals new roles for many genes. Cell 154, 452–464. doi: 10.1016/j.cell.2013.06. 022 Robinson, A. M., Takahashi, S., Brotslaw, E. J., Ahmad, A., Ferrer, E., Procissi, D., et al. (2020). CAMSAP3 facilitates basal body polarity and the formation of the central pair of microtubules in motile cilia. Proc.Nat. Acad. Sci. U. S. A. 117, 13571–13579. doi: 10.1073/pnas.1907335117 Wiwatpanit, T., Lorenzen, S. M., Cantú, J. A., Foo, C. Z., Hogan, A. K., Márquez, F., et al. (2018). Trans-diﬀerentiation of outer hair cells into inner hair cells in the absence of INSM1. Nature. 563, 691–695. doi: 10.1038/s41586-018-0570-8 Rosenhall, U., and Engstrom, B. (1974). Surface Structures of the Human Vestibular Sensory Regions. Acta oto-Laryngologica. 77, 3–18. doi: 10.1080/ 16512251.1974.11675749 Xue, J., and Peterson, E. H. (2006). Hair bundle heights in the utricle: diﬀerences between macular locations and hair cell types. J. Neurophysiol. 95, 171–186. doi: 10.1152/jn.00800.2005 Rostosky, C. M., and Milosevic, I. (2018). Gait analysis of age-dependent motor impairments in mice with neurodegeneration. J. Vis. Exp. 136:57752. doi: 10. 3791/57752 j Yang, M., and Crawley, J. N. (2009). Simple behavioral assessment of mouse olfaction. Curr. Protoc. Neurosci. 8:8.24. REFERENCES A., Chen, J., et al. (2019). Mouse screen reveals multiple new genes underlying mouse and human hearing loss. PLoS Biol. 17:e3000194. doi: 10.1371/journal.pbio.3000194 Okawa, A., Nakamura, I., Goto, S., Moriya, H., Nakamura, Y., and Ikegawa, S. (1998). Mutation in Npps in a mouse model of ossiﬁcation of the posterior longitudinal ligament of the spine. Nat. Gene. 19, 271–273. doi: 10.1038/956 Jiang, K., Faltova, L., Hua, S., Capitani, G., Prota, A. E., Landgraf, C., et al. (2018). Structural Basis of Formation of the Microtubule Minus-End-Regulating CAMSAP-Katanin Complex. Structure 26, 375–382.e4. doi: 10.1016/j.str.2017. 12.017 Palit, S., and Kendrick, J. (2014). Vascular calciﬁcation in chronic kidney disease: role of disordered mineral metabolism. Curr. Pharm. Design. 20, 5829–5833. doi: 10.2174/1381612820666140212194926 Jiang, K., Hua, S., Mohan, R., Grigoriev, I., Yau, K. W., Liu, Q., et al. (2014). Microtubule minus-end stabilization by polymerization-driven CAMSAP deposition. Dev. Cell 28, 295–309. doi: 10.1016/j.devcel.2014.01.001 Pan, N., Jahan, I., Kersigo, J., Kopecky, B., Santi, P., Johnson, S., et al. (2011). Conditional deletion of Atoh1 using Pax2-Cre results in viable mice without diﬀerentiated cochlear hair cells that have lost most of the organ of Corti. Hearing Res. 275, 66–80. doi: 10.1016/j.heares.2010.12.002 Jones, S. M., and Jones, T. A. (2014). Genetics of peripheral vestibular dysfunction: lessons from mutant mouse strains. J. Am. Acad. Audiol. 25, 289–301. doi: 10.3766/jaaa.25.3.8 Jones, S. M., Vijayakumar, S., Dow, S. A., Holt, J. C., Jordan, P. M., and Luebke, A. E. (2018). Loss of α-Calcitonin Gene-Related Peptide (αCGRP) Reduces Otolith Pau, H., Hawker, K., Fuchs, H., De Angelis, M. H., and Steel, K. P. (2004). Characterization of a new mouse mutant, ﬂouncer, with a balance defect and June 2022 \| Volume 16 \| Article 876805 Frontiers in Cellular Neuroscience \| www.frontiersin.org 13 CAMSAP3 Is Important for the Vestibular Kinocilium Formation O’Donnell and Zheng inner ear malformation. Otol Neurotol. 25, 707–713. doi: 10.1097/00129492- 200409000-00010 Tanaka, N., Meng, W., Nagae, S., and Takeichi, M. (2012). Nezha/CAMSAP3 and CAMSAP2 cooperate in epithelial-speciﬁc organization of noncentrosomal microtubules. Proc.Nat. Acad. Sci.U. S. A. 109, 20029–20034. doi: 10.1073/pnas. 1218017109 inner ear malformation. Otol Neurotol. 25, 707–713. doi: 10.1097/00129492- 200409000-00010 Perez-Garcia, V., Fineberg, E., Wilson, R., Murray, A., Mazzeo, C. I., Tudor, C., et al. (2018). Placentation defects are highly prevalent in embryonic lethal mouse mutants. Nature 555, 463–468. doi: 10.1038/nature26002 Testa, G., Schaft, J., van der Hoeven, F., Glaser, S., Anastassiadis, K., Zhang, Y., et al. (2004). REFERENCES doi: 10.1002/0471142301.ns0824s48 Saito, H., Matsukawa-Usami, F., Fujimori, T., Kimura, T., Ide, T., Yamamoto, T., et al. (2021). Tracheal motile cilia in mice require CAMSAP3 for the formation of central microtubule pair and coordinated beating. Mole. Biol. Cell. 32:ar12. doi: 10.1091/mbc.E21-06-0303 Zenker, J., White, M. D., Templin, R. M., Parton, R. G., Thorn-Seshold, O., Bissiere, S., et al. (2017). A microtubule-organizing center directing intracellular transport in the early mouse embryo. Science 357, 925–928. doi: 10.1126/ science.aam9335 Sekerka, G., Richter, C.-P., and Bartles, J. (2011). Roles of the Espin Actin-Bundling Proteins in the Morphogenesis and Stabilization of Hair Cell Stereocilia Revealed in CBA/CaJ Congenic Jerker Mice. PLoS Gene. 7:e1002032. doi: 10. 1371/journal.pgen.1002032 Zheng, J., Furness, D., Duan, C., Miller, K. K., Edge, R. M., Chen, J., et al. (2013). Marshalin, a microtubule minus-end binding protein, regulates cytoskeletal structure in the organ of Corti. Biol. Open 2, 1192–1202. doi: 10.1242/bio. 20135603 Skarnes, W. C., Rosen, B., West, A. P., Koutsourakis, M., Bushell, W., Iyer, V., et al. (2011). A conditional knockout resource for the genome-wide study of mouse gene function. Nature 474, 337–342. doi: 10.1038/nature1 0163 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. Sobkowicz, H. M., Slapnick, S. M., and August, B. K. (1995). The kinocilium of auditory hair cells and evidence for its morphogenetic role during the regeneration of stereocilia and cuticular plates. J. Neurocytol. 24, 633–653. doi: 10.1007/BF01179815 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. Spoon, C., and Grant, W. (2011). Biomechanics of hair cell kinocilia: experimental measurement of kinocilium shaft stiﬀness and base rotational stiﬀness with Euler-Bernoulli and Timoshenko beam analysis. J. Exp. Biol. 214, 862–870. doi: 10.1242/jeb.051151 Copyright © 2022 O’Donnell and Zheng. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). REFERENCES 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. Spoon, C., and Grant, W. (2013). Biomechanical measurement of kinocilium. Met. Enzymol. 525, 21–43. doi: 10.1016/B978-0-12-397944-5.00 002-X Takahashi, S., Mui, V. J., Rosenberg, S. K., Homma, K., Cheatham, M. A., and Zheng, J. (2016). Cadherin 23-C Regulates Microtubule Networks by Modifying CAMSAP3’s Function. Sci. Rep. 6, 28706. doi: 10.1038/srep28706 June 2022 \| Volume 16 \| Article 876805 Frontiers in Cellular Neuroscience \| www.frontiersin.org 14
W4382045388.txt	https://journalsfp.org/article/75086.pdf	en		Volume 3 Issue 1 2019, Complete	Journal of solution focused practices	2,019	cc-by	36,648	Journal of Solution Focused Practices Volume 3 Issue 1 Article 12 10-2019 Volume 3 Issue 1 2019, Complete Follow this and additional works at: https://digitalscholarship.unlv.edu/journalsfp Recommended Citation (2019) "Volume 3 Issue 1 2019, Complete," Journal of Solution Focused Practices: Vol. 3: Iss. 1, Article 12. Available at: https://digitalscholarship.unlv.edu/journalsfp/vol3/iss1/12 This Article is protected by copyright and/or related rights. It has been brought to you by Digital Scholarship@UNLV with permission from the rights-holder(s). You are free to use this Article in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you need to obtain permission from the rights-holder(s) directly, unless additional rights are indicated by a Creative Commons license in the record and/ or on the work itself. This Article has been accepted for inclusion in Journal of Solution Focused Practices by an authorized administrator of Digital Scholarship@UNLV. For more information, please contact digitalscholarship@unlv.edu. et al.: Volume 3 Issue 1 2019, Complete Volume 3, Number 1 – 2019 IN THIS ISSUE Editorial — Sara Smock Jordan, David Hains Peer-reviewed articles Changing How We Think About Change ¾ Evan George A Brief, Informal History of SFBT as Told by Steve de Shazer and Insoo Kim Berg ¾ Peter De Jong Leaving No Footprints ¾ Chris Iveson It Will Never Be the Same Again ¾ Alexey Mikhalsky, Plamen Panayotov, & Alasdair MacDonald Solution Focused Work as an Aesthetic ¾ Mark McKergow Solution Focused Therapy for Trauma Survivors: A Review of the Outcome Literature ¾ Ray Eads & Mo Yee Lee Invited Papers The 3.0 version of “Reflections on Mark’s paper SFBT 2.0 - The new generation of SFBT has already arrived” ¾ Harry Korman JSFBT Journal of Solution-Focused Brief Therapy Response to Harry Korman’s Reflections on SFBT 2.0 paper ¾ Mark McKergow Published by Digital Scholarship@UNLV, 2020 1 Journal of Solution Focused Practices, Vol. 3 [2020], Iss. 1, Art. 12 Volume 3, Number 1¾ 2019 EDITORIAL Sara Smock Jordan ..............................................................................................................1 David Hains..........................................................................................................................4 PEER-REVIEWED PAPERS Changing How We Think About Change...............................................................................7 Evan George A Brief, Informal History of SFBT as Told by Steve de Shazer and Insoo Kim Berg.................9 Peter De Jong Leaving No Footprints.........................................................................................................17 Chris Iveson It Will Never Be the Same Again..........................................................................................27 Alexey Mikhalsky, Plamen Panayotov, & Alasdair MacDonald Solution Focused Work as an Aesthetic..............................................................................35 Mark McKergow Solution Focused Therapy for Trauma Survivors: A Review of the Outcome Literature.......47 Ray Eads & Mo Yee Lee The 3.0 version of “Reflections on Marks’s paper SFBT 2.0 - The new generation of SFBT has already arrived”..................................................................................................................66 Harry Korman Response to Harry Korman’s Reflections on SFBT 2.0 paper.................................................74 Mark McKergow https://digitalscholarship.unlv.edu/journalsfp/vol3/iss1/12 2 et al.: Volume 3 Issue 1 2019, Complete Journal of Solution Focused Brief Therapy (JSFBT) Editor: ISSN 2203-6784 Dr. Sara Smock Jordan, Program Director, Couple and Family Therapy, Associate Professor, University of Nevada, Las Vegas Published by: Associate Editors: Australasian Association for Solution Focused Brief Therapy Inc. (AASFBT) Rayya Ghul, Programme Director, Post-graduate Certificate in Learning and Teaching in Higher Education and Senior Lecturer in Occupational Therapy, Canterbury Christ Church University, UK Suite 401 51 Rawson Street Epping NSW 2121 Australia www.solutionfocused.org.au Dr. Frank Thomas, Professor of Counseling and Counselor Education, College of Education, Texas Christian University, Fort Worth, USA Editorial Board: Dr. Mark Beyebach, Assistant Professor, Departmento Psicologia y Pedagogia, Universidad Publica de Navarra, Pamplona, Spain Brian Cade, Private practice, Cirencester, UK Rights and permissions: All articles published in JSFBT become the property of the journal. To obtain rights to reproduce any material published in the journal, please email admin@solutionfocused.org.au Authors have full rights to alter and/or republish their own material with appropriate acknowledgement. NOTE: All views expressed in papers published in this journal reflect the opinions of the individual author(s) and are not the views of the Editorial Board or of the Australasian Association for Solution Focused Brief Therapy. Dr. Susan Colmar, Program Director, School Counselling and School Psychology, Faculty of Education and Social Work, University of Sydney Australia Dr. Peter De Jong, Emeritus Professor, School of Social Work, Calvin College, Michigan, USA Kirsten Dierolf, Director, Solutions Academy, Germany Dr. Cynthia Franklin, Stiernber/Spencer Family Professor in Mental Health, The University of Texas at Austin, USA Dr. Adam Froerer, Assistant Professor, Illinois School of Professional Psychology, Argosy University, Chicago USA Brent Gardiner, Programme Leader Counselling & Guidance Institute of Education, Massey University, Palmerston North, New Zealand Dr. Wei-Su Hsu, Professor, Department of Educational Psychology and Counseling, National Taiwan Normal University, Taiwan Chris Iveson, Co-director, BRIEF, London, UK Dr. Micheal Kelly, Associate Professor, School of Social Work, Loyola University, Chicago, USA Dr. Harry Korman, Director, SIKT, Malmo, Sweden Dr. Mo Yee Lee, Professor, College of Social Work, Ohio State University, Columbus, USA Dr. Alasdair Macdonald, Consultant Psychiatrist, Dorset Healthcare University Foundation NHS Trust UK ii – Journal of Solution Focused Brief Therapy Published by Digital Scholarship@UNLV, 2020 Journal of Solution Focused Brief Therapy – iii 3 Journal of Solution Focused Practices, Vol. 3 [2020], Iss. 1, Art. 12 Dr. Mark McKergow, Director, Centre for Solutions Focus at Work, and Visiting Research Fellow, University of Hertfordshire, London, UK Karin Thorslund, Co-director, Gotesborgs Institut for Losningsfokus, Gotesbords, Sweden Dr. Andrew Turnell, Director, Resolutions Consultancy, Perth, Australia Dr. Svea van der Hoorsn, Visiting Senior Lecturer and Examiner, Graduate School of Business, University of Capetown, South Africa Mr. John Wheeler, Director, Solution Focused Trainers, and External Lecturer in Social Work, Durnham University, UK EDITORIAL POLICY The Journal of Solution Focused Brief Therapy is a scholarly journal that aims to support the Solution Focused community through the publication of high-quality research in outcome, effectiveness or process of the Solution focused approach and the publication of high quality theoretical and/or case-study related material in the area of Solution Focused practice. The journal invites submissions as follows: Research reports – We are committed to helping expand the evidence base for Solution Focused Brief Therapy and Solution Focused Practices. The journal seeks scholarly papers that report the process and results of quantitative and/or qualitative research that seeks to explore the effectiveness of Solution Focused Brief Therapy or seeks to explore the aspects of the Solution Focused process. We are also committed to research reports being “userfriendly” and so invite authors submitting research-based papers to address specifically the implications of relevance of their research findings to Solution Focused practitioners. Theoretical papers – The Solution Focused approach raises many issues relating to psychotherapy theory, to our basic assumptions of working therapeutically and to the philosophical stance adopted by Solution Focused practitioners. The journal welcomes papers that explore these issues and which offer novel arguments or perspectives on these issues. Case study/Practice-related papers – We are committed to the journal being related to Solution Focused PRACTICE. Therefore, we invite papers that explore the experience and perspective of practitioners. This might be a single case study, with significant analysis and reflection on the therapeutic process and which the distills some principles or insights which might be replicable, or it might be a paper which explores a series of clinical/practical cases and which seeks to draw out overarching principles which might be used by others. Please discuss your ideas with the Editor (sarasmockjordan@gmail.com). Not just “therapy” – The Journal recognizes that many useful and interesting manifestations iv – Journal of Solution Focused Brief Therapy https://digitalscholarship.unlv.edu/journalsfp/vol3/iss1/12 of the Solution Focused approach occur in settings that are not to do with therapy. Nonetheless, Solution Focused interventions are all concerned with helping to facilitate change. The journal is called the Journal of Solution Focused Brief Therapy, at least in part in homage to our heritage. Nonetheless, the journal welcomes submissions that explore the use of Solution Focused ideas in other settings. SUBMISSION OF MANUSCRIPTS Manuscripts Manuscripts should be sent to the Editor as Microsoft Word or Apple Pages word processing documents. Please do not submit your manuscript elsewhere at the same time. Please send the manuscript double spaced with ample margins and a brief running head. The title of the paper should appear on the first page. Since all manuscripts will be blind reviewed, please include names, affiliations, etc. of the author or authors on a SEPARATE first page. Please also include on this (or a next) page details of any grants that have supported the research, and conference presentations relating to the paper, any potential (or even perceived) conflicts of interest. Solution Focused Brief Therapy and Solution Focused may be abbreviated to SFBT and SF after the first mention. References should follow the format of the American Psychological Associations (Publication Manual of the American Psychological Association, 6th ed.). Papers should include an abstract of no more than 150 words. Any tables, figures or illustrations should be supplied on a separate pages (or in separate computer files) in black and white and their position indicated in the main document. For any images or photographs not created by the author, the submission must include written permission to reproduce the material signed by the copyright holder. We would expect that papers will ordinarily me a maximum of 5,000 words; however, this limit is negotiable if the content of the paper warrants more. Clinical/client material This journal’s policy is that any actual clinical details in a paper (including but not limited to, therapy transcripts, client/patient history, descriptions of the therapy process) should have signed consent from the clients/patients for the material to be published. If a paper includes clinical material or descriptions, please include a declaration, signed by the first author, either that signed consent of clients/patients, specifically for the publication of their clinical information in this journal, has been obtained and is available for review OR that clinical material has been altered in such a way as to disguise the identity of any people. Fictional case examples can be used to illustrate techniques/ideas if consent from real clients in your practice can’t be obtained. Journal of Solution Focused Brief Therapy – v 4 et al.: Volume 3 Issue 1 2019, Complete Peer Review Manuscripts will be reviewed by at least two members of the Editorial Board or ad hoc reviewers, who will be asked to recommend that the paper be accepted, revised, or rejected for publication; however, a final decision about publication rests with the Editor. Reviewers will also be asked to indicate what kinds of changes might be needed in order for the paper to be published. Where reviewers have indicated that the changes are required or recommended, we are happy to work with authors to address the reviewers’ comments. When the reviewers recommend that the paper not be accepted, and the Editor accepts this/these recommendation, a final decision of reject is made by the Editor and no further consideration of the paper will begiven. When the reviewers (and the Editor) suggest that your paper, while it may have merit, does not meet the requirements for this journal, we will endeavor to suggest other journals to which the author might submit the paper; however, we are under no obligation to help achieve publication in our journal or in other journals. Where one or more authors of a paper is a member of the Editorial Board, that person will take no part in the review process and the review process will still be anonymous to the author or authors. Send manuscripts to: sarasmockjordan@gmail.com vi – Journal of Solution Focused Brief Therapy Published by Digital Scholarship@UNLV, 2020 Journal of Solution Focused Brief Therapy – vii 5 Journal of Solution Focused Practices, Vol. 3 [2020], Iss. 1, Art. 12 ACKNOWLEDGEMENTS We would like to thank our generous donors for making this issue possible. A New Chapter Australasian Association for Solution-Focused Brief Therapy Sara Smock Jordan Editor-in-Chief, Journal of Solution Focused Brief Therapy and Franceschi Foundation http://www.fondazionefranceschi.org/ viii – Journal of Solution Focused Brief Therapy https://digitalscholarship.unlv.edu/journalsfp/vol3/iss1/12 As a doctoral student, I had the opportunity to be an editorial assistant of a top-tier scholarly journal. Within a few weeks, I knew I wanted to be an editor someday. At that time, JSFBT didn’t exist. I remember asking myself “What journal would be interested in an editor who focused on furthering the evidence-base of SFBT?” Time passed and I entered my first academic position as an assistant professor. More time passed and I finally obtained promotion and tenure. For the first time in a while, I revisited my interest in becoming an editor. Several years ago, I was asked to join the editorial board for the new Journal of SolutionFocused Brief Therapy. I was very excited and honored to be part of JSFBT. As a member of the JSFBT editorial board, I was impressed with the quality of JSFBT over the years. I eagerly awaited the arrival of a new issue. After a lapse in publication, I became concerned. The value of this journal is so great I didn’t want to see it dissolve. This spring, I received an email from David Hains stating that AASFBT was looking for a new editor. I have to be honest, I thought “Could this be true? Could I be someone they might be interested in for the role?” I emailed David back, trying not to sound too eager, letting him know I was interested in the position. One day, David emailed me saying that he would like me to step into the JSFBT editorship. I agreed. We began to talk about possibilities for how JSFBT could be restructured. The excitement began to grow within me. I remember thinking to myself “Is this really happening? Am I really the new editor?” My excitement about the JSFBT editorship was directly connected to the importance of the journal to the SF community. In academia, publishers ask “why do we need a new journal?” In the case of JSFBT, the question was “why should JSFBT continue?” JSFBT is important to the international community because it provides an outlet for scholars, clinicians, and practitioners from various disciplines to share their work with others. Steve and Insoo passed before JSFBT was launched, however there is no doubt in my mind that they would support and encourage the continuation of the journal. Both Steve and Insoo understood the need for SFBT to be recognized as an evidence-based practice, as well as the importance of maintaining the validity of SF. I honestly believe that JSFBT fulfills their vision Journal of Solution Focused Brief Therapy – 1 6 Editorial et al.: Volume 3 Issue 1 2019, Complete of disseminating quality SF work and widening the evidence-base of SFBT. When asked to be the editor of JSFBT, I expressed my apprehension about JSFBT’s sustainability. Early in our discussions, David and I began to brainstorm ideas for increasing the support for the journal. One idea was to include co-sponsors. During a zoom call about the possibility of co-sponsors, I started to become emotional; I began to cry. I became so overwhelmed with the hope and opportunity to collaborate with various organizations on an international level. Across groups, cultures, practices, and schools of thought, differences exist. Could the re-birth of JSFBT be a common cross-cultural thread that would join and unite individuals and groups from around the world? The excitement began to grow even more. My vision of JSFBT was now more than generating quality materials but bridging a community of like-minded people. First, we developed a plan to publish a crowdfunding issue of JSFBT. Our intent was to generate funds for JSFBT’s production. We also discussed platforms for JSFBT. We wanted a low cost, easily manageable option, and eventually decided on an open-access platform. We discussed how we would financially support the costs associated with the journal. The AASFBT had produced the journal on a shoestring budget, but the JSFBT could not be sustained or grow on such a tight budget. Through many conversations, we developed a plan to have cosponsoring organizations fund the journal. We started by asking the larger SFBT organizations to be our main co-sponsors, and over the next few months we plan to open the invitation to other smaller organizations who wish to support JSFBT. Needless to say, it’s been a busy summer. As JSFBT continues to evolve, my best hopes are the following. First, that the journal becomes more widely accessible to individuals and groups around the world. Starting in 2020, the journal will be published using an online open-access platform. This will promote a worldwide readership. In addition, our hope is to translate abstracts into various languages. Second, my hope is to widen the scope of JSFBT’s articles, including SF manuscripts from a wide variety of topics, populations, and disciplines. This widened scope will foster further innovations for the SF approach. Third, my hope is that the journal will further promote SFBT’s recognition as an evidence-based practice. Maintaining a SF journal will greatly increase the amount of SF research generated and published. Fourth, the journal plans to be more intentional about mentoring new scholars and non-native speakers. The hope is to develop a mentoring program that will encourage individuals and groups to submit their creative ideas and work. So, what small steps need to happen to make these best hopes possible? You! It will take a community of SF individuals who are willing to writing up their new ideas, serve on the Editorial Board, and volunteer to be ad hoc reviewers. It will also require groups/associations to donate or co-sponsor financially. I am so honored and excited to be the new editor of JSFBT. Over the next few months, the editorial board and I will unveil more small steps to accomplishing these best hopes. The future looks bright and I’m thrilled to be part of this international, collaborate effort to leave 2 – Journal of Solution Focused Brief Therapy Published by Digital Scholarship@UNLV, 2020 Editorial a legacy of SF materials! Sara Smock Jordan Email: sarasmockjordan@gmail.com Journal of Solution Focused Brief Therapy – 3 7 Journal of Solution Focused Practices, Vol. 3 [2020], Iss. 1, Art. 12 The Day I Triumphed Over Hofstadter's Law (but not Impostor Syndrome) David Hains President, Australasian Association for Solution Focused Brief Therapy Solution Focused Brief Therapy, Impostor Syndrome, and Hofstadter’s Law. I can link all three things, but only inside my head. I can’t find any previous writing or research how these 3 things may be linked. While SFBT is my friend, the other two are my greatest enemies. If the JSFBT gets published in 2019 then I have finally gotten one-up on Hofstadter’s Law. (I can’t find anything previously written about the link between Hofstadter’s Law and SFBT, but I note that Frank Thomas referenced Douglas Hofstadter back in 1996). Hofstadter's Law states that important or worthwhile tasks will always take longer than you expect, even when you take into account Hofstadter's Law. It doesn’t really matter what I do, Hofstadter’s Law slows me down…until now. From here on Hofstadter’s Law will consume me no more. I’m not sure what it is about SFBT that helped me conquer Hofstadter’s Law. I never consciously thought about applying SF principles when trying to revive the JSFBT. Perhaps SF is just ingrained in me to the point that I can only focus on my preferred future? Maybe it is because talking about SFBT is so much more fun than the boring (non-SF) stuff I usually do? In retrospect, perhaps it was my other nemesis, Impostor Syndrome, that finally pushed me through? Whatever it was, I admit that the last 12 months have been pretty cool! Most mornings I wake up, check my inbox and find emails from all around the world – Sara, Mark, Harry, Chris, Evan, Alasdair, Frank, Peter, Gale, Kirsten, Rayya; I’ve seen your videos, I’ve read your books and your papers, but now you are emailing me? Ok, I admit that I am a solution focused nerd; your emails make me feel as excited as a kid in a candy shop. Why are they contacting me? I am just a nurse from Adelaide with a long history of suffering from Impostor Syndrome. As it turns out, my international colleagues heard a rumour that the Journal of Solution-Focused Brief Therapy was coming back to life, and they wanted to make sure that it wasn’t just a rumour. What have I learned over the past 12 months? There’s an enormous amount of international support for a peer-reviewed solution-focused journal, and 4 – Journal of Solution Focused Brief Therapy https://digitalscholarship.unlv.edu/journalsfp/vol3/iss1/12 Editorial a lot of people want to be a part of its success. The story so far … The Australasian Association for Solution Focused Brief Therapy (AASFBT) was started in 2013. AASFBT’s original aim was to publish an academic journal as a service to the worldwide Solution Focused community. Michael Durrant, the founding President of AASFBT and Editor of the JSFBT, was the driving force. Michael assembled an editorial board consisting of scholars and practitioners from 20 different countries. Over the course of four years, four issues were published. It is fair to say that Michael did most of this work on his own. The journal was his passion. He invested countless hours establishing the JSFBT. He solicited manuscripts, assembled papers, arranged peer reviews, completed copy edits, formatted content, printed and mailed hard copies of the journal, and all of this without payment. Amazingly he was able to produce a world-class publication for as little as AU$3,000 per edition. The AASFBT, and the International Solution Focused community are indebted to Michael for the work he has done. Given the demands of maintaining an academic journal, the workload was too much for one person, or perhaps he too was defeated by Hofstadter's Law? Back in July 2018 I took over as President of the AASFBT. One of my goals was to get the journal back into publication. It was clear to me that in order for JSFBT to succeed it would need more resources than what the AASFBT could provide, so we began to discuss ways to restructure the production of JSFBT. The first step was to find a new editor, someone who could build on the foundation set by Michael, someone who could expand and maintain the journal through the worldwide community. I was VERY happy that Sara Smock Jordan applied for the role as editor of JSFBT. It only took a couple of emails before I knew we had the right person for the job. Sara first volunteered to serve as editor on May 1, and was unofficially appointed on May 23. Now, less than 5 months later, our new team has published volume 3, issue 1! In addition, we are establishing a permanent new platform for future issues of the journal in 2020. Our first issue with our new online platform is planned for July of 2020. Take that Douglas Hofstadter! Sara really needs no introduction. She is one of the most well-respected people in the international solution-focused world. From her early days as a graduate student, Sara had the opportunity to learn the model from Steve, Insoo, and key members of the original Milwaukee team. Sara has studied, practiced, researched, and published extensively on SFBT. She brings an amazing amount of both clinical and academic expertise to her new role. Sara is a founding member of Solution Focused Brief Therapy Association, and past president of the association. Currently, she serves on SFBTA’s board of directors and is a member of the research committee. We are excited to have Sara as our new Editor and have been working with her to rebuild the journal. This special edition is the culmination of 5 months’ worth of work by Sara, her editorial assistant Kaitlin Andrewjeski, and the editorial board. I would like to personally thank everyone involved for their work, dedication, enthusiasm, and encouragement in getting the journal up and running again. Journal of Solution Focused Brief Therapy – 5 8 Editorial et al.: Volume 3 Issue 1 2019, Complete International collaboration: it takes a community to sustain a journal! From the very beginning, it was clear to me that the journal had to be supported by more than one association. While we are proud and grateful for what Michael and the AASFBT established, my desire was to have a fully international publication. Associations around the world are beginning to commit to the journal and we would like to invite other groups to join us as cosponsors. In 2020 we will be establishing an open-access publication, providing free issues of the journal. While this platform will provide full international exposure of SFBT, costs of producing the journal still exist. Therefore, we call upon the solution-focused community organisations, conferences, collectives, and individuals - to financially support the work. So, welcome to this special edition of the Journal of Solution-Focused Brief Therapy. I hope you are as excited as I am to be reading this special edition (I haven’t actually seen a copy yet!). Thank you for purchasing a copy. The money raised from this edition will be used to help fund the 2020 editions as we move to a full production schedule of two issues per year. From here, I will hand over the journal to Sara and wish her and her team every success in rebuilding the journal. As the President of AASFBT, I will stay involved in a managerial/oversight role along with our co-sponsors to build a truly international publication. Please do not hesitate to contact me should you have any questions, suggestion, or submissions regarding the journal, SFBT in Australasia, or even just to say g’day, or to feed my impostor syndrome. David Hains Email: david@leftturnsolutions.com.au https://www.solutionfocused.org.au/journal Reference Thomas, F. (1996). Solution-focused supervision. In Miller S., Hubble M., Duncan B. (eds) Handbook of solution-focused brief therapy. Wiley. 6 – Journal of Solution Focused Brief Therapy Published by Digital Scholarship@UNLV, 2020 Changing How We Think About Change Evan George BRIEF: The Centre for Solution Focused Practice What happened to the word “change?” I have recently completed four really enjoyable days of training in Liverpool and what the training has left me thinking about, amongst many other things, (including how much I like the city of Liverpool, of course), is the small word “change.” In fact, I have come to realize just how uncomfortable I feel with the word. The realization came to me during a discussion. We were talking about what we can ask when someone is finding the ‘best hopes’ question hard to answer. One of the groups, very appropriately, suggested that we might reframe the question. What she might ask, I wondered, and her first thought was something on the lines of “so what do you want to change?” I immediately knew that this was not a question that I would ever want to ask. The first reason for my certainty was simple. If you ask, “so what do you want to change?” inevitably it invites the client into a problem-framed response, “well it is their behaviour that I want to change.” The question is not a million miles from “so, what brings you here today?”, again a question that will pretty well guarantee a problem-based answer. The second reason was trickier to articulate. I just don’t use the word change! Now isn’t that strange? A therapist (or counsellor or coach or consultant) who feels uncomfortable using the word change. So, what is going on I began to wonder. It seems to me that the idea of change is fading into the background of the way that we conceptualise as Solution Focused practitioners. Obviously, the mechanics of the change process disappeared long ago; certainly in the BRIEF version of the approach. We do not ask prospective strategy questions, “How are you going to do that?” questions. We take the mechanics for granted linguistically jumping over them, focusing on the difference that the change makes. This is in some ways a development of the miracle question. The client wakes up and the change has happened. We are not interested in inviting the client to focus on how it is going to happen; what they must do to have it happen. We merely invite the client to describe their miracle day, how they will be able to know that the miracle has happened. I Journal of Solution Focused Brief Therapy – 7 9 Evan George Journal of Solution Focused Practices, Vol. 3 [2020], Iss. 1, Art. 12 notice that I tend to do something similar with the scale question. “Imagine that you move one point up on the scale and find yourself at 4” (for example). “How will you know that you are at 4?” People are invited to “find” themselves one point higher in my questions; the process of the change has disappeared. We do not invite the client to be interested in the prospective ‘how’. Now, this disappearance of the prospective process of change, which in some ways we can date back to the early days of the approach (even though Steve de Shazer regularly [and rhetorically in my view] insisted on asking clients “so how are you going to do that?”), seems to have strengthened its hold on the way that many of us use the approach. Our friends and colleagues Elliott Connie and Adam Froerer are I think to be credited with the appearance of the word “version” in our conversations with clients. For example, we might ask, “imagine that you wake up tomorrow the confident and optimistic version of you – what is the very first thing that you will notice?” What interests me about the word ‘version’ is that any sense of change disappears. The “confident and optimistic version” is already there, latent in the client’s life. It is not an unrealized possibility that the client has to put in place or construct. It really is already there. Clients can wake up living the life of that version of themselves. If clients answer the question, they are implicitly accepting the premise. This small, but in my view significant, tweak to the way that we think and the way that we construct our questions builds on the clarity that BRIEF’s Chris Iveson brought to our thinking some years ago. Chris challenged whether we are in the business of change. We are, he counter-proposed, in the business of perspectives. We invite clients to look into the shadows of their lives, the hidden corners, and to notice the elements of the preferred future that are already in place, just not noticed, invisible and perhaps unseeable while the light is shining so strongly on the problems and difficulties that have come to occupy the fore-ground, the frontstage, of our clients’ lives. The brighter the light that shines on the problems the deeper the shade that obscures the instances and exceptions. As we look into a bright light our eyes, quite literally, find it harder to see in the gloom, the behind, the beyond. And in our lives our perception seems to operate rather like our eyes. So, people are not having to make changes, we are merely inviting them to shift their gaze. This really is quite a step from the early days of our approach when Steve de Shazer emphasized that one of the characteristics of “wellformed” or “workable” goals is that they should be perceived by the clients as involving their “hard work”’ (de Shazer, 1991, p.112). Even the word ‘goal’ has disappeared! We have come a long way in our thinking. Reference de Shazer, S. (1991). Putting difference to work. New York: Norton. Evan George Email: evangeorge@brief.org.uk 8 – Journal of Solution Focused Brief Therapy https://digitalscholarship.unlv.edu/journalsfp/vol3/iss1/12 A Brief, Informal History of SFBT as Told by Steve de Shazer and Insoo Kim Berg Peter De Jong International Microanalysis Associates This account of the origins and development of SFBT is not based on a rigorous historical analysis of key events, recovered unpublished documents, or the formal writings of de Shazer, Berg, and their colleagues at the Brief Family Therapy Center (BFTC). Rather, it is taken from the author’s records of two experiences he had with de Shazer and Berg: 1) notes from a lecture de Shazer gave about the history of SFBT just one month before his death, and 2) a recorded interview with Berg in 1998 about the origins of BFTC and SF techniques. The article concludes with a few reflections by the author. In the latter part of the summer of 2005, I (Peter De Jong) came to Milwaukee, Wisconsin from Michigan for a couple of weeks as I had done every summer since 1990. I came mainly to work with Insoo Kim Berg on our joint projects and related writings. I would, however, also sit in on the workshops Insoo and Steve conducted each summer, and stay at their home, so we could discuss the workshop content and the participants’ responses to the material. These discussions helped inform Insoo’s and my writing about how to make SFBT more accessible to learners. The workshops were always run by Insoo. I would sit next to Steve, both of us drinking coffee, until Insoo asked one of us to do something; such as a demo of a miracle question conversation or address a particular topic. Insoo began that 2005 summer workshop as she always did, asking the participants what they would like to have heard, seen, or done by the end of the workshop. Predictably, among the requests was the wish to hear about how Steve, Insoo, and their colleagues at BFTC developed their innovative SF techniques. So, part way through the workshop Insoo asked Steve to talk about the history of the approach, and I took notes on what he said. The version of BFTC’s history contained in this article is reconstructed from those notes. It is supplemented with quoted comments from Insoo taken from a recorded interview I did with her in 1998 about the origins and development of SFBT. I conclude the article with my reflections from Steve’s lecture and Insoo’s comments. Steve & Insoo’s Telling of the History The story begins in the 1970’s at the Mental Research Institute (MRI) in Palo Alto, Journal of Solution Focused Brief Therapy – 9 10 Peter De Jong et al.: Volume 3 Issue 1 2019, Complete California. There, the likes of Don Jackson, Jay Haley, and John Weakland had been developing a form of brief therapy since the inception of MRI in 1958. Steve and Insoo, both from Milwaukee but unknown to each other at the time, came to Palo Alto wanting to learn more about the ideas and practices of MRI. They learned, among other things, that MRI's practice was, in part, inspired by the work of the psychiatrist Milton Erickson. Some of MRI's therapists including Haley, would visit Erickson in Arizona and talk to him about his cases. Haley, especially, has written about Erickson's work describing many of his cases (Haley, 1986). Erickson's practice with clients was short term, sometimes included hypnosis, and always involved Erickson doing something to bring about change. Steve became fascinated by Erickson’s work. While Erickson did not develop and write about a detailed model of doing therapy, he did describe many cases and what he did with them. For example, there was the case of a twenty-one-year-old woman who came to Erickson saying she was thinking of ending her life. She said she wanted a husband and children but had never had a boyfriend and felt she was too unattractive to attract a man. She said she worked as a secretary at a construction firm and kept to herself. There was a young man at work who she found attractive, who showed up at the drinking fountain when she did, and who seemed interested in her; however, she never spoke to him. She had no friends and believed she was “too inferior to live.” She decided to see a psychiatrist before ending her life, telling Erickson she would work with him for three months before carrying out her plan. Erickson thought the woman was pretty but dressed very unattractively and her hair was stringy and unevenly cut. The woman told him her main physical defect was a gap between her two front teeth which she self-consciously covered with her hand when she talked. Erickson responded to the woman by assigning two main tasks. First, he told her that since she was going downhill anyway, she might as well have one “last fling.” She was to go to an assigned store and get help selecting an attractive outfit and then to an assigned beauty shop to have her hair styled. Erickson said she accepted the task because she did not interpret doing the task as improving herself but only having a “last fling.” Second, she was to go home and, in her bathroom, practice filling her mouth with water and then squirting the water through the gap in her front teeth. She was to practice until she could squirt the water up to six feet and do so with accuracy. Erickson said she thought this was a silly task, but its silliness apparently prompted her to go home and practice it conscientiously. When the woman returned and was attractively dressed, her hair newly styled, and skillful at squirting water through the gap in her front teeth, Erickson proposed another task. This time she was to play a practical joke at the office. The next time the young man appeared at the fountain, she was to fill her mouth with water, turn, squirt it at him, run toward him a bit, and then immediately turn away and “run like hell down the corridor.” At first, the woman rejected this proposal as ridiculous, but later decided it could be part of having one “last fling.” So, the next day she went to the office dressed in her new outfit and looking very attractive. When she approached the water fountain the young man predictably appeared. She filled her 10 – Journal of Solution Focused Brief Therapy Published by Digital Scholarship@UNLV, 2020 A Brief, Informal History of SFBT mouth with water and squirted it at him. He yelled an expletive at her which made her laugh; she then turned and ran down the hallway. The young man chased her, caught her, and to her astonishment, kissed her. The next day, the young woman nervously approached the water fountain. The young man was hiding nearby and jumped out squirting her with a water pistol. Steve studied scores upon scores of Erickson’s cases trying to figure out his way of working with clients. Insoo had this to say about Steve’s study of Erickson’s work: He (Steve) is the type that when he’s interested in something he just reads and reads and reads …. That’s what he did with Erickson’s work; he just immersed himself. And he’s always looking for patterns that connect. So, he looked a lot really into Ericksonian patterns – what is it about his way, how can he describe the patterns – that seem so out of nowhere. Steve came to see at least two patterns that connected in Erickson’s way of treating cases. First, Erickson heard and tied his tasks and proposals for the client to the client’s goal. In the case of the young woman, he heard that she wanted a husband, a family, and friendships. Second, he creatively drew on qualities and skills that the client possessed and could be put to use in reaching the client’s goal(s) (the principle of utilization). In the case of the young woman, she had a space between her two front teeth and she mastered squirting water through that space. Steve also noticed that Erickson’s brief way of doing therapy -- often in just a few sessions -- was radical because it came at a time when therapy was indefinite. Many therapists believed clients regularly needed one-hundred or more sessions. When research studies at the time showed the average number of sessions for clients was about four sessions, therapists would bemoan this and say things like: “the client’s progress is only a temporary flight into health,” or “the client’s quick progress did not address the real or underlying problems,” or “leaving therapy so soon is a defense mechanism; it is a sign of client resistance to getting better.” In thinking about these common therapist explanations in the 1970’s for the average few number of sessions, Steve and his colleagues saw a “big disconnect” between these explanations and what their clients were telling them. As Insoo described it: In that time (early to mid-1970’s), families kept dropping out and dropping out of treatment. Families don’t tend to stay in treatment very long. Couples don’t tend to stay in treatment very long. And I didn’t know that at the time. So, I kept getting this uncomfortable feeling that this isn’t right, this isn’t right. The clinical phenomenon and what the theory says didn’t go together. So, I was in search for something – there must be an answer for this …. And I think that another thing that was interesting was that these ‘failure cases’ because by their (most therapists’) criteria – anything less than people who stay in treatment for less than a year – was a failure case -- because they are dropouts. Yeah, (but) dropout cases were sending their best friends, their family members … I only saw them three times and they must have thought that I helped ... because they are Journal of Solution Focused Brief Therapy – 11 11 Peter De Jong Journal of Solution Focused Practices, Vol. 3 [2020], Iss. 1, Art. 12 sending their sister, their mother – so I thought something isn’t right, something isn’t right, but I didn’t know what. As a consequence of this “disconnect,” Steve and Insoo began looking more carefully at the existing data about number of sessions. They saw that mental health facilities at the time were taking as many as six sessions to do extensive assessments before they began treating the assessed problems. Clients often “dropped out” before the assessments were completed. Nevertheless, clients said coming to the sessions had been helpful. Other data indicated 80 to 90 percent of therapy was less than 20 sessions; yet, most clients said therapy was useful. Steve then realized that clients were using therapy differently than most therapists thought they should. With Erickson’s work and the data about client number of sessions as background, Steve thought: Let’s build a brief therapy around the client goals; they obviously are using therapy that way anyway. So, he decided to listen more intentionally to and believe what clients say they want and say is useful for them. Meanwhile, by this time which was in the later 1970’s, Steve had returned to Milwaukee and joined the large family service agency where Insoo worked. Insoo had put a one-way mirror into her large office so she and colleagues could observe practitioners working with clients. They and their colleagues soon discovered that they could not simply ask the client: “What is your goal?” When they did that, the client would respond “to stop drinking,” or “to stop fighting with my teenage son,” or “to be less depressed.” These client responses were more like problem statements rather than goals. Accepting these responses as goal statements was not useful because practitioners know the hardest way to change is to try to stop something. Soon, Steve and Insoo observed and recognized that the clients who were making progress had discovered something else to do instead of the problem behaviors. So they began experimenting with questions like: “What are you going to do instead of the drinking?” “When you are not drinking, what will be there instead?” “What will be happening when you are not drinking anymore?” “What will others notice you doing when you are not drinking anymore?” These questions, too, were difficult for clients to answer. Clients would often first respond, “I don’t know.” So, Steve, Insoo, and their colleagues (who by now had formed BFTC in 1978) kept working at ways to ask questions about client goals in order to give clients maximum opportunity to construct useful goals for themselves. And then, in the early 1980’s, Insoo had the case of a woman who came to her saying she was depressed and was contemplating killing herself. She had several children with many problems themselves and a husband who drank too much alcohol and was out of work. Insoo began goal work with the woman asking questions like: “so what needs to happen here for you to say our meeting was useful?” And, “what do you want different by the end of our work together?” To these and several similar questions, the client responded “I don’t know” and she continued to give more details about the problems of her family and herself. Then, at one point the woman added the words “unless a miracle happens” to her “I don’t know’s.” Insoo, by this time intentionally attendant to client words, picked up the phrase and asked: “Okay, so suppose a miracle happens and all these 12 – Journal of Solution Focused Brief Therapy https://digitalscholarship.unlv.edu/journalsfp/vol3/iss1/12 A Brief, Informal History of SFBT problems are over, what would be happening instead?” The woman then began to answer, “my husband would stop drinking and have a job,” and “I would have more energy.” Insoo continued with “what else would be different?” The woman responded with ”my kids would be doing better in school.” As Insoo continued following up on each client answer and getting more details, she and the team noticed the woman became more animated and seemed less depressed and more hopeful. The differences in the woman from the beginning of the session to the end impressed the observing team so much that they decided, as they had been doing with each promising new technique, to ask the “miracle question” of all clients for the next several months and see what difference that made in the rates of client progress. The “miracle question” turned out to be so useful that BFTC made asking it a standard practice of their developing new form of brief therapy. Insoo has commented that the observing team at BFTC did not invent the miracle question; instead, it came from listening carefully to what clients say and then using that: You know, I think clients say that stuff all the time: “do you have a magic pill?” Or, “do you have an answer to this?” “I need an answer from you.” “I need a miracle from you.” “Or a magic wand from you.” I think clients say that all the time… But sometimes when the event, the case, and the circumstances come together, you hear them! (italics added) And I think that out of desperation (laughing), when the case seems so hopeless; out of desperation you hear them. That’s what happens a lot; and you get new ideas. It comes from that, not that we are so brilliant or so smart. But I think that, “oh my gosh, what do we do now?” creates that kind of crossroad, and then something opens up I think. Steve says the same sort of careful listening to the client and then building on what he heard led to the use of scaling questions in SFBT. Steve had a case shortly after the release of the American film “10” starring Bo Derek as a beautiful young woman and Dudley Moore as a middle-aged composer. Moore’s character, experiencing a mid-life crisis, becomes infatuated with the young woman whom he rates as “11” on a scale that only goes up to 10. The film was very popular and the practice of rating things on a 10 point scale was finding its way into popular culture. Steve’s case was a man who was returning for a later session and Steve asked him how he was doing. The man said: “I’m doing better.” Steve asked: “How much better?” The man replied, “Well, I’m not a perfect 10; but I’m about an 8.5.” Then Steve asked: “So what tells you it’s ‘about an 8.5’?” The client went on to describe the progress he had been making. After that case, and through discussion and reflection with Insoo and his colleagues, Steve came to realize scaling was so useful because the client had to be scaling himself relative to his own goal(s), not some professional, supposedly objective, criterion of success. And, in asking the client to provide the details for the number he gave, the client and Steve became clearer about what it is the client wanted different in his life which, in turn, made deciding what to do next easier. Once it dawned on Steve that the client’s goal was implicit in the scaling numbers for Journal of Solution Focused Brief Therapy – 13 12 et al.: Volume 3 Issue 1 2019, Complete Peter De Jong progress, he also began to notice that many clients who were making progress came into later sessions already describing what was better. Implicit in these descriptions was what clients wanted different in their lives, i.e. their developing goals. So, Steve and his colleagues began asking “what’s better?” at the beginning of follow-up sessions and then lots of follow-up questions to get the details of what was better. This began in the early 1980’s and was a new direction because in the late 1970’s and into the early 1980’s, BFTC practitioners were still influenced by practice at MRI and would give clients tasks intended to bring change. As the tasks were intended to change clients and/or their situations, it was natural to begin later sessions by asking clients whether they had done the tasks and what the results were. In shifting toward conversations about what was better and away from asking whether clients completed their tasks, Steve and his colleagues discovered most clients, if asked, could identify something better. As BFTC practitioners pursued this new line of questioning, figuring out more and more ways to keep the conversation about what was better going, even when some clients would start out by saying “nothing,” they found over 90 percent could identify something better. The more they stayed with the “what’s better” opening in their follow-up sessions, the less they focused on asking about tasks and designing intricate tasks based on family systems thinking as they had been doing earlier. Steve says a bonus to shifting toward asking “what’s better” was the discovery that what clients described as “better” often had nothing to do with the original problem(s) that brought them to therapy. The bottom line here, Steve says, is that clients define success differently than most practitioners who try to help clients by assessing and solving their problems. He came to realize that in listening ever more intentionally to what clients want and inventing with them more and more ways to invite them to describe in detail what they want and the progress they are making, clients were teaching the practitioners at BFTC to move away from problem solving in favor of building solutions in partnership with them. Reflections I (the author) am struck by three things in Steve’s brief telling of the history of BFTC and SFBT. The first is his focus on just a few years of that history; namely, from the mid-1970s to the mid-1980s. I wonder if this was the period, in his mind, when the key discoveries were made at BFTC. It was the period when BFTC began abandoning the theories and practices of the field of psychotherapy in general and differentiated itself in practice and thinking from MRI. It is also the brief period during which the unique solution-focused questions and practices were invented at BFTC that have endured to the present as heart of SFBT. Second, I am struck by Steve’s emphasis throughout that the team at BFTC learned to listen to clients in a different way. While the rest of the field was using professionally constructed categories to assess client problems and then move to helping clients with related interventions, BFTC practitioners were learning to listen to clients on their terms versus the lens of the field. Steve and Insoo first noticed the “big disconnect” between the field’s view 14 – Journal of Solution Focused Brief Therapy Published by Digital Scholarship@UNLV, 2020 A Brief, Informal History of SFBT about how much therapy clients needed and how clients were using just a few therapy sessions and finding that useful. BFTC believed the clients about the usefulness of just a few sessions and began listening more intentionally to what clients said they wanted and what progress they were making. In a sense, BFTC closed the textbooks about how to do therapy in favor of listening to their clients. And, as Insoo said, “…sometimes when the event, the case, and the circumstances come together, you hear them (the clients)!” The increasing BFTC capacity to hear clients on their terms rather than through professional categories, led to the signature SF questions and practices. Third, I am impressed by the approach to investigation and knowing that BFTC adopted. While early on Steve and Insoo experimented with practices drawn from family systems theory, they soon set that approach aside in favor of direct observation of therapy sessions. Insoo put a one-way mirror in her office at the family service agency in the 1970’s. One-way mirrors and direct observation and review of recorded sessions remained a central feature of practice, research, and learning at BFTC until Steve and Insoo’s passing. The colleagues at BFTC consistently observed for which clients were making progress and what those clients and their practitioners were doing together that might be contributing to that progress. When they noticed a client and practitioner collaborating in a new and potentially useful way (such as Insoo picking up on her client saying “unless a miracle happens”), they incorporated the innovation into their practice and formed a research study to measure its usefulness. Employing rigorous observation of real time and recorded sessions is what contributed most to listening to clients in a new way and the invention of SF techniques. Steve, in a book published in the 1990’s reaffirms the importance of such observation: Therapists are interested in the doing of therapy and, at least in a certain sense, only the observation of sessions or watching videotapes of therapy sessions can give them the ‘data’ they need [to learn SF and improve their practice skills] (de Shazer, 1994, p. 65). Reviewing these notes from Steve’s 2005 lecture and the 1998 interview with Insoo has gotten me thinking that we may have more to learn from this version of the history of SFBT than I first realized. Many of us who teach workshops and write about SF practices, tell our learners that SF is “simple but not easy.” That is to say, it is simpler to describe, understand, and teach the SF approach in concept than it is to actually conduct a SF conversation. On reflecting once again on Steve and Insoo’s history described in this article I wonder if we have ignored some of their genius in our teaching. I know that for the nearly thirty years that I have been teaching and practicing the SF approach, I have focused mainly on teaching the SF questions invented at BFTC together with the outlook about clients and practice embedded in those questions. This question-based approach largely ignores how SF questions were invented. In contrast, Steve, Insoo, and their colleagues themselves first “learned” the SF approach through direct observation of therapy sessions and listening to and learning to hear clients on their own terms. Having recorded sessions allowed them to revisit the words of what clients said and stay close to those words so as to reduce the natural tendency (often Journal of Solution Focused Brief Therapy – 15 13 Peter De Jong Journal of Solution Focused Practices, Vol. 3 [2020], Iss. 1, Art. 12 unintentional and below the level of awareness) to transform what clients say into the practitioner’s preferred or professional categories. Perhaps, I am thinking more and more, SF learning would be enhanced by consistently having our learners record their SF interactions from the outset of their learning. That is easier than ever to do with smart phones, laptop computers, and role playing. The teaching can then be organized around inviting learners to observe for what their “clients” are saying and what they and their clients are doing together that contributes to clients constructing detailed visions of what they want and measuring progress toward these goals as the clients define progress. In organizing SF learning around learners becoming keen observers of their own SF conversations, they will be reinventing the SF model for themselves. Doing it this way originally worked well for the BFTC team; perhaps shifting our teaching in that direction will produce similar results for today’s SF learners. Leaving No Footprints Chris Iveson BRIEF: The Centre for Solution Focused Practice Peter De Jong Email: pdejongsft@gmail.com Abstract In this paper, clients’ experiences of therapy are used to examine two essential paradoxes: neutrality and influence, in the minimalist version of Solution Focused Brief Therapy developed by the author and his colleagues at BRIEF. Both concepts are linked to trust, a radical trust in each client to know what is best for their future, and a trust that decisions about this future are solely the business of the client. Maintaining this trust in the face of our own ideas and good wishes towards our clients requires a discipline which may not suit all Solution Focused practitioners. Dressed to Kill Angela stepped in from the pages of Vogue; cool, assured and with an air of authority. She had been planning to kill herself when she read about Solution Focused Brief Therapy in The Times and decided to give it a go before carrying on with her plan. She had been drawn by the future focus and the apparent lack of need to talk about the past. She said she knew perfectly well the source of her problems but had no intention of talking about it. Angela’s adult life had been one of extremes. Having performed well at school she went so far off the rails that in her late teens she became a homeless heroin addict. A suicide attempt brought her to the attention of the mental health services and a successful rehabilitation programme. Picking up the threads of her life, Angela continued her education and became an accountant. Unfortunately, the past, as she put it, crept up on her and after a disastrous spell in a psychiatric hospital she once again became a homeless heroin-addict. It was barely possible to equate the ultra-fashionable, expensively dressed woman before me with the “bag lady” she had been through her late twenties. It was an attempted rape that “woke her up” a second time. She fought off her attacker and in doing so was reminded of her determination to survive. 16 – Journal of Solution Focused Brief Therapy https://digitalscholarship.unlv.edu/journalsfp/vol3/iss1/12 Journal of Solution Focused Brief Therapy – 17 14 Chris Iveson et al.: Volume 3 Issue 1 2019, Complete This time she went cold turkey’ alone, came off the heroin, cleaned herself up and began the long climb back into a more liveable life. Over the next few years, she built a successful career in the burgeoning financial services industry eventually securing a senior position in a small private investment company. With an expensive apartment, successful career and looks that had survived the ravages of homelessness and heroin she seemed to have her life in control until, once again, the past caught her out. Hearing music from her neighbor’s radio Angela found herself at his front door screaming abuse in his face. The next morning, on a crowded underground she punched a commuter for accidentally pushing her. She knew the signs: this was the road back to paranoia, drugs, homelessness, and death. This time rather than try to cope once again with the horrors of post-traumatic stress, Angela decided to go straight to death. She could not face the whole awful process yet another time. It was only the newspaper article that held her back. All this she explained in the first few minutes of our meeting before expressing a hope that she might just get on with her life without the past dragging her back. Once we have desired outcome, especially one that can begin immediately, we are likely to dispense with the miracle and begin with the less dramatic, less memorable, “Let’s imagine you wake up tomorrow. . .?” However, Angela seemed to be asking for something she was doubtful could happen and like many clients who have suffered childhood abuse she believed only the eradication of the past would give her back a future. In these cases, the deployment of a “small miracle” can sometimes open a very large door: Let’s imagine that tonight, while you are asleep, a miracle happens; it’s only a small miracle, it doesn’t get rid of the past, but what it does is stop the past messing with your future. What’s the first thing you’ll notice as you wake up that tells you are free to get on with your life without the past dragging you back? This was the opening into a rich and detailed description of Angela’s everyday life as she hoped to live it. Angela made good progress and according to brief therapy principles the time between sessions was stretched to accommodate this movement. The third session had been scheduled with a gap of two months, but after four weeks Angela asked for an emergency session. She arrived thinking that she might need to be admitted to hospital. While this was not good news, it was a step up from suicide. I asked her what was it about this crisis and the way she was dealing with it that was keeping her on the side of life, no longer thinking of suicide. She said that the advances she had made in the course of our work together had been a huge and pleasant surprise. Stopping her self-harming behaviour (something she had never mentioned until this moment!) and managing to hang on to her job had been big factors, but much more importantly, her friends had noticed big changes. She was more engaged with life, easier to be with, more outgoing and, as her closest friend put it, “softer.” Angela had always appeared super-confident but for the first time she was beginning to feel it. Until the previous day when she had lost her cool once again. Thinking that her boss was being patronising, she had shouted at him in front of the whole office. She was afraid the paranoia was coming back, 18 – Journal of Solution Focused Brief Therapy Published by Digital Scholarship@UNLV, 2020 Leaving No Footprints but after a little reflection, she decided that he actually was patronizing, and this had been troubling her for some time. On further reflection, she was even pleased that she had called him out, even though she might lose her job. We are always on the look-out for new behavior, even behaviors which at first sight might seem problematic. Any standing up to abuse, real or imagined is worth investigating. I asked Angela what had made the difference, what had decided her to stand up to her boss she said she had no idea but then told the following story. I went to visit some old friends at the weekend. They live out in the country and had been trying to get me to stay with them for years. For some reason I decided to go, and it turned out to be a lovely experience. The woman was an old school friend and reminded me what a popular girl I had been. We had long walks and long chats and I slept better than I can ever remember. It was just lovely. I was planning to leave on Monday morning, but they begged me to do them a favour by looking after their baby while they did the week’s shop. So, there I was with a baby plonked in my arms! Angela was sitting with her arms held stiffly out, mimicking her discomfort at having a child in them. I didn’t know what to do so I just sat there, then, I saw him looking at me. He just kept looking with his big round eyes and I couldn’t help looking back. It seemed to go on for ages, and gradually, I started to realize that this was a little human being, as perfect as anyone could be. As Angela continued her story her arms lost their rigidity and began to fold around the baby. I had such a strong feeling, like I’ve never had before. He was so beautiful, so perfect and I wondered ‘How could anyone harm an innocent child like you’. It was such a strange experience I wanted to cry. It was a moving story and perhaps a turning point since Angela then went on to say that she felt a new confidence in herself and did not think we would need to meet again! Then just before leaving Angela, looking uncomfortable, blurted out, “Before I go there’s something I’ve got to say to you!” I thought I was going to be told off and began running the past hour through my mind to find the cause! In fact, it was the reverse. She went on, “I know that if I had not come here I would now be dead, but I want you to be clear that you have not touched my life at all!” Rather than be offended as Angela expected I felt I had been afforded the most wonderful of compliments and responded, “And I have to say that no one has ever paid me such a compliment, and you haven’t touched my life either. But I’ll always remember you!” “And I’ll always remember you!” She said and stepped back into the pages of Vogue. These had been three very straightforward, description-focused sessions. The “small Journal of Solution Focused Brief Therapy – 19 15 Journal of Solution Focused Practices, Vol. 3 [2020], Iss. 1, Art. 12 Chris Iveson miracle” opened the door to a detailed description of Angela’s everyday life. As routine and hum-drum as the “behind-the-scenes” of most lives: making coffee, washing up, waiting for trains, sitting at computer screens, chatting to colleagues, meeting a friend and so on. Then a few minutes to begin looking at how much of this small miracle might already be happening. No summary, compliments, or tasks just a disciplined neutrality about whatever she decides to do tomorrow. Subsequent sessions were forensic examinations of progress. “What’s better?” with the emphasis on “How?” and following the ripples and counter-ripples of each achievement. Angela’s final words were not the only comment on the value of detail. At one point during the last session, she remarked on the fact that she could never remember any of my questions. My half-joking response was that maybe it was because her answers were more interesting, which, of course, they are. A question such as “What might you notice as you are making the coffee?” places the client in a most inconsequential moment. If, as we hope sometimes happens, the client’s answer opens a new door to possibility, it is the door, not the question, that will be remembered. If the answer is as inconsequential as the question, both are likely to be forgotten. In this way, the client’s own words take centre-stage adding to the experience of it being all their own work. Angela’s experience was not that our conversations had had no impact, she knew that they had saved her life. Nonetheless, like many clients she could not see the direct connection because everything she had done to turn her life around had come from her. The therapist had no part except that of a catalyst, an instigator of change. I might have walked with Angela, ahead, beside or behind, but every step she took was her own and only her own. From Goals to Hopes Several significant steps in the development of BRIEF’s work led towards this “hands-off, footprint-free” approach. One of the early, language-created short-comings of Solution Focused Brief Therapy was its use of the word “goal”. It is a word that carries with it the notion of something specific to be achieved and can easily divert the therapist’s attention towards overly specific outcomes and problem-solving. This has led to a confusion in de Shazer’s writing where sometimes he refers to “well-formed” and “achievable” goals (de Shazer, 1991, p. 112) and describes simple “problem-solving”, or complaint-focused, strategies by which they might be achieved (de Shazer, 1988, p. 93-96; 1991, p. 115-118). However, at other times, especially when describing 10 on a scale he defines the outcome as “the day after the miracle” (de Shazer, 1994, p. 231). This latter definition encompasses not just the specific goal, or problem resolution, but the whole way of life with which it is associated. It was the “way of life” outcome that most attracted the interest of my colleagues, Harvey Ratner and Evan George, and myself to BRIEF. We began to see the “miracle” not as the resolution of the problem, nor the achievement of a specific outcome; but more as the context or “way of living” within which the problem will resolve itself or the specific outcome will appear. This led naturally to an 20 – Journal of Solution Focused Brief Therapy https://digitalscholarship.unlv.edu/journalsfp/vol3/iss1/12 Leaving No Footprints outcome-led start to the process, and from this, the word “hope” began to appear in our work. Not as a deliberate intervention, but as a sign of trust in our clients that they must be sitting with us for some good reason (Ratner et al., 2011). A typical example of the process in which we move from a specific “goal” to a contextual or “way of living” outcome would be: Therapist What are your best hopes from our talking? Client I want my daughter to come home on time This is a specific outcome which could be tackled in many ways within a broad Solution Focused framework. For example, by looking for and amplifying exceptions or by describing a “miracle” in which the daughter does come home and extrapolating from this a plan of action aimed at encouraging “miracle” behavior. This would represent a “goal-directed” or “problemsolving” approach rather than the “way of life” approach the therapist in this case uses. The question most often used to begin the expansion from a specific outcome, being home on time, to a ‘way of life’ outcome, having a good relationship, is a “What difference?” question (Shennan & Iveson, 2008). For example: Therapist What difference would that make? Client I wouldn’t be fighting with her all the time Therapist What would you be doing instead? Client Not screaming at each other! Therapist What difference do you think that would make? Client We just wouldn’t always be arguing. Therapist So what difference would that make, if you weren’t fighting, screaming and arguing? Client Then maybe we could get along like we used to. We were always very close, well, we still are – occasionally! Therapist If somehow, our meeting led you and your daughter to get along more like you used to, and bring more of that closeness, would that mean it had been useful? Client Definitely! We now have the hoped-for outcome within which the client can find her own way to the initial, more specific goal, and can move on to a description of one way this outcome might Journal of Solution Focused Brief Therapy – 21 16 et al.: Volume 3 Issue 1 2019, Complete Chris Iveson unfold. The client’s preferred future. Therapist So let’s imagine that you wake up tomorrow and somehow you and your daughter are getting along in just the right way, with more of the closeness you still sometimes have, what might you notice is different as you began waking up into a new day? Client I wouldn’t be dreading having to wake her up for school. Therapist What might you be feeling instead? Client Maybe that it would be nice to see her – she always used to be very sweet in the morning. A Preferred Future As the session above continues a very detailed description of the morning unfolds and is followed by some further description of the client’s day. Then more description of the motherdaughter relationship after school but stopping short of the potentially troublesome evening. Finally, a few minutes to sketch out a scale: “How much of this miracle is already happening?” There is no attempt to address the specific issue of the coming home time. The assumption, borne out by client follow-up, is that the description will lead to an improved relationship between mother and daughter. They will do what every family must do – come to an agreement about mutual behaviours and boundaries. There was a similar description in the last session with Angela when she expressed worry about losing her job and wondered how she might approach her boss. Instead of focusing on the way she might approach him we focused on the likely consequence of a successful approach. As Angela began to answer, “He’d ring me about –” she suddenly broke off with a gasp “He already has! He rang me while I was on my way here to check if I was still up for a meeting we’d planned for tomorrow!” However, frequently it happens that clients bump into the fact that something they are hoping for has already happened without them noticing! Clues (de Shazer, 1988) became BRIEF’s bible when it was first published in 1988 and, like the Bible, it is full of contradictions. The “specific” rather than “vague” goals that he argues for (p. 93) are confused with detailed descriptions or “pictures in words” (p. 187) to describe “life without the problem” which might consist of tens, or even hundreds, of differences far too many to be realistically thought of as goals. They can be more aptly described as “ways of living.” It was this realisation that led us at BRIEF to talk of the client’s “preferred future” (Ratner et al., 2011), rather than the client’s goals, and paved the way to move from “What brings you here?” (a request for problem information which is redundant to the therapeutic endeavour) to “What are your best hopes from our work together?” (a question designed to discover the client’s hoped-for outcome). From this perspective, as illustrated in the case examples above, what de Shazer would 22 – Journal of Solution Focused Brief Therapy Published by Digital Scholarship@UNLV, 2020 Leaving No Footprints have criticized as “vague goals” have become the preferred starting point for our work. Thus, a client who wants to give up drugs might wish to “have a normal life.” The therapist could ask “Let’s imagine you wake up tomorrow beginning to move towards the normal life that you are seeking.” A client who is isolated and depressed might want a future in which he is more self-confident. A suicidal client might want to wake up with the sense of a future, or, as in Angela’s case, “free to get on with her life.” All vague, even global outcomes, but ones that give the opportunity for the client to describe a way of living which might lead to problems being resolved “organically” without the therapist needing to know what they are. Small changes are big changes De Shazer was quite right when he said, “Goals need to be achievable but perhaps not so right when he said they also need to be hard to attain” (1988, p. 93; 1991, p. 112). The purpose of a very detailed preferred-future description is to make sure every aspect is well within the client’s range of possibilities. The more the hoped for future (or the miracle) can be located in the everyday routine of the client’s life, the more possible it would seem to be. Asking Angela, “What might you notice as you are making your coffee?” will elicit an answer very close to what has been happening for months, or years, yet, it will also describe part of a more desirable way of living. Looked at from this perspective, the essentially linear idea that a small change can lead to a big change might be replaced by the idea that each small change is, in fact, part of the big change already happening. In the case of the mother wanting a better relationship with her daughter, the imagined “good morning” is not a small thing that might lead to a larger thing, but a small thing which is a consequence of the large thing (e.g. the miracle) already having happened. For instance, a young mother struggling with serious and chronic eating difficulties described her experience of a single session and clearly demonstrates that the “small” is also the “large”: I have been through every sort of therapy since I was 14, and though this sounded different, I wasn’t really hopeful. When I was asked about a miracle my heart sank because I knew a miracle wasn’t going to happen, but when I started answering the questions, I felt a glimmer of real hope because my answers were things I could easily do. So, I set a sort of test. Every time I answered a question, I asked myself “Can you do that?”. If the answer was “yes” I’d carry on but once I said ‘no’ I would know it wasn’t going to work for me. Because all my answers were “yes” I knew for the first time that it was possible to overcome anorexia; I’m not sure that I’ll manage that, but now I know it’s possible I’m going to give it my best shot! Therapist Neutrality This detailing of preferred futures has been described many times elsewhere (Connie, 2013; Journal of Solution Focused Brief Therapy – 23 17 Chris Iveson Journal of Solution Focused Practices, Vol. 3 [2020], Iss. 1, Art. 12 Ratner et al., 2011), but what has not been emphasized so much is the value of the therapist’s neutrality towards these descriptions. This is not a new idea. The Milan Group (Palazzoli et al., 1982) were heavily criticized for appearing to place neutrality above responsibility. At BRIEF, neutrality refers to a therapeutic stance which is overridden if there is a serious concern for the safety of the client, or others, or if any likely actions of the client are potentially harmful in other ways. When these concerns become paramount, the therapy ceases and the therapist becomes an “agent of society” exercising (depending on their role) their legal, professional or citizen responsibilities. It is never our right to dictate how others should behave. But we do have the right and responsibility to guard the outer boundaries of what is permissible in our society. We may not tell a parent how to raise a child, but we have a duty to do what we can to prevent harm to that child. Being neutral is not easy. And what we write and what we do, as we at BRIEF are constantly reminded, are not always aligned. Our writing tends to reflect our ambitions more than our practice. And, the same was true of de Shazer who also aspired to a form of neutrality: Frequently, by the end of a session clients are beginning to know their way about or at least are starting to have some confidence that they can find their way about. Thus, there is no need to overwhelm clients by making lots of suggestions or inventing [tasks]; rather, the therapist simply needs to support clients’ going in their own chosen direction with the confidence that once they get where they want to be they will then know their way about (de Shazer, 1994). But whatever de Shazer’s aspirations to neutrality it is lacking in the cases described throughout all his books (de Shazer, 1985, 1988, 1991, & 1994). Time and again, client’s descriptions are turned into goals and action plans and hence become, in some way, the property of the therapist. At BRIEF, we have come to see these descriptions as just one set of possibilities which we have no right to expropriate or recommend as future actions. Instead, we see them as creating a realisation that more preferred ways of living are within the client’s range even if, by the time tomorrow arrives, these preferred ways of living turn out to be different from those imagined today. Put very simply, what each of our clients does tomorrow is none of our business. Each client is responsible for the decisions they make. If we believe our own soft-spoken words, that each client is in the best-placed position to make their own decisions, we cannot attribute expertise to the client only on the condition that it fits with our view of the best way forward. This neutrality is a discipline and one which may not sit comfortably with many Solution Focused practitioners. Nor, given de Shazer’s practice as cited above, is it essential. However, it is one which fits well with the underlying philosophy of de Shazer’s writings, and clearly demonstrates the therapist’s trust in the client as well as guarding us against our ‘better knowing’. But as a behavioral discipline it requires hard work and constant attention. There can be nothing robotic or unconcerned about the discipline of neutrality. Instead, it must sit 24 – Journal of Solution Focused Brief Therapy https://digitalscholarship.unlv.edu/journalsfp/vol3/iss1/12 Leaving No Footprints side by side kindness and wishes for the well-being of others; as well as side by side with our need to be successful as therapists. Ultimately, neutrality is a pragmatic decision. Does it work? Does it fit with brevity? Our experience is that it does. It is the same with “footprints.” Every time we sit down with a client, we must fervently wish that it will be a life-changing event, that the client will begin a new course towards a better future. We back up this wish by using techniques that we hope, a hope based on evidence, will create transformation. We also have to know that whatever the client does tomorrow has a history going back through generations and that tomorrow has always been possible. All we have done is ask the questions that bring that possible tomorrow, and its history, into focus. This raises the question of just how “co-constructed” is the future our clients aspire to and how much of our own lives are similarly “co-constructed” during our conversation with them. Hopefully, we are not too changed by every encounter so, at the end of a busy day, we can return to our families and friends not too different from how we set off in the morning. Whereas, we hope rather the opposite for our clients. To return to Angela, her words suggest that she experienced whatever happened as entirely her own work. With every idea and action coming only from herself. How could it be otherwise, and what could be better than this realisation? What we hope Angela also experienced, as we do for every client, was a complete trust in her ability to make her own choices without pressure, however subtle, however well-meant, from the therapist to make those choices that best fit the hoped-for outcome. Solution Focused Brief Therapy, as with every other talking therapy, provides a set of guidelines for managing the therapeutic conversation. To follow these guidelines requires discipline and discipline can only be maintained with constant practice. One of the obvious disciplines of Solution Focused Brief Therapy is to avoid questions which seek an explanation of the problem. Information from these questions does not further the Solution Focused process. This does not mean that the answers to these questions are uninteresting, especially within a culture that privileges explanations, just that they are not useful within the Solution Focused model. Therefore ,we must guard against our natural (culturally determined) curiosity and wish to understand. Devising questions that are content-free is also a discipline which is hard, perhaps impossible, to maintain. We do this by seeking only descriptions of future possibilities. Though we might be less neutral about past achievements, the history of the preferred future, we do our best to follow the client with regard to what constitutes success. Using the client’s words, guarding against introducing our own words, interpretations, and ideas, and looking at the world through our client’s eyes, rather than our own, are all part of this discipline. Similarly, the “sister” discipline of neutrality requires constant practice. Our good wishes for our clients provide an all too easy excuse for trying to influence the decisions they make, even just by summarizing what we think are the important parts of what they have said. Such good intentions are one of the most used excuses for the abuse of power. Whether it be by Journal of Solution Focused Brief Therapy – 25 18 et al.: Volume 3 Issue 1 2019, Complete Chris Iveson therapist to client, or state to citizen. If we were to ask our clients, “Would you prefer your therapist to guide you or to trust you?” What might they answer? And how might their answer influence our practice? Let us give the last words to another client who puts it all in a ten-word nutshell! Client (At the end of a single session) It’s the questions, isn’t it? It’s the questions! Therapist Well, maybe it’s not so much the questions as the answers. Client I know, but I would never have had those answers without the questions! (Iveson et al., 2014) References Connie, E, (2013). Solution building in Couples Therapy. New York, NY: Springer. de Shazer, S. (1985). Keys to Solution in Brief Therapy. New York, NY: Norton. de Shazer, S. (1988). Clues: Investigating Solutions in Brief Therapy. New York: Norton. de Shazer, S. (1991). Putting Difference to Work. New York, NY: Norton. de Shazer, S. (1994). Words were Originally Magic. New York, NY: Norton. Palazzoli, S. M., Boscolo, L., Cecchin, G. & Prata, G. (1980). Hypothesizing, Circularity and Neutrality: three guidelines for the conductor of the session. Family Process, 19, 3-12. Ratner, H., Iveson, C. & George, E. (2012). Solution Focused Brief Therapy: 100 Key Ideas and Techniques. London, UK: Routledge. Iveson, C., Ratner, H. & George, E. (2014). Love is all around. In M. Hoyt, & M. Talmon (Eds.), Capturing the moment. Bethel, UK: Crown House Iveson, C. & McKergow, M. (2015). Brief therapy: Focused description development. Journal of Solution-Focused Brief Therapy, 2, 1-17. Shennan, G. and Iveson, C. (2008). What difference would that make? Journal of Family Psychotherapy 19, 97-101. Shennan, G., & Iveson, C. (2011). From solution to description: Practice and research in tandem. In C. Franklin, & T. Trepper, et al., (Eds.), Solution-focused Brief Therapy: A Handbook of Evidence-based Practice. New York: OUP. It Will Never Be the Same Again Alexey Mikhalsky , Plamen Panayotov, and Alasdair MacDonald** Systemic Family Therapy Center, Moscow, Russia. Solutions Centre, Rousse, Bulgaria; Angel Kanchev University, Rousse, Bulgaria. *Solution Focused Approaches, United Kingdom. Abstract Chris Iveson This paper describes the Conversations Led by Clients (CoLeC) model as practiced at several Bulgarian and Russian helping institutions as a development of Solution-Focused Brief Therapy (SFBT). The model emphasizes therapeutic conversations as something that has to follow step by step from the clients’ enquiries. The questions include the mind-activating Question (MAQ): What do you think is the most useful question you can hear from me now? (or at our next session?); the time-oriented question: What do you think is most useful to talk about now: past, present or future?; the multiple-choice question: Which of these questions do you think is best for you right now? (from a list provided by the therapist); and the delayed-answers question: If client has no current answer, ask them to think about the question until the next session and propose self-questioning activities. CoLeC, being a step beyond some therapeutic models, is an initial effort to assist helping professionals find answers to this quite different question, “How should this client’s conversation with me be?” This makes it the beginning of a qualitatively new attitude to what we, as professionals, do. We hope that many, young and new to the profession, will not only join in, but also help further this way of thinking and doing as we see these types of conversations with clients, useful. Email: chrisiveson@brief.org.uk Keywords: client, conversations, help, questions, usefulness, therapy Introduction This paper is about what we believe could be the future of helping professionals’ conversations with clients – which are conversations organized by professionals and led by clients. It is a step beyond traditional SFBT, and shares its basic belief in clients, their abilities to be their own best helpers, their good will and resources for change. Our approach is a step 26 – Journal of Solution Focused Brief Therapy Published by Digital Scholarship@UNLV, 2020 Journal of Solution Focused Brief Therapy – 27 19 Alexey Mikhalsky, Plamen Panayotov, and Alasdair MacDonald Journal of Solution Focused Practices, Vol. 3 [2020], Iss. 1, Art. 12 beyond SFBT because it emphasizes the client’s feelings and personal knowledge pertaining the best time for therapeutic-interventions. The client chooses the appropriate time to discuss any issue, the most useful questions to be asked by the therapist, and the answers they need when creating future change. However, our approach still shares the basic SFBT beliefs about clients’ abilities to be their own best helpers through their good will and resources for change. Once Upon a Time… If we take a closer look at therapeutic conversations, we shall notice that therapy can be understood as a specific kind of socially constructed Language game (Wittgenstein, 1953), which is very similar to somatic medicine. Going to a doctor, patients expect many and seemingly different activities from him, all aimed at discovering the nature of their problem, which is intended to create a pathway for effective interventions. Most of these activities, however, are in fact improvisations of the same game: asking questions. The conversation usually includes a discussion about the patient’s medical history and reason for visit. This kind of conversation is usual for directive and/or problem-focused health care approaches. Even when we ask clients about their best hopes from therapy in a solution-focused way we start to play our roles as professionals in asking questions, thus reflecting the traditional Language game of asking. If we look at the ancient roots of this approach, we find Socratic debate as a method for finding the hidden knowledge leading the opponent to an insight when he finally contradicts himself, through a series of questions. Started as a medical activity by a neurologist, Sigmund Freud, psychotherapy has been traditionally perceived and practiced in the cause-and-effect medical frame of thinking. Since rational and cognitive therapies started to develop, the problem in therapy was often seen as a clients’ enmeshment in illogical beliefs. So the therapist takes a stance of a curious questioner and leads the client out. Though this Socratic method model is widely known and accepted in many modern therapy approaches, it sometimes leads to possible ignorance of clients’ intentions, nuances, causing resistance and “non-participative” style of therapy where clients feel themselves as not understood. Even within this framework, however, Dr. Albert Schweitzer noted (in Norman Cousins’ Anatomy of an Illness as Perceived by the Patient, 2005, p. 78), “Each patient carries his own doctor inside him. They come to us not knowing that truth. We are at our best when we give the doctor who resides within each patient a chance to go to work.” It Will Never Be the Same Again professional. Sometimes we just forget the fact that a majority of therapy is initiated by clients (or others important persons in their lives – relatives, neighbors etc.), and less often by caring professionals. We should clearly realize that every professional encounter is a result of a series of steps previously taken by clients: they have asked themselves “Do I need to talk to someone about my situation?”, then obviously replied “Yes” to it; then they asked themselves “Who should I talk to?”, and in response to this have made a preliminary study, asking other people, preferably ex-clients in their families, among friends, at their workplace, at the hairdresser’s, on the Internet, in diverse media... in this way they find the professional they would like to discuss their situation with. And then they ask the next question (appearing to the professional as if being the first one), quite often containing its answer: “Can I come to see you on Wednesday at 10:30, as this is the most convenient time for me? A wish to speak within a session is a session is a client’s personal initiative. Some Tools in Brief In CoLeC, the professional is engaged in what we call now the Questioning for Most Useful Questions (QUQu). It is a set of techniques that try to keep, maintain, and support the clients’ lead by helping them develop their own abilities in the art of asking useful questions. This has not been overlooked by solution-focused therapists, so we are supposed to make some additional accent to these points. We also suggest the use of clients’ self-questioning. We often invite the client to think specially about those questions that involve most clients’ interest, and about possible answers. We can use these self-questioning techniques in different ways, in direct or paradoxical manner (changing and reformulating them in a creative way). The process of asking oneself brand new and unexpected questions and trying to find answers is a process that develops dialectically balanced views (Mikhalsky, 2014). Now… The therapeutic tradition is maintained today by many professionals who believe that ‘therapy’ should be the therapist’s job just like a violinist’s job is to play the violin. In SFBT, for example, there is a metaphor that compares a therapist to a taxi-driver. This is because a taxidriver asks his passenger “Where to?” when inquiring about the final destination (Bannink & McCarthy, 2014), implicitly admitting that the person behind the wheel is the real worker, the 28 – Journal of Solution Focused Brief Therapy https://digitalscholarship.unlv.edu/journalsfp/vol3/iss1/12 Journal of Solution Focused Brief Therapy – 29 20 Alexey Mikhalsky, Plamen Panayotov, and Alasdair MacDonald Table 1. CoLeC Questioning Techniques Eliciting clients’ own questions What questions did you want to ask me in this session? Answering what questions would be most useful for you today? What questions did you ask to yourself often but not finding the answers? Mind-activating question What do you think is the most useful question you can hear from me now (or at our next session)? Time-oriented question What do you think is most useful for you to talk about: your past, present or future? Multiple-choice question Which of one of these questions, if any, do you think is best for you to discuss right now? (From a list provided by ConCon) Best therapist question When the dialogue gets stuck, it could be very useful to ask in this way: ‘What question would the best therapist ask now?’ Delayed-questions question If the client doesn’t have questions or hesitates which will be useful, we can ask him to think about the Most Useful Question for the next session. Delayed-answers question If clients have no current answer, ask them to think about the question until the next session, or until they find a high quality and useful answer. As Dr. Alasdair Macdonald (2019) states: “The Delayed Answers question above draws on the original MRI model which often mentioned ‘going slow’. A useful variation mentioned by Steve de Shazer was ‘Think about possibilities but don’t do anything until we next meet.’ Telling clients to do nothing often leads to some action. If clients ask ‘Why does this happen?’ Then ask them ‘What do you think is the simplest possible and most useful explanation of this situation?’ If they have no answer Suggest they choose from this list: it happens first and then becomes a habit; because you love each other (in cases of conflict); because you are a living person; it is your energy; because you say so. et al.: Volume 3 Issue 1 2019, Complete It Will Never Be the Same Again Discussion Encouraging clients to ask questions and to develop new ones considerably changes both the usual narrative form these situations are described, as well as clients’ perceptions of them. The ongoing tough life situation becomes under question, in other words, manageable, and clients become much more creative trying to state whatever they want to say in a question form. As Steve de Shazer (1994, personal communication) said, “If a client has already stated her situation for seventeen times in the same way, try not to be the eighteenth person hearing the same story, but do everything possible to be the first one who hears at least a little bit different story!” Stating one’s situation (no matter if past, present, or desired) as a narrative is dramatically different from questioning it. This move from narration to dialogic coconstruction based on self-questioning changes its framing and its perception by clients and professionals. Conversation Led by Clients (CoLeC) tries to challenge expert-centered, medical and Socratic habits of structuring human conversations, thus tending to be perceived as strange and unusual within the existing ‘normal’ psychotherapy setting. These changes, however, require non-automatic use of their language from clients, helping them to develop their own new style of communication and thinking. As a client recently noted at the end of her singlesession consultation, “I never expected that we shall talk in my algorithms and not in yours.” Practicing the Questioning for Useful Questions (QUQu) also touches upon George Herbert Mead's (1913) concept of the internalized conversation. G. H. Mead argues that we regularly engage in unspoken internalized conversations as we reflect upon practical issues in our everyday lives. The questioning practices described here, promises to expand clients' skills and options in conducting their internal conversations, far beyond their encounters with a therapist or a counselor. What do we need second-order changes for? Each and every client’s question has at least three advantages over any therapist’s question: 1. It is stated in the client’s own language, so it is understandable to her. Any client can misunderstand every therapist’s question, while we all (clients included) seem to understand our own utterances. Miller (June 7, 2016) in personal communication, however, proposes another important aspect of focusing on clients’ own questions: I would say that clients' own language appears to be understandable to them (this is a good reason for asking them to form their own questions) but that upon further reflection clients might discover that they misunderstood what they were asking. I think the key word here is discovery. When the client asks a seemingly understandable question that turns out to have really been a misunderstanding on the client's own part, it is a discovery and potential 30 – Journal of Solution Focused Brief Therapy Published by Digital Scholarship@UNLV, 2020 Journal of Solution Focused Brief Therapy – 31 21 Alexey Mikhalsky, Plamen Panayotov, and Alasdair MacDonald Journal of Solution Focused Practices, Vol. 3 [2020], Iss. 1, Art. 12 source of insights into one's self that immediately expands clients' sense of personal agency, knowledge, skills, and perhaps strengths (Miller, personal communication, June 7, 2016). 2. It is always on time! A therapist’s question may happen to be on time, and may happen to be out of time (since therapists cannot know what time it is now for the client), while a client’s own question is obviously fitting her timeline. 3. The more clients practice asking useful questions, the better they become in this. The ability to do it effectively helps clients not only find solutions to their present problems, but also to deal effectively with tough situations in their future. As Einstein is supposed to have said (as cited in Quote Investigator, 2014): If I had an hour to solve a problem and my life depended on the solution, I would spend the first 55 minutes determining the proper question to ask, for once I know the proper question, I could solve the problem in less than five minutes (Quote Investigator, 2014). To summarize, the benefits are: empowering of clients, appropriate timing of therapeutic interventions, solving the problem with misunderstanding, helping clients not only find solutions to their current dilemmas, but also preparing them for handling future difficulties. Since effective help is based on the asking of useful questions, the more capable clients become in this, the less help they will need in the future. Conclusion Therapeutic conversations cannot evade the times of radical shifts in meaning. CoLeC is just one of the many possible and coming changes in therapeutic conversations. These changes do not come too fast. People often have their own pace for making changes. They can usually tell you what pace they prefer, which can inform expectations about progress in your work together. The above-described sequence of clients’ activities clearly defines them as the initiators, owners, and main change-agents in therapeutic conversations to follow. In the Conversations Led by Clients (CoLeC) approach, we use the metaphor of the professional who acts as the Conversations Conductor (ConCon). He is not supposed to sound, but to organize the best possible sounding of the conducted musicians. The professional tries not only to keep the clients’ ownership and leadership, but also helps the client develop her language skills. The ConCon therapist does not focus on algorithms, and instead of asking himself “How should my conversations with clients look like?” tries to ask “How should this client’s conversation with me look like?”, “How can I help the client to formulate his questions and answers?” If we define our approach to helping activity as a kind of assisted self-help therapy, the professional's role drifts towards following, encouraging, and assisting the already started process of clients asking themselves useful questions, finding answers to these, and helping to develop on this basis their next steps. The steps can lead towards their desired situations 32 – Journal of Solution Focused Brief Therapy https://digitalscholarship.unlv.edu/journalsfp/vol3/iss1/12 It Will Never Be the Same Again (Switek, 2014) or thoughts and emotions, or states of mind and body, whatever. In our opinion, the final goal of this assisted self-help Conversations Led by Clients (CoLeC) is not only in helping clients in co-constructing solutions to their current hardships, but also in preparing and equipping them with specific language tools they can use in the future for managing other tough situations. Practicing the art of asking useful questions within their sessions with us, clients learn how to handle other difficulties they will further encounter. That sounds similar to the original intention of Solution Focused Brief Therapy to focus on clients as able to create their own solutions and to live the lives they truly want to live (de Shazer, 1994). References Atkinson, B., Atkinson, L., Kutz, P., Lata, J., Lata, K. W., Szekely, J., & Weiss, P. (2005). Rewiring neural states in couples therapy: Advances from affective neuroscience. Journal of Systemic Therapies: Special Issue: Psychotherapy and Neuroscience, 24(3), 3-16. Bannink, F. & McCarthy, J. (2014). The solution-focused taxi. Counseling Today. Retrieved from the Internet: https://ct.counseling.org/2014/05/the-solution-focused-taxi/# Brabazon, J. (2005). Albert Schweitzer: Essential writings. New York, NY: Orbis Books. Cousins, N. (2005). Anatomy of an illness as perceived by the patient. New York, NY: W. W. Norton. de Shazer, S. (1985). Keys to solution in brief therapy. New York, NY: W. W. Norton. de Shazer, S. (1988). Clues: Investigating solutions in brief therapy. New York, NY: W. W. Norton. de Shazer, S. (1994). Words were originally magic. New York, NY: W. W. Norton. Ghul, R. (2017). The power of the next small step. Keller, TX: Connie Institute. Greenberg, G. S. (1998). Brief, change-delineating group therapy with acute and chronically mentally ill clients: An achievement-oriented approach. In W. A. Ray, & S. de Shazer (Eds), Evolving brief therapies. Iowa City, IA: Geist and Russell (pp 142–232). Iveson, C. & McKergow, M. (2016). Brief therapy: Focused description development. Journal of Solution-Focused Brief Therapy, 2(1), 1-17. James, W. (1890). The principles of psychology. New York, NY: Henry Holt and Company Macdonald, A. J. (2019). Solution-focused therapy: The story so far. In Making waves: solution focused practice in Europe: 25th anniversary conference book (pp 13-31). Maier, H. W. (1987). Developmental group care of children and youth: Concepts and practice. New York, NY: Haworth. Mead, G. H. (1913). The social self. Journal of Philosophy, Psychology and Scientific Methods, 10, 374-380. Journal of Solution Focused Brief Therapy – 33 22 Alexey Mikhalsky, Plamen Panayotov, and Alasdair MacDonald et al.: Volume 3 Issue 1 2019, Complete Metcalf, L., & O'Hanlon, B. (2006). The miracle question: Answer it and change your life. Bancyfelin, Carmarthen, UK: Crown House Publishing Limited. Mikhalsky, A. (2014). Psychology of constructing the future. Moscow City University of Psychology and Education, Moscow. Mikhalsky, A. (2018, September). Positive woodpecker: A solution-focused self-questioning technique. Master-class presented at the European Brief Therapy Association Conference, Sofia, Bulgaria. Mikhalsky, A. (2019). The ecology of social practice in language, communication, and constructing the future. In: Handbook on research on ecosystem-based theoretical models of learning and communication. IGI-Global. Mullan, S. J. (1914). The spiritual exercises of St. Ignatius of Loyola. P. J. Kenedy & Sons. New York. Panayotov, P. A. (2011). Simple therapy. PIK-BS. Sofia. Panayotov, P. (2012). Solution is only a smile away. Retrieved from http://en.solutions-centrerousse-bulgaria.org/files/simple_therapy.pdf (accessed 12/01/2018) Panayotov, P. & Strahilov, B. (2018). From therapy towards conversations led by clients (CoLeC). Retrieved from http://en.solutions-centre-roussebulgaria.org/files/ftt_colec.pdf Panayotov, P. & Strahilov, B. (2018). The ultimate self-helper. Retrieved from http://en.solutions-centre-rousse-bulgaria.org/files/self_helper_en.pdf Schweitzer, A. (2009). Out of my life and thought: An autobiography. Johns Hopkins University Press. Baltimore. Shilts, L. (2008). The WOWW program. In P. DeJong & I. K. Berg (Eds), Interviewing for solutions (3rd ed). San Francisco, CA: Brooks/Cole. Simon, F., Stierlin, H., & Wynne, L. (1985). Language of family therapy: A systemic vocabulary and source book. New York, NY: Family Process Press. Switek, T. (2014). The situations focused model: A map of solution-focused brief therapy used as an open systems approach with clients and in human services. International Journal of Solution-focused Practices, 2(2), 40-51. Watzlawick, P., Weakland, J., & Fisch, D. (1974). Change: Principles of problem formation and problem resolution. New York, NY: W. W. Norton & Company. Wittgenstein, L. (2001). Tractatus logico-philosophicus. London, UK: Routledge. Young, S. & Holdorf, G. (2003). Using solution-focused brief therapy in referrals for bullying. Education Psychology in Practice, 19, 271–82. Alexey Mikhalsky, Plamen Panayotov, and Alasdair MacDonald Email: amikhalsky@gmail.com; plamenpan@mail.bg; macdonald@solutionsdoc.co.uk 34 – Journal of Solution Focused Brief Therapy Published by Digital Scholarship@UNLV, 2020 Solution Focused Work as an Aesthetic Mark McKergow The Centre for Solutions Focus at Work Abstract The current paper looks at Solution Focused (SF) work in a novel way – as an aesthetic (what makes it beautiful?) as opposed to a method (how do you do it?). This term comes from the art world, where different schools of painting can be described as having different aesthetics. Starting with a definition of the term, I propose five elements of an SF aesthetic: brevity, client autonomy, radical acceptance, staying at the surface and valuing small differences. While these are not present in every piece of SF work, they are things that we strive for, qualities that bring me (at least) satisfaction, cheer and reasons to continue to support, promote and develop SF. Keywords: Solution-Focused, beauty, aesthetic, satisfaction, qualities Introduction This paper brings a different way to look at Solution Focus (SF) – what do we as SF practitioners think is “beautiful” in our work? This look might be extended to other forms in the brief therapy tradition, but I want to focus on SF in particular here. This examination might go some way to shedding light on the long-term resistance and ignorance of SF from those in other schools. If we are working to a different aesthetic, then they won’t be valuing the same things and will be confused, angry and baffled by what we do (and possibly vice versa, of course). I was immediately and passionately engaged with SF work when I first discovered it in 1993, for reasons that were not completely clear to me at the time. Over the past three decades or so, I have continued in this commitment, and have come to realise that these aesthetic aspects are very important to me. I am now wondering if these (not often discussed and often assumed) aspects are shared, and to what extent. This paper seeks to present these aspects explicitly. It is an overview of why I personally love SF practice. What about you? What is an ‘aesthetic’? Journal of Solution Focused Brief Therapy – 35 23 Mark McKergow Journal of Solution Focused Practices, Vol. 3 [2020], Iss. 1, Art. 12 Looking at the dictionary (Aesthetic, n.d.), we find definitions of ‘aesthetic’ in the following terms: Adjective: Concerned with beauty or the appreciation of beauty. Example usage: ‘the pictures give great aesthetic pleasure’ Noun: A set of principles underlying the work of a particular artist or artistic movement. Example usage: ‘the Cubist aesthetic’ The term originates from the Greek word aisthētikos, meaning “perception connected with the senses”. Although sensory perception is clearly important, the idea of aesthetics means more than this. David Hume, 18th century philosopher and a good cook as well, wrote that delicacy of taste is not merely "the ability to detect all the ingredients in a composition", but also our sensitivity "to pains as well as pleasures, which escape the rest of mankind." (Hume, 1987, p. 5). This is about considered affective and emotional responses as well as sensory distinctions. One way into this topic is to look at the world of art and in particular painting. For centuries, artists strove to produce renditions of (say) landscapes which were detailed, clear and representational. Look at Canaletto’s famous paintings of Venice for a fine example, or the British artist Thomas Gainsborough. In these cases, the artists made extensive preparatory sketches and then worked up a final highly detailed artwork in their studios, constructing the composition to be pleasing (aesthetically) rather than a snapshot of a particular moment. Even today, the results are sensational. In the second half of the 19th century however, painters started to explore with greater vigour what happened when they ventured outside the studio and worked “en plein air”, in the open air at the location. Painters such as John Constable and JMW Turner had begun to explore this in the early 19th century, but it was taken to new extremes by painters such as Claude Monet, Pierre-August Renoir, Alfred Sisley and others in the early 1860s. Their work focused on light and the immediacy of the moment, and was being routinely rejected from the Salon de Paris, the accepted leading curated art show which favoured painters of the classical style. Emperor Napoleon III saw the rejected works and decreed that the public should be allowed to judge for themselves. As a result, a “Salon de Refusés”, an exhibition of the refused, was organised. While many came to laugh, this exhibition was a key rallying point for those keen on the new “impressionist” aesthetic which took its name from Monet’s Impression, Sunrise. As has often been the case through history, the name came as an insult from critic Louis Leroy who in his article The Exhibition Of The Impressionists referred to Monet’s work as at best a sketch, nothing like a finished work. (For more details on the development of impressionism the reader is referred to the Metropolitan Museum, New York’s excellent website; Samu, 2004). The impressionist aesthetic is much more about capturing an “impression” of a moment in 36 – Journal of Solution Focused Brief Therapy https://digitalscholarship.unlv.edu/journalsfp/vol3/iss1/12 Solution Focused Work as an Aesthetic time; the brushwork is bigger and bolder, the effect more spontaneous. If we look at an impressionist painting with a classical aesthetic, we see what Leroy and others saw – unfinished, incomplete daubs of little lasting consequence. If we look at Caravaggio and Gainsborough with an impressionist aesthetic, we see stylised, overworked ”perfection” which says little about the artist’s (or the viewer’s) response. (It is interesting that impressionism emerged around the same time as photography, and can be seen as a conscious alternative to it). Both of these aesthetics are, of course, interesting and valuable. What I am seeking to point to here is the way that an alternative paradigm, a new way of looking at things, can be seen through the aesthetic lens; what makes something beautiful? What is valued, prized, admired, noticed, applauded? It’s something to do with what makes you go “Yes!!” and what makes you go “Blurghhh!” In this article I will seek to explore what makes me cry “Yes!” in SF work, and thereby shed a little more light on what it means to work in an SF aesthetic. What follows is my list of the things that I love and value about SF practice. The list is not complete, of course, and it’s a personal one. I hope that many SF practitioners may find a recognition and a resonance in at least some of these items. Brevity SF was originally called Solution-Focused Brief Therapy (SFBT) for a reason – it’s brief! The original version of interactional brief therapy emerged from the Brief Therapy Centre of the Mental Research Institute. Palo Alto, led by John Weakland, Paul Watzlawick and Dick Fisch. This version of practice, called MRI model or Problem Solving Brief Therapy, is still around, and uses a 10 session framework as its basis (Segal, 1991). At the time (in the 1970s) this was startlingly brief, compared to the years of weekly treatment considered normal by practitioners working in the psychodynamic tradition. When Steve de Shazer and Insoo Kim Berg moved from hanging around MRI to setting up their own centre in Milwaukee in the late 1970s and early 1980s, they wanted to build on this work and developed the idea that brief therapy should be ‘as brief as possible, and not one session more’ (de Shazer, in his Foreword to Dolan, 1991). This is quite a step onwards from even a 10 session basis – every session could be the last, and is carried out with this possibility in mind. The choice of continuing is at least partly with the client; we will return to this aspect of power sharing later. The very idea that therapy can be effective at all in one or two sessions is still considered outlandish by some. Indeed, workers in the psychoanalytic tradition have developed the concept of the “flight into health,” where the client’s claims that they are suddenly and completely cured is seen as a defensive reaction to the treatment, and therefore a sign of the need for even more therapy. This can be seen as a kind of Catch-22 bind; if the client says they are better, they need more treatment. And if they say they aren’t better, then of course they need more treatment! Journal of Solution Focused Brief Therapy – 37 24 et al.: Volume 3 Issue 1 2019, Complete Mark McKergow Why is brevity an important end in itself? Of course, effectiveness – the results of the treatment – is important. There are influential findings (Wampold, 2001) saying that all forms of therapy, at least those based on the five “common factors”, are as effective as each other. Wampold doesn’t look at efficiency, the amount of time it takes to reach an effective outcome, nor does he take account the disturbance to the client of participating in the treatment. If all therapies are equally effective, then surely it’s better to choose one that takes less time? It helps the client get on with their life sooner, and it frees up the practitioner to help new clients. The advantages of this are particularly clear when resources are limited, as in the UK National Health Service (NHS). The efficiency of a service is directly proportional to both the number of practitioners and their average length of treatment, assuming that each practitioners has the same number of sessions every week. According to the 2013 report We Still Need To Talk (MIND, 2013), over one in ten people had been waiting over a year to see a therapist, and approximately 50% had waited more than three months. Brevity, via effective treatments, is clearly valuable to a statutory service. It remains something of a mystery why the popular view of longer equals better still seems to hold so much sway. Another benefit of brevity is that a short treatment will mean that the client is back living their own life sooner. There is a clear distinction between those practitioners who see the act of seeing a client as valuable in itself, and those who seek to get out of the client’s life as soon as they can. The goal of SF is, as Steve de Shazer (quoted by his long-term collaborators at BRIEF, 2019), used to say, echoing an old aphorism sometimes attributed to Edna St. Vincent Millay, to return the client to a life of “one damn thing after another”. This is everyday life as we know it – not a flawless and effortless glide but a series of ups and downs, handled by the client without professional help. In contrast, those who seek treatment usually do so because their lives have become “the same damn thing over and over” – something keeps happening that they don’t want, or doesn’t happen when they seek it. The former case isn’t seen in the SF aesthetic as grounds for treatment: the latter is. The same holds true for MRI Problem Solving Brief Therapy - when the same damn thing isn’t happening any more, that’s enough for now. Brevity is not a simple matter of a small number of sessions or a limited time. It’s about the work being as brief as possible - subject to a satisfactory conclusion, or onward referral. This is not to say that every person only has one problem in their lives. As an SF coach to business leaders, I sometimes get contracted for a series of sessions (ten, for example). However, I don’t consider this to be outside the brief aesthetic, as we are not using the ten sessions to tackle a single issue. Each conversation is usually about a new issue, something that’s fresh on the client’s mind, or perhaps reflects a developing situation that we’ve discussed before. And the client can decide that they’ve had enough of a topic, or indeed of me – which leads us onto the next aspect of an SF aesthetic, the valuing of client autonomy. Client autonomy In the normal everyday world, people get to make decisions about their own lives; what to 38 – Journal of Solution Focused Brief Therapy Published by Digital Scholarship@UNLV, 2020 Solution Focused Work as an Aesthetic do, who to be with, where to go. These decisions are never the only factor in determining what happens. There are always other context and forces at work. One of my favourite quotes is from biologist Steven Rose (1997) who, paraphrasing Karl Marx, observed that “we create our own futures, but not in circumstances of our own choosing” (Rose, 1997, p. 309) It is worth considering, then, that in therapeutic work it has been normal for practitioners to take decisions for, and sometimes in opposition to, their clients. This comes from an old version of the basic doctor/patient relationship, where in decades gone by the doctor’s word was law, with the patient’s role being to play a grateful and willing recipient of the doctor’s expertise. Of course, if about to undergo brain surgery, then we would want someone with expertise in charge of it, and we would listen seriously to their advice. The risk is that this relationship can become unbalanced, one-sided and potentially abusive. If the doctor becomes an unchallengeable authority figure, and perhaps even one who is getting paid by making the relationship continue, then the risks of over-long treatments and disempowered clients are clear. The SF position has been that, broadly, it is the client who makes many of the decisions. What are their hopes from therapy? What are they going to do about it? When are things good enough to stop coming? In everyday life these questions are clearly for us as individuals. In the therapy world, however, it can be seen as a paradigm-busting revolution. While the conventional doctor/patient relationship can be characterised as parent/child (in the Transactional Analysis tradition, see for example Berne, 1958), the SF worker/client relationship is much more adult/adult. Both have responsibilities, both have parts to play, both have priorities, and these are to be kept as balanced as feasible. In the 1990s, I attended a workshop with Bill O’Hanlon in which he urged us to find the healthy adult within - in a pointed repost to the fashionable and non-SF urge to see our clients as wounded children or whatever. Of course, client autonomy is not automatic and over-riding in all circumstances. If the client seems to be putting themselves, or others, at risk then clearly the practitioner has some choices to make. Should they inform others? Should they instigate safeguarding processes? These are matters of professional judgement for the practitioner and are not to be underestimated. There are other situations where outside constraints – for example court orders, probation agreements or other matters of law, impinge on the client’s freedom of action. These can be taken into account in various ways with the client’s autonomy bounded rather than removed. In all these situations, the limits on client autonomy are seen as topics for discussion and ideally agreement with the client in terms of the next steps to be taken. There is an interface here with brevity as discussed above. The client’s autonomy includes their choice to decide when the treatment is over, or that they wish to see someone else. SF has found a healthy home in the world of coaching over the past couple of decades. One reason for this may be that SF therapy looks more like coaching than many other forms of practice, and so the fit is clear and natural from the start. Coaching clients are not usually seen as Journal of Solution Focused Brief Therapy – 39 25 Journal of Solution Focused Practices, Vol. 3 [2020], Iss. 1, Art. 12 Mark McKergow vulnerable people needing protection – rather, they are informed individuals who are making their own decisions about seeking support. The International Coach Federation (2019) defines coaching as “partnering with clients in a thought-provoking and creative process that inspires them to maximize their personal and professional potential”. The focus on partnering with clients seems to me to be a clear fit with the aesthetics of SF. Radical acceptance If we are viewing the client as making their own decisions, then we should also think very carefully about trying to argue with them. This skill of not arguing is sometimes called “radical acceptance” (de Shazer, 1997). He argued in the same article that: The client's answer needs to be accepted fully and literally” which is an art rather than a science and “difficult for many people. It requires a lot of self-discipline and a good deal of close listening. It is not easy to give up making judgments about how high the [scaling] number should be or how unreasonable and unrealistic the initial response to the miracle might be (de Shazer, 1997, p. 378). A key word here is the “initial” response to a question. One of the concerns I come across from people learning SF is that their clients may seek something impossible from the miracle; the amputee seeking the return of their lost limb, or the bereaved child wanting their parent to come back. And yes, it can be unsettling in some way when this happens. It is also a very obvious and heartfelt wish, and can therefore be accepted quite easily. Who wouldn’t want these things? The key point is that accepting doesn’t mean blindly agreeing and moving on. It can look like nodding gently, exhaling and waiting; waiting is often a good strategy, to allow the client to continue their thinking. It can look like quietly saying “Yes, of course…” and pausing to see what comes next. What comes next - sometimes after a very considerable silence - might be something like ”I’d be visiting my friends more” or ”I’d be happier going to school”. These are more tractable and can be picked up and expanded in the conversation. It turns out that in any actual client response to a question there will be multiple elements, and it is impossible to respond to all these elements equally or indeed at all. In a particular favourite example of this, Swedish psychiatrist and veteran SF practitioner Harry Korman (personal communication, 2009) mentioned (in a workshop some years ago) the woman who told him, “I want to be a better mother, but I’m such a worthless, worthless person.” There are clearly two parts to this statement. A problem focused practitioner might want to ask about being worthless. An SF practitioner will clearly see the first part, about being a better mother, as more obviously connected to what she wants. Harry Korman (2009), being the highly experienced SF practitioner that he is, found a way to accept both and yet promote the solution-focused element: So you think you’re a worthless person… but you want to be a better mother? (rising inflection at the end, making it a question and an invitation to carry on with this thread). 40– Journal of Solution Focused Brief Therapy https://digitalscholarship.unlv.edu/journalsfp/vol3/iss1/12 Solution Focused Work as an Aesthetic Radical acceptance comes down to not arguing with the client, even when to do so would be quite normal and acceptable. In his book Preventing Suicide: The Solution-Focused Approach (Henden, 2008), John Henden covers ten ways to accept an apparent wish for suicide, up to and including: If you decided to go ahead with the last resort option: (a) What method would you use? (i.e. pills, rope, razor blades, vacuum cleaner tube, firearms, etc.) (b) How prepared are you should you decide? (Henden, p. 129). In the context of an ordinary conversation, this sounds appalling – we might be seen to be urging the person on. But an SF conversation is not an ordinary one. There has been conversation before this, beginning to explore the client’s situation and build trust and connection within the first 10 minutes of the session. And indeed, Henden himself says that if he uses this question it is to get a better idea of how serious the person is – many people will back off at great speed when asked this question. With those who don’t, the practitioner can at least be more aware of what is happening, while continuing to take the client seriously and radically accept what they say. De Shazer shared this outlook with his long-term mentor and friend John Weakland. In a joint interview from 1994 (Hoyt, 2001) de Shazer speaks about taking the client seriously, whatever they say about their situation. A client tells you they’ve got a problem, then they’ve got a problem and you’d better take it seriously. You’d also better take it seriously if they tell you they ain’t got a problem… someone sent him because he drinks too much. He says he doesn’t drink too much and it’s not a problem. Leave it alone. Take it seriously (p. 21). Of course, there are other ways to take this kind of conversation forward without arguing with the client, such as asking about how come the referrer sent then along. And as the conversation develops, the client’s view of what they want may well develop and change. But if we don’t take them seriously to start with, an argument immediately ensues, enabling the practitioner to label the client as “resistant” or “in denial” which contributes little to progress and much to continuing stuckness. “Reading between the lines” is a distraction; listening very carefully to the lines and formulating appropriate responses is the name of the game. Radically accepting, not arguing with our clients and not reading between the lines leads to another key element of the SF aesthetic; staying at the surface. Staying At The Surface In more traditional methods of therapy, counselling and allied practices, it is quite normal to see the client’s behaviour as the outward manifestation of some kind of hidden internal causal mechanism. There are various forms of these hidden mechanisms, ranging from emotional to neuroscientific to ancient experience. The therapist’s task is to go deep to discover Journal of Solution Focused Brief Therapy – 41 26 Mark McKergow et al.: Volume 3 Issue 1 2019, Complete these causes and assist the client to deal with them. Indeed, the practitioner may claim to be the first to notice these causes and their importance, which the client must then address in order to satisfy their practitioner. In SF work, as I have written before (McKergow & Korman, 2009), we step around these potential questions by looking at the “in-between” – the interaction of the client with their environments, including other people. Sometimes newcomers don’t notice initially that SF questions are always framed with a person (often the client, but sometimes others in the client’s world) and their interactions, rather than any “internal” drivers. “What would be the first tiny signs you noticed that things were getting slightly better?” is a typical example. It is addressed to a person, about their interactions. It is not inviting them to introspect, to speculate over their feelings or other causal matters. Rather, we seek to focus our clients’ attention towards the outside, towards the world and towards what’s better in the past, present and future (Jackson & McKergow, 2002; 2007). This focus can be traced back to the Interactional view of the Mental Research Institute, Palo Alto (Watzlawick & Weakland, 1977) where Steve de Shazer and Insoo Kim Berg met and were first enthused about the brief therapy tradition. The MRI method of problem solving brief therapy seeks to get out of the mental box by looking for specific, concrete, descriptive information - who does what to whom and when? Their purpose in asking these questions was to seek patterns of behaviour that were holding the problem in place and then get the client to break or disrupt them. In SF, which followed on in the same tradition, the same questioning techniques were used to look for specific exceptions to the problem, so that these patterns could be amplified, and later to describe days after the miracle when the problem had mysteriously vanished overnight. These descriptions have become even more important as SF work has developed in recent years (Iveson & McKergow, 2016). Both the MRI team and later SF workers were striving to take mental health out of the clients’ heads and into their interactions with others. There has long been a conceptual muddle generated by those who want mental illness to ape physical illness. In the latter, something is amiss within the patient’s body, which must be diagnosed and cured. It’s an easy assumption to think of mental illness in the same way – something, perhaps depression or schizophrenia, is lurking within the client’s body or brain, and therefore diagnosis accompanied by either internal reflection or drugs are required to cure it. I am not anti-drugs by any means, but I am not in favour of putting people on drugs when they can be helped by a few sessions of conversation. Note that staying at the surface, like radical acceptance, does not mean that one client utterance is the end of the story. Different things will emerge as the therapeutic conversation goes on, and indeed the task of the SF practitioner is to frame questions to help this process. However, I don’t think this is about matters coming to the surface, as if they were there all the time, lurking in the depths and waiting the right moment to pop up. Rather, meaning and awareness shift during the conversation, new and overlooked things become more or less 42 – Journal of Solution Focused Brief Therapy Published by Digital Scholarship@UNLV, 2020 Solution Focused Work as an Aesthetic relevant, and fresh ideas emerge from the interaction. It’s interesting to notice how client autonomy and radical acceptance sit happily alongside staying at the surface. These are all important parts which add up to a dramatic new take on what it means to be human. Value of small differences This final, for now, part of the SF aesthetic is slightly different to the others – the value we place on small differences, detailed descriptions and tiny as opposed to huge signs. It is normal and logical to assume that large changes to the client’s life and circumstances will require big plans, big efforts, total commitment, and utter transformation. One part of this is why it is assumed that long treatments must always be superior to short ones, despite evidence to the contrary. Small signs of progress are seen as very valuable, the potential forerunners of more change, and signs that the client is on the way to a good enough life, where they feel able to tackle things under their own steam. From my very first SF training workshop (with Jane Lethem of the then Brief Therapy Practice in 1994), the idea has been present that small changes in one area of the client’s life can expand both through natural processes, but also as a ripple effect into other areas of life. The client notices changes and becomes more aware of how they are contributing to these changes, which then spread into other domains. It’s not hard to see how - slightly better relations at home can spill over into more confidence at work, more openness to relations with children and parents, less stress, more time to enjoy life and so on. This kind of connectedness fits well with both Buddhist philosophy, in which Steve de Shazer was interested (see for example de Shazer 1994, p. 9), and also with the science of complexity (see for example Waldrop, 1993), which emerged in the early 1990s. (I discovered both SF and complexity at the same time, and made some initial connections of my own.) Complexity shows how novel and unpredictable outcomes can emerge from small differences in unplanned and unexpected ways. The 1980s Chaos Theory offers a more sophisticated version of the butterfly effect - how the tiniest change to a weather system, such as the flapping of a butterfly’s wings in Brazil can lead to a tornado in Kansas or a storm in the Philippines. It doesn’t always lead to these outcomes – but small changes with amplification can lead to more impact than large plans, which stall and lead nowhere. SF questioning has led more and more to discussions of small, indeed tiny, details and differences which are part of better for the client. Follow-up questions like “what’s the first sign someone else would notice?”, and “what else?” help us to build more and more detail. Similarly, in follow-up sessions we ask about “what’s better?” and invite our clients to expand on whatever has emerged – whatever that might be. Of course, many of them start by saying “nothing is better…” which can be radically accepted and built on by a skilful practitioner. Sometimes when I have worked with people from other traditions, I have noticed that while Journal of Solution Focused Brief Therapy – 43 27 Journal of Solution Focused Practices, Vol. 3 [2020], Iss. 1, Art. 12 Mark McKergow Solution Focused Work as an Aesthetic they may be happy to discuss better in big picture, abstract noun, $5,000 words terms, they can become very nervous when I start asking about tiny details. One very experienced facilitator complained that they thought I was forcing people into action by doing this. Well, as long as that’s what they are paying me for, I make no apology! It is certainly interesting and under-discussed how talk about tiny details seems to lead smoothly and quickly to new possibilities for action. My book chapter from the SF World Conference (McKergow, 2019) will start to address this question. interested back in 1993 and has kept me at it for all the years since. For a new way of things to be more elegant is precious enough. For the new way to be more efficient as well is truly extraordinary. If SF delivers brief, respectful, humane treatment and progress at work, at school, in the hospital and the therapy room, then that’s worth nearly 30 years of my life. Conclusion Berne, E. (1958). Transactional analysis: A new and effective method of group therapy. The American Journal of Psychotherapy, 12, 735-743. BRIEF. (2019). One thing after another? Retrieved from https://www.brief.org.uk/resources/faq/one-thing-after-another de Shazer, S. (1994). Words were originally magic. New York, NY: W. W. Norton. de Shazer, S. (1997). Commentary: Radical acceptance. Families, Systems, & Health, 15(4), 375-378. http://dx.doi.org/10.1037/h0090136 Dolan, Y. M. (1991). Resolving sexual abuse: Solution-focused therapy and Ericksonian hypnosis for adult survivors. New York, NY: W. W. Norton. Henden, J. (2008). Preventing suicide: The solution focused approach. Chichester, UK: Wiley Hoyt, M. F. (2001). On the importance of keeping it simple and taking the client seriously: A conversation with Steve de Shazer and John Weakland. In Conversations with brief therapy experts. Routledge. Retrieved from http://web.uvic.ca/psyc/bavelas/De%20Shazer_Weakland%20interview.pdf Hume, D. (1987). Essays moral, political, literary. Indianapolis, IN: Literary Fund. International Coach Federation. (2019). Definition of coaching. Retrieved from https://coachfederation.org/about Iveson, C. & McKergow, M. (2016). Brief therapy: Focused description development. Journal of Solution-Focused Brief Therapy, 2(1), 1 - 17 Jackson, P. Z. and McKergow, M. (2002). The solutions focus. London: Nicholas Brealey Publishing (2nd revised edition 2007). Korman, H. (2009). Personal communication. Aesthetic. (n.d.). In Lexico.com. Retrieved from https://www.lexico.com/en/definition/aesthetic McKergow, M. (2019). Stretching the world: A friendly explanation of SF practice. Chapter in Solution Focused Practice Around The World, edited by Kirsten Dierolf, to be published by Taylor and Francis 2019. McKergow, M. W. & Korman, H. (2009). Inbetween - neither inside nor outside: The radical simplicity of Solution-Focused Brief Therapy. Journal of Systemic Therapies, 28(2), 34 – 49. Rose, S. P. R. (1997). Lifelines: Life beyond the gene. New York: Oxford University PressSamu, M. (2004). “Impressionism: art and modernity.” In Heilbrunn Timeline of Art History. If we look at these five aspects of an SF aesthetic, we can see a very telling contrast from the classical psychotherapeutic norm. Table 1. Comparing the classical therapeutic and SF aesthetics. Classical aesthetic SF aesthetic Long treatments are necessary Brevity – as brief as possible – is desirable Power is with the practitioner Client autonomy is to be respected wherever possible Read between the lines Radical acceptance Go deep Stay at the surface Valuing large and dramatic transformations Valuing small differences Note that none of these are about miracle questions, contracts, scaling, compliments and so on as such. These techniques seem to me to be more like corollaries of the aesthetic, natural ways of working which follow from these basics. There may well be a clue here about how come SF gets such short shrift from those accustomed to a more classical/traditional way of working. In the same way that Monet and Matisse were laughed at by the Parisian art audience, so SF is seen as a bit of a joke by those used to valuing length and depth. It may well be this total shift to a new paradigm that got me 44 – Journal of Solution Focused Brief Therapy https://digitalscholarship.unlv.edu/journalsfp/vol3/iss1/12 References Journal of Solution Focused Brief Therapy – 45 28 Mark McKergow et al.: Volume 3 Issue 1 2019, Complete New York: The Metropolitan Museum of Art, 2000Retrieved from https://www.metmuseum.org/toah/hd/imml/hd_imml.htm Segal, L. (1991). Brief therapy: The MRI approach. In A. S. Gurman & D. P. Kniskern (Eds.), Handbook of family therapy, 2, 171-199). Philadelphia, PA, US: Brunner/Mazel. Tate Gallery. (2019). Impressionism. Retrieved from https://www.tate.org.uk/art/artterms/i/impressionism Waldrop, M. (1993). Complexity: The emerging science at the edge of order and chaos. New York, NY: Pocket Books. Wampold, B. E. (2001). The great psychotherapy debate: Models, methods, and findings. New York, NY: Routledge Watzlawick, P. & Weakland J. (Eds). (1977). The interactional view: Studies at the Mental Research Institute, Palo Alto, 1965-1974. New York, NY: W. W. Norton. Solution Focused Therapy for Trauma Survivors: A Review of the Outcome Literature Ray Eads and Mo Yee Lee The Ohio State University Mark McKergow Email: mark@sfwork.com Abstract Directly confronting and processing past trauma can be distressing for clients and may contribute to the high dropout rates among leading trauma treatments. Solution-focused therapy (SFT) primarily focuses on the present and future and has been proposed as a strengths-based alternative for treating trauma survivors. This review systematically evaluated the existing outcome literature for the effectiveness of SFT for trauma survivors. Multiple databases were searched using search terms to identify results for solution-focused therapy as a treatment for trauma survivors. Eligible studies included experimental, quasi-experimental, or pre-post designs that reported outcome measures following SFT-based treatment. A total of five studies met inclusion criteria and were evaluated and summarized. Four out of the five studies included data on within-subjects changes in the SFT treatment group, reporting statistically significant improvements on trauma symptoms, recovery, self-esteem, and parenting, with moderate to large effect sizes. Three studies compared SFT with treatment-asusual (TAU) or no treatment and found mixed results. Compared to control groups, SFT showed statistically significant improvements with large effect sizes on post-traumatic growth and sleep issues, but effect sizes for trauma symptoms were small and not statistically significant or varied greatly between different reporters. The existing literature provides initial evidence of overall improvement for trauma survivors who received SFT, but the effectiveness of SFT at addressing trauma symptoms requires further investigation. More high quality, controlled studies are needed to evaluate SFT as a trauma treatment. Solution Focused Therapy for Trauma Survivors: A Review of the Outcome Literature Trauma is a significant public health issue with wide-ranging consequences for individuals and communities (Magruder, McLaughlin, & Elmore Borbon, 2017). Up to 70% of people 46 – Journal of Solution Focused Brief Therapy Published by Digital Scholarship@UNLV, 2020 Journal of Solution Focused Brief Therapy – 47 29 Ray Eads and Mo Yee Lee Journal of Solution Focused Practices, Vol. 3 [2020], Iss. 1, Art. 12 experience some form of trauma in their lifetime, with an average of up to three traumas per person (Kessler et al., 2017). The risk of trauma exposure varies widely across different countries due to variations in experiences related to war, crime, and disasters (Burri & Maercker, 2014), but many traumatic experiences are more common to everyday life—such as interpersonal violence, sexual assault, and sudden loss of loved ones (Kessler et al., 2017). Traumatic experiences that cause symptoms such as hyperarousal, flashbacks, and intense psychological distress may lead to a diagnosis of post-traumatic stress disorder (PTSD) (American Psychiatric Association [APA], 2013), but only if the traumatic event meets narrow and controversial criteria related to threat of death, severe injury, or sexual violence (Pai, Suris, & North, 2017). Qualitative research drawing on the lived experiences of participants indicates that PTSD criteria encompass only a small portion of problematic symptoms secondary to trauma, and instead suggests a complex relationship among relational distress, individual distress, and resilience (Coulter & Mooney, 2018). In response to the limitations of the PTSD diagnosis, there has been increased attention in the research literature to complex trauma and developmental trauma, which include repeated traumatic exposures and trauma beginning in early developmental stages (Denton, Frogley, Jackson, John, & Querstret, 2017; WamserNanney & Vandenberg, 2013). Effects of Trauma Traumatic experiences are associated with a variety of co-occurring disorders and disproportionately affect vulnerable populations (Mørkved et al., 2018; Slack, Font, & Jones, 2017). The effects of childhood trauma continue to reverberate through later life. Adverse childhood experiences (ACE) are associated with problematic changes in brain structure, mental and physical health problems in adulthood, and even early death (Brown et al., 2009; Herzog & Schmahl, 2018). The experience of childhood trauma is also associated with mental illness and substance use disorders, and increased exposure to repeated childhood trauma is related to increased rates of psychosis (Mørkved et al., 2018). There is also a relationship between child abuse and more severe psychosis; trauma from psychological abuse is associated with increased hospital admissions, and sexual abuse doubles the likelihood of attempting suicide (Álvarez et al., 2011). Trauma and PTSD are both found at high rates among youth in foster care, with males more likely to experience interpersonal violence and females more likely to experience sexualized violence (Salazar, Keller, Gowen, & Courtney, 2013). Trauma Treatment Considering the high prevalence and lasting impacts of trauma, effective interventions are needed to address symptoms and promote healing following the experience of trauma. There has been significant focus on evaluating effective treatments for PTSD among adults, children, and people with serious mental illnesses (Bisson, Roberts, Andrew, Cooper, & Lewis, 2013; Gillies, Taylor, Gray, O’Brien, & D’Abrew, 2012; Sin, Spain, Furuta, Murrells, & Norman, 2017). 48 – Journal of Solution Focused Brief Therapy https://digitalscholarship.unlv.edu/journalsfp/vol3/iss1/12 Solution Focused Therapy for Trauma Survivors In general, research supports the effectiveness of psychotherapy for improving symptoms related to trauma among adults and children (Bisson et al.; Gillies et al., 2012). However, the evidence is weaker for the treatment of PTSD symptoms in persons who also have diagnoses of serious mental illnesses (Sin et al., 2017). The most tested interventions for PTSD symptoms are trauma-focused cognitive behavioural therapy (TF-CBT), exposure therapy, eye movement desensitization and reprocessing (EMDR), and non-trauma focused cognitive behavioural therapy (CBT; Bisson et al.). While the overall evidence supports the effectiveness of psychotherapeutic approaches, there is weaker evidence that these treatments are significantly more effective than other psychotherapies (Bisson et al., 2013). The most commonly tested trauma treatments—TF-CBT, EMDR, and exposure therapy (Bisson et al.)—reflect a linear perspective that trauma treatment must directly address the traumatic event to be effective. However, there is growing interest and evidence for present-centered therapy (PCT) as an effective alternative to “active” treatments focused specifically on trauma (Belsher et al.). Drawbacks of a Trauma-focused Approach Trauma can be a difficult subject for clients to discuss. By the nature of PTSD’s diagnostic criteria, clients with PTSD likely already experience flashbacks, hypervigilance, and psychological distress (APA, 2013), even without the added stress of having to recall traumatic memories during therapy. Incompetence or lack of empathy among helping professionals can result in a client’s re-traumatization rather than healing (Newgent, Fender-Scarr, & Bromley, 2002). The potential drawbacks of a trauma-focused approach are evident in the high dropout rates for PTSD treatments, which include reported dropout and non-response rates as high as 50% (Schottenbauer, Glass, Arnkoff, Tendick, & Hafter Gray, 2008). One meta-analysis found that various trauma treatments showed similar dropout rates when compared with each other, with the exception that PCT showed notably lower dropout rates than trauma-focused therapies (22% for PCT compared to 36% for trauma-focused; Imel, Laska, Jakupcak, & Simpson, 2013). This has contributed to increased interest in PCT as a frontline treatment for trauma, but the authors of a Cochrane Review Protocol point out that PCT was originally designed only as a comparator condition for TF-CBT, and thus its design can likely be improved upon (Belsher et al., 2017). Solution-Focused Therapy Solution-focused therapy (SFT) originated at the Brief Family Therapy Center in Milwaukee, Wisconsin, with an emphasis on the construction of solutions rather than assessment of problems (de Shazer et al., 1986). Like PCT, SFT does not focus on the past, except to elicit past successes and exceptions to problems (De Jong & Berg, 2013). Unlike PCT, SFT has an intentional design based in constructivist philosophy, systems theory, and observations from real-world practice with clients and families (de Shazer et al., 1986). The fundamental shift from a problem-solving approach to a solution-building approach eschews Journal of Solution Focused Brief Therapy – 49 30 et al.: Volume 3 Issue 1 2019, Complete Ray Eads and Mo Yee Lee the need for detailed discussion of past events, and instead necessitates a present- and futurefocused orientation to generate change that is meaningful from the client’s point of view (De Jong & Berg). SFT techniques such as praise, exploring past successes, and looking for exceptions to problems reflect a strengths-based orientation that may help with problems such as client “resistance” or treatment drop out (De Jong & Berg). Solution-Focused Therapy for Trauma SFT has been applied to clients managing a variety of different forms of trauma (Froerer, von Cziffra-Bergs, Kim, & Connie, 2018), with an emphasis on post-traumatic success rather than PTSD symptoms or the trauma itself (Bannink, 2008). Trauma can produce overwhelming feelings of helplessness and hopelessness (Sklarew & Blum, 2006), but SFT offers a number of strategies for empowering clients and building hope (De Jong & Berg, 2013). First, the exploration of exceptions can help clients identify the times when they are already able to manage the symptoms or effects of their trauma and could generate hope that these moments of exception can increase in the future. Second, the emphasis on small changes—which will reverberate through client systems to become larger change (De Jong & Berg)—may seem more realistic and manageable for trauma survivors than attempting to directly confront their worst trauma. The miracle question may not be appropriate for clients who have experienced severe trauma, as this does involve picturing the trauma completely gone and may be too much for some clients to imagine (Coulter, 2014). SFT has demonstrated effectiveness across a variety of populations and problem areas (Gingerich & Peterson, 2013; Kim, 2008). Research has also supported the utility of resourcebased and future-oriented processes in SFT techniques (Franklin, Zhang, Froerer, & Johnson, 2017), which are key to the conceptual case for SFT as a trauma treatment. SFT has been applied to work with populations with a high prevalence of trauma history, such as child welfare (Sabalauskas, Ortolani, & McCall, 2014). Growing evidence supports the effectiveness of SFT among foster care youth; SFT has demonstrated improved results in placement stability (Koob & Love, 2010), self-efficacy (Cepukiene, Pakrosnis, & Ulinskaite, 2018), and behaviour problems (Cepukiene & Pakrosnis, 2011). Systemic group therapy—with a similar orientation to SFT—outperformed a psychoanalytic group for adult survivors of childhood sexual abuse (Lau & Kristensen, 2007), though the treatment effects diminished over time (Elkjaer, Kristensen, Mortensen, Poulsen, & Lau, 2014). With a strong conceptual argument for SFT’s applicability to trauma (Bannink, 2008; Coulter, 2014), current application of SFT for trauma treatment (Froerer et al., 2018), and evidence of effectiveness in populations where trauma is likely (Cepukiene & Pakrosnis, 2011; Cepukiene et al.; Koob & Love), a review of the evidence for SFT for trauma survivors is warranted. Method The present study aimed to conduct the first systematic review of the outcome literature 50 – Journal of Solution Focused Brief Therapy Published by Digital Scholarship@UNLV, 2020 Solution Focused Therapy for Trauma Survivors for the effectiveness of SFT for trauma survivors, and to evaluate the methodological rigor and fidelity of existing studies. For the purposes of the review, studies needed to clearly identify the presence of trauma history among the entire treatment group or employ a direct measure of trauma symptoms. Due to the systemic nature of SFT—where change in one area is expected to cause change throughout the system—additional outcome measures unrelated to trauma were assessed as part of the effectiveness of SFT so long as the entire sample consisted of trauma survivors. As a result of the variety of outcome measures included, the authors decided not to employ meta-analytic techniques as part of the review. Selection Criteria The study aimed to obtain as much useful information as possible regarding a topic that has never previously been the subject of a systematic review. For this reason, the study sought all available outcome literature on the effectiveness of SFT for treatment with trauma survivors. For the purposes of the review, we included any research study—published or unpublished—that 1) utilized identifiable SFT techniques with a treatment group, 2) identified the entire sample as trauma survivors or directly measured the effect of SFT on trauma symptoms, and 3) reported quantitative outcome measures. Unpublished dissertations met inclusion criteria but masters theses found in database searching were excluded. Study designs could include randomized controlled trials (RCTs), quasi-experimental designs, or one group pre-post designs; single subject designs and case studies were excluded. Though randomized controlled studies are considered the most rigorous evidence (Engel & Schutt, 2017), we decided to include a broader range of methodologies to allow for the most comprehensive review possible of the literature regarding SFT for trauma survivors. Search Process The search process began with database searches to identify studies related to the treatment of trauma survivors or trauma symptoms that used a solution-focused approach. Since there has been no prior review on the topic area, we searched the time period up to and including June 2019. The search included the following databases: EBSCOHost (Criminal Justice Abstracts with Full Text, MEDLINE, PsycINFO, Social Work Abstracts, SocINDEX with Full Text), PubMed, Web of Science, ProQuest Dissertations and Theses, Campbell Collaboration, and Cochrane Library. In each database, we searched for SFT studies by searching titles, abstracts, and keywords for “Solution focused” OR “SFBT” OR “SFT,” and narrowed results to trauma survivors by adding an additional title, abstract, and keyword search for “trauma” OR “PTSD” OR “post-traumatic” OR “abuse” OR “victim” OR “violence” or “survivor.” In addition to database searching, the grey literature was assessed by looking for studies on ClinicalTrials.gov, as well as by contacting SFT researchers. We also reviewed the reference lists of included studies and identified one potential study from the reference list of a systematic review evaluated during the full-text review process. Studies written in languages Journal of Solution Focused Brief Therapy – 51 31 Ray Eads and Mo Yee Lee Journal of Solution Focused Practices, Vol. 3 [2020], Iss. 1, Art. 12 other than English were included in the review and were assessed based on their English abstract; no studies in other languages proceeded to full-text review. The search process identified 676 total records for screening and review. Solution Focused Therapy for Trauma Survivors Screening and Eligibility Review From the initial pool of 676 records, we eliminated 275 duplicate results so that 401 records progressed to the screening process (see Figure 1). We then conducted title and abstract reviews and excluded a further 333 records that did not meet study selection criteria. The remaining 68 articles and dissertations warranted full-text review to determine if they met all inclusion criteria. During full-text review, we determined that 37 results did not met criteria for being an outcome study, and a further 5 studies did not meet the criteria for using an SFTbased intervention. The final phase of eligibility screening involved determining whether the study used SFT as a treatment for trauma survivors. Twelve studies in the full-text review did not relate sufficiently to trauma and were excluded. Another 3 articles used solution-focused approaches as part of macro interventions for trauma-informed agencies, and 6 studies used SFT with offenders or couples in domestic violence situations; these studies were excluded as they were not interventions targeting the survivors of trauma. In total, 63 studies were excluded during full-text review, and 5 studies met all inclusion criteria and were included in the analysis. Data Analysis Strategy Figure 1. Systematic Review Process 52 – Journal of Solution Focused Brief Therapy https://digitalscholarship.unlv.edu/journalsfp/vol3/iss1/12 For the five studies meeting all inclusion criteria, data were abstracted from the articles regarding the study design, intervention, sample size, population, and outcome measures. We then assessed each article for its methodological quality and SFT fidelity, adapting a format used in a prior SFT review by Gingerich and Peterson (2013). The present study used an adapted version of the SFBT Model Adherence Checklist (Smock et al., 2008) to assess for seven SFT components and techniques: scaling questions, miracle question, exceptions, goalsetting, focus on solutions, break for consultation, and compliments/praise. For methodological quality, seven common components of high-quality studies were assessed for each study: use of a control group, randomization to treatment conditions, clear treatment fidelity procedures, large sample size for the treatment group (n > 20), active treatment comparison condition, and peer-reviewed publication process (Engel & Schutt, 2017; Gingerich & Peterson, 2013). Finally, the present study compiled and summarized the findings of each study regarding the effectiveness of SFT for trauma symptoms and/or trauma survivors, and the comparative effectiveness of SFT against control groups. When possible, we included effect sizes in terms of Cohen’s d that were published by the included studies’ authors, that we converted from other published effect sizes into Cohen’s d, or that we calculated ourselves from information provided in the included studies’ results sections. Journal of Solution Focused Brief Therapy – 53 32 et al.: Volume 3 Issue 1 2019, Complete Ray Eads and Mo Yee Lee Table 1. Study Outcomes and Effect Sizes Study Design (Control condition) Solution Focused Therapy for Trauma Survivors Results Sample Size Sample Population Outcome Measures Effect Size Within Group Treatment / Between Group Control Kim, Brook, & Akin (2018) Experimental (vs. TAU) 64 Child welfare parents Trauma Symptom Checklist-40Ɨ (TSC40) TSC-40: .76* / .62* TSC-40: .29 Liu (2017) [dissertation] Experimental (vs. TAU) 41 Children with sleep problems and trauma history CRTES-R: .82** / -.09 CTS: IDR SSR: IDR CRTES-R: 1.00* CTS: .06 SSR: 1.05* Zhang, Yan, Du, & Liu (2014) Quasiexperimental (vs. No treatment) 43 Mothers of ASD children Child Reaction to Traumatic Events Scale-Revised (CRTES-R) Connecticut Trauma Screen (CTS) Sleep Self Report (SSR) Post-traumatic Growth Inventory (PTGI) [Chinese version] PTGI: IDR Hiebert-Murphy & Richert (2000) One Group Pretest-Posttest (N/A) 29 Mothers with CSA history Rosenberg SelfEsteem Scale Parenting Sense of Competence Scale Kansas Parental Satisfaction Scale Index of Parental Attitudes Kruczek & Vitanza (1999) One Group Pretest-Posttest (N/A) 41 Teen girls with CSA history Self-esteem: .68** Parenting efficacy: .47* Parenting self-esteem: .81** Attitude toward children: .53* Recovery: 2.62 SMT = IDR PTGI: Post-test: 1.26 6-month follow up: .92** N/A The Solution Focused N/A Recovery Scale for Survivors of Sexual Abuse The Skill Mastery Test (SMT) Note. TAU = Treatment as usual; N/A = Not Applicable; ASD = Autism Spectrum Disorder; CSA = Childhood Sexual Abuse; Effect Size = Cohen's d IDR = Insufficient data reported to calculate effect size ƗFor Kim et al., the entire sample did not have established trauma history, so only the trauma symptom measure was assessed statistically significant at p < .05; statistically significant at p < .01 Positive effect size indicates desired direction (improvement or favoring treatment), negative effect size indicates change in undesired direction 54 – Journal of Solution Focused Brief Therapy Published by Digital Scholarship@UNLV, 2020 Five studies met all criteria for inclusion in the review. The studies consisted of two RCTs (including one dissertation), one quasi-experimental design, and two single group pretestposttest designs. Total sample sizes ranged from 29 to 64, and SFT treatment conditions ranged from 18 to 41 participants each. The studies were all assessed to be adequately powered, which was supported by the later observation that each study produced at least one statistically significant effect size. Four of the five studies had samples comprised entirely of trauma survivors, which included mothers and adolescent girls with history of childhood sexual abuse, mothers whose children had received an autism spectrum disorder (ASD) diagnosis, and children with sleep problems and assessed trauma history. Kim, Brook, and Akin (2018) did not specify trauma history for their sample of child welfare parents with substance use problems—though a high prevalence of trauma is expected for this population—so only the outcome measure directly assessing trauma symptoms was included in the review. In addition to the trauma histories among studies’ participants, four out of the five studies also included outcome measures related to trauma symptoms, post-traumatic growth, or recovery following sexual abuse. Table 1 shows the study designs and samples, as well as outcome measures and effect sizes. Intervention Outcomes As shown in Table 1, the included studies employed a variety of outcome measures capturing symptoms and recovery directly related to trauma, as well as additional benefits of SFT treatment on the lives of trauma survivors. The inclusion of indirect as well as direct effects of SFT on trauma reflects the systemic perspective underlying SFT. Among the direct measures related to trauma, two studies used outcome measures specifically assessing trauma symptoms, which included: Trauma Symptom Checklist-40 (TSC-40); Child Reaction to Traumatic Events Scale-Revised (CRTES-R; child report); and Connecticut Trauma Screen (CTS; parent report). Additionally, two studies directly measured growth or recovery following the experience of trauma, which included: Post-traumatic Growth Inventory (PTGI; Chinese version) and The Solution Focused Recovery Scale for Survivors of Sexual Abuse. In addition to the outcomes directly related to trauma, included studies also measured additional benefits of SFT for trauma survivors, including sleep problems (Sleep Self Report [SSR]), self-esteem (Rosenberg Self-Esteem Scale), parenting (Parenting Sense of Competence Scale [PSOC], Kansas Parental Satisfaction Scale [KPS], and Index of Parental Attitudes), and knowledge of positive coping strategies (The Skill Mastery Test [SMT]). Since included studies measured outcomes in terms of within-subjects improvement over time, improvement compared to no treatment, and improvement compared to treatment-asusual (TAU), it is important to analyze various categories before discussing the overall evidence of SFT effectiveness. Journal of Solution Focused Brief Therapy – 55 33 Ray Eads and Mo Yee Lee Journal of Solution Focused Practices, Vol. 3 [2020], Iss. 1, Art. 12 Within-subjects findings. All five included studies reported results of within-subjects changes over the course of treatment, though, for several measures (CTS, SSR, PTGI, SMT) there was not sufficient data reported to calculate an effect size. On direct measures of trauma symptoms (TSC-40, CRTES-R), subjects in SFT treatment groups showed statistically significant improvements in their trauma symptoms with moderate to large effect sizes (d = .76 – .82). On the TSC-40, the control group also showed statistically significant within-group improvements with moderate effect size (d = .62), but on the CRTESR the control group showed slight regression (d = -.09). The reporting in Liu (2017) did not allow within-subjects effect sizes to be calculated on the CTS, but visual inspection of reported results showed notable improvements for both the SFT and control groups. As the CRTES-R (child report) and CTS (parent report) represent trauma symptom measures from two sources within the same study, it is unclear why the control group in Liu’s study varied so significantly between child and parent reports; however, the SFT group showed improved PTSD symptoms on both child and parent reports. On direct measures of post-traumatic growth or recovery, the SFT group in Kruczek and Vitanza (1999) showed statistically significant improvements in symptom recovery with a very large effect size (d = 2.62). Zhang, Yan, Du, and Liu (2014) did not report sufficient data to report within-subjects effect sizes on the PTGI, but visual inspection showed notable improvement in the SFT group and no significant change in the control group. For the additional indirect benefits (not directly related to trauma) from SFT with trauma survivors, three studies reported data on additional benefits but only Hiebert-Murphy and Richert (2000) reported sufficient data to calculate effect sizes. The SFT treatment for mothers with history of childhood sexual abuse showed statistically significant improvements related to self-esteem (d = .68 – .81) and parenting (d = .47 – .53). The authors also published significant results on parental satisfaction from the PSOC but noted that the improvement in parental satisfaction on KPS was not significant (p = .11) without reporting the data, so the effect size on parental satisfaction was excluded from this review. For sleep problems (SSR), visual inspection showed improvements for both the SFT and control groups, and for knowledge of coping strategies (SMT). Kruczek and Vitanza (1999) noted visual evidence of improvement that did not achieve statistical significance. Between-group findings. Three of the included studies used control groups that allowed statistical testing between the treatment and control conditions. For post-traumatic growth, Zhang et al. (2014) tested SFT against a no-treatment control condition. The PTGI scores were significantly better for the SFT group at both post-intervention and 6-month follow-up, with a very large effect size in favor of SFT at post-intervention (d = 1.26, p < .01) and a large effect size favoring SFT at 6-month follow-up (d = .92, p < .01). Two other studies compared SFT to a TAU control group and tested direct measures of trauma symptoms. Liu (2017) compared solution-focused art therapy provided during a summer youth program to a control group receiving only the summer youth program. The findings on the effectiveness of SFT compared 56 – Journal of Solution Focused Brief Therapy https://digitalscholarship.unlv.edu/journalsfp/vol3/iss1/12 Solution Focused Therapy for Trauma Survivors to the youth program differed between child and parent report of PTSD symptoms. Based on CRTES-R (child report) scores, SFT significantly outperformed TAU in reducing PTSD symptoms with a very large effect size (d = 1.00, p < .05). However, based on CTS (parent report) scores, there was no meaningful difference between SFT and TAU (d = .06). Liu (2017) also tested SFT for sleep problems (SSR) among trauma survivors against TAU and found a large effect size (d = 1.05, p < .05) favoring SFT. Finally, Kim et al. (2018) compared SFT to a TAU condition consisting of other research-supported treatments used by agency clinicians, which mostly consisted of CBT, TF-CBT, and motivational interviewing. The study found a small effect size in favor of SFT (d = .29) for improved TSC-40 scores at post-treatment, but the effect was not statistically significant. Based on this finding, Kim et al. concluded that SFT showed comparable effectiveness with other evidence-based treatments. The overall evidence for SFT versus TAU for trauma symptoms varies widely, with effect sizes ranging from very small to large (d = .06 – 1.00) in favor of SFT. Harms from SFT treatment? None of the five included studies indicated evidence of harm caused by SFT with trauma survivors. In fact, all within-subjects changes mentioned by study authors showed some improvement following SFT even if the trend was not statistically significant, and none of the control groups outperformed SFT when compared on outcome measures. Treatment Fidelity and Study Quality In addition to compiling the empirical evidence for SFT for trauma survivors, the present study sought to evaluate the quality and methodological rigor of included studies. The included studies provided SFT-based interventions through a number of modalities, including individual counselling, group treatment, and art therapy (see Table 2). This review assessed the SFT treatment fidelity of each included study, and also evaluated the quality of the study design. SFT fidelity. To determine whether the treatments delivered in each study met criteria for being solution-focused, the author assessed each study for evidence of seven solution-focused techniques: scaling, miracle question, exceptions, goal-setting, focus on solutions, consultation break, and compliments/praise (Smock et al., 2008; SFBTA, 2013). All three of the controlled studies included six out of the seven SFT components, indicating a high level of fidelity to SFT principles and techniques. Both RCTs also included formal fidelity procedures and measures, while the quasi-experimental study employed expert content developers. The two older prepost designs employed four and one SFT components respectively, with no formal fidelity process, indicating moderate to poor SFT treatment fidelity. Journal of Solution Focused Brief Therapy – 57 34 et al.: Volume 3 Issue 1 2019, Complete Ray Eads and Mo Yee Lee Table 2. Intervention Fidelity and Study Quality Study Interventi on SFBT Fidelity SFBT Componentsa Quality Componentsb Kim, Brook, & Akin (2018) SFBT individual counselin g 40 hours SFBT training for clinicians, SFBT Fidelity Instrument, sessions reviewed at random by clinical directors S, E, G, F, B, C C, R, P, F, L, A, O Liu (2017) [dissertation] Solutionfocused art therapy S, M, E, G, F, C C, R, F, L, A, O Zhang, Yan, Du, & Liu (2014) SFBT group counselin g S, M, E, G, F, C C, P, O Hiebert-Murphy & Richert (2000) Solutionfocused parenting group Author developed Solution-Focused Art Therapy Manual and Fidelity measure Intervention content develop by 10 experts on SFBT, group counseling or raising children with Autism Spectrum Disorders Authors give an outline of a 12 session-group based on a solutionfocused approach to intervention Authors developed treatment protocol based on solutionfocused and Ericksonian interventions E, G, F, C P, L, O Kruczek & Vitanza (1999) Solutionfocused / Ericksonia n group therapy Study quality. The present review also assessed the methodological quality of the included studies using seven components of design quality: control groups, randomization, peerreviewed publication process, formalized treatment fidelity process, large treatment group sample size (n > 20), active treatment control condition, and objective outcome measures (Engel & Schutt, 2017; Gingerich & Peterson, 2013). Only one study (Kim et al., 2018) included all seven quality components, representing a high level of methodological quality and rigor. The other RCT (Liu, 2017) included six out of seven quality components, but was an unpublished dissertation that did not go through a peer-review process. Also, the wide variation in the control group’s post-test PTSD scores between child and parent reports raises concerns about the study’s measurement validity. The remaining three studies each met three out of seven quality criteria, with only Zhang et al. (2014) including a control group, representing lower methodological quality susceptible to various threats to internal validity (Engel & Schutt). These three studies all lacked formal fidelity processes, which weakens the conclusions that can be drawn regarding the effectiveness of SFT for their reported outcome measures. In the case of Kruczek and Vitanza (1999) in particular, it is questionable whether the intervention tested truly represents SFT. Discussion F P, L, O Note. SFBT = Solution-focused brief therapy S = scaling questions, M = miracle question, E = exceptions, G = goal-setting, F = focus on solutions, B = break for consultation, C = compliments b C = control group, R = randomization, P = peer review, F = fidelity process, L = large sample (treatment group > 20), A = active treatment control O = objective outcome measures a, b Evaluation of components adapted from Gingerich and Peterson (2013) a Solution Focused Therapy for Trauma Survivors The present study conducted the first systematic review of the effectiveness of SFT for the treatment of trauma survivors. The evidence base for SFT for trauma is still in an emerging developmental state, with only five studies meeting inclusion criteria for SFT outcome studies for trauma survivors. Despite the small number of studies and dearth of high-quality studies, the review provides valuable insights into the potential benefits of SFT with trauma survivors. Effectiveness of SFT for Trauma Survivors The existing outcome literature provides initial evidence of the overall effectiveness of SFT for treating survivors of trauma. In particular, within-subjects treatment effects showed moderate to large effect sizes on direct measures of both trauma symptoms and recovery, as well as indirect benefits on outcome measures including self-esteem and parenting. The withinsubjects tests meet two criteria for causal validity—time order and association—but cannot rule out additional explanations for the change in scores, such as maturation (Engel & Schutt, 2017). In fact, two measures of direct trauma symptoms also showed notable improvements in the control group, and the unpublished dissertation reported large time effects in repeated measures ANOVA tests (Liu, 2017). However, the effectiveness of SFT for trauma survivors was also supported by between-group tests, particularly for post-traumatic growth and benefits for sleep problems. SFT showed large effect sizes for post-traumatic growth (compared to no treatment) and for sleep problems (compared to TAU). The use of control groups in both studies and randomization in the latter study lend greater confidence to the evidence of benefits from SFT for trauma survivors. Though the overall evidence is weakened by fidelity 58 – Journal of Solution Focused Brief Therapy Published by Digital Scholarship@UNLV, 2020 Journal of Solution Focused Brief Therapy – 59 35 Journal of Solution Focused Practices, Vol. 3 [2020], Iss. 1, Art. 12 Ray Eads and Mo Yee Lee and rigor concerns and the small number of studies, there is some evidence that SFT provides both general benefit to trauma survivors and specifically encourages post-traumatic growth and recovery. SFT for Alleviating Trauma Symptoms A primary concern among many studies of trauma treatments is the reduction of PTSD symptoms (Bisson et al., 2013). In this area, the existing evidence regarding the effectiveness of SFT is mixed, particularly when compared with TAU. Though all trauma symptom measures showed significant improvements following SFT in within-subjects tests, this evidence is weakened by similar improvements in control groups. In the highest quality study, SFT outperformed TAU that included established trauma treatments, but the effect size was small (d = .29) and not statistically significant. In the other RCT, the large effect size favoring SFT over TAU on child-reported PTSD symptoms vanished when comparing parent-reported PTSD symptoms, suggesting possible measurement issues. More well-controlled studies are needed to establish the effectiveness of SFT for alleviating trauma symptoms. Appropriateness of SFT for Trauma Treatment The application of SFT to trauma survivors draws from compelling conceptual arguments that a solution-focused approach could be an effective means of treating trauma without subjecting clients to the stress of directly focusing on traumatic memories. Notably, the included studies in this review did not show evidence of harms from SFT, and no evidence suggested SFT was less effective than TAU. Furthermore, the benefits seen from SFT with trauma survivors on a variety of direct and indirect outcomes provide support for the systemic assumptions underlying the SFT treatment approach. The initial evidence supports the appropriateness of SFT for trauma survivors, and it is notable that SFT produced favorable treatment effects without a direct, past-focused approach to trauma treatment. Therefore, it is plausible that some of the clients who drop out of trauma-focused treatments could benefit from the SFT approach. The present study did not analyze retention or dropout rates, though the comparative dropout rates for SFT versus trauma-focused treatments would be a rich area for future research. Limitations The small number of studies and lack of high-quality controlled studies significantly limits the conclusions that can be drawn regarding the effectiveness of SFT for treating survivors of trauma. Many of the conclusions noted in this review are based on within-subjects findings, which are especially susceptible to multiple sources of bias. The decision to include weaker methodological designs added to the scope of the review but lowers the quality of research evidence summarized in this review. Also, the search process did not include hand searching of trauma journals, so it is possible that some studies could have been missed; however, the 60 – Journal of Solution Focused Brief Therapy https://digitalscholarship.unlv.edu/journalsfp/vol3/iss1/12 Solution Focused Therapy for Trauma Survivors final list of studies was sent to leading SFT researchers who felt it was comprehensive. We also opted to exclude studies that used SFT as a treatment for perpetrators of trauma as well as macro-level responses to traumatized populations, which may have omitted valuable insights on the systemic applications of SFT in the field of trauma. This review did not analyze included studies’ dropout rates, which would help bolster the case for SFT as an alternative to traumafocused treatments with high dropout. Implications This systematic review of the outcome literature for SFT for trauma survivors has important implications for future research and practice. First, policymakers, agencies, and clinicians should consider adding SFT to the evidence-supported treatments offered to clients who have experienced trauma. While the evidence for SFT for trauma is in an early developmental stage, there is no evidence of harm from SFT or lower effectiveness compared to other treatments. More importantly, SFT offers a distinctly different approach than the direct, trauma-focused approaches that may contribute to the high dropout rates seen for PTSD treatments (Imel et al., 2013). Some traumatized clients who would otherwise drop out of traditional treatment may find SFT a more acceptable alternative. Even as the evidence base continues to build for SFT as a trauma treatment, clients who prefer a present-focused or strengths-based approach should be given the option of receiving SFT as part of an approach that allows clients to discuss their traumatic experiences if they choose, but without pressuring them to do so. Future Research The current review’s findings indicate the need for additional research on the effectiveness of SFT for trauma survivors. The current evidence suffers from a small number of studies and low-quality research designs, so additional studies with randomized, experimental designs would add considerably to the quality of the evidence for SFT for trauma treatment. In particular, more research is needed regarding the effectiveness of SFT at alleviating trauma symptoms when compared with other treatments. Also, the conceptual basis for SFT for trauma treatment warrants additional research on the comparative retention rates between SFT and trauma-focused treatments. This review did not analyze dropout rates, but future research studies and systematic reviews should seek to determine whether SFT involves lower dropout than trauma-focused approaches. Finally, the search process uncovered a number of studies regarding SFT with perpetrators of trauma and couples experiencing domestic violence – this alternative approach to use SFT to prevent future trauma may warrant its own systematic review. Conclusion This study conducted the first systematic review of the effectiveness of SFT for the treatment of trauma survivors. Though based on a small number of studies with limited quality, Journal of Solution Focused Brief Therapy – 61 36 Ray Eads and Mo Yee Lee et al.: Volume 3 Issue 1 2019, Complete the evidence provides initial support for the benefits of SFT for trauma survivors without needing to directly focus on past trauma. Additional research is needed in this area, especially regarding the effectiveness of SFT for alleviating trauma symptoms when compared with other treatments. The conceptual basis for SFT for trauma suggests that SFT may involve a lower dropout rate than trauma-focused treatments, but this was not a focus of the review. Future studies should seek to replicate the positive effects of SFT with trauma survivors, and also test retention rates for SFT versus trauma-focused treatment. References References marked with an asterisk indicate studies included in the systematic review. Álvarez, M. J., Roura, P., Osés, A., Foguet, Q., Solà, J., & Arrufat, F. X. (2011). Prevalence and clinical impact of childhood trauma in patients with severe mental disorders. Journal of Nervous and Mental Disease, 199(3), 156–161. https://doi.org/10.1097/NMD.0b013e31820c751c American Psychiatric Association. (2013). Diagnostic and statistical manual of mental disorders (5th ed.). Arlington, VA: American Psychiatric Publishing. Bannink, F. P. (2008). Posttraumatic success: Solution-Focused brief therapy. Brief Treatment and Crisis Intervention, 8(3), 215–225. https://doi.org/10.1093/brieftreatment/mhn013 Belsher, B., Beech, E., Evatt, D., Rosen, C. S., Liu, X., Otto, J., & Schnurr, P. P. (2017). Presentcentered therapy (PCT) for post-traumatic stress disorder (PTSD) in adults (Protocol). Cochrane Database of Systematic Reviews, 2017(12), 1–13. https://doi.org/10.1002/14651858.CD012898 Bisson, J. I., Roberts, N. P., Andrew, M., Cooper, R., & Lewis, C. (2013). Psychological therapies for chronic post-traumatic stress disorder (PTSD) in adults (Review). Cochrane Database of Systematic Reviews, (12),1–242. https://doi.org/10.1002/14651858.CD003388.pub4.www.cochranelibrary.com Brown, D. W., Anda, R. F., Tiemeier, H., Felitti, V. J., Edwards, V. J., Croft, J. B., & Giles, W. H. (2009). Adverse childhood experiences and the risk of premature mortality. American Journal of Preventive Medicine, 37, 389–396. https://doi.org/10.0.3.248/j.amepre.2009.06.021 Burri, A., & Maercker, A. (2014). Differences in prevalence rates of PTSD in various European countries explained by war exposure, other trauma and cultural value orientation. BMC Research Notes, 7(1). https://doi.org/10.1186/1756-0500-7-407 Cepukiene, V., & Pakrosnis, R. (2011). The outcome of solution-focused brief therapy among foster care adolescents: The changes of behavior and perceived somatic and cognitive difficulties. Children and Youth Services Review, 33, 791–797. Cepukiene, V., Pakrosnis, R., & Ulinskaite, G. (2018). Outcome of the solution-focused selfefficacy enhancement group intervention for adolescents in foster care setting. 62 – Journal of Solution Focused Brief Therapy Published by Digital Scholarship@UNLV, 2020 Solution Focused Therapy for Trauma Survivors Children and Youth Services Review, 88 (November 2017), 81–87. https://doi.org/10.1016/j.childyouth.2018.03.004 Coulter, S. (2014). The applicability of two strengths-based systemic psychotherapy models for young people following type 1 trauma. Child Care in Practice, 20(1), 48–63. https://doi.org/10.1080/13575279.2013.847057 Coulter, S., & Mooney, S. (2018). Much more than PTSD: Mothers’ narratives of the impact of trauma on child survivors and their families. Contemporary Family Therapy, 40(3), 226–236. https://doi.org/10.1007/s10591-017-9408-z De Jong, P., & Berg, I. K. (2013). Interviewing for solutions (4th. ed.). Belmont, CA: Brooks/Cole. De Shazer, S., Berg, I. K., Lipchik, E., Nunnally, E., Molnar, A., Gingerich, W., & Weiner-Davis, M. (1986). Brief therapy: Focused solution development. Family Process, 25(2), 207– 221. https://doi.org/10.1111/j.1545-5300.1986.00207.x Denton, R., Frogley, C., Jackson, S., John, M., & Querstret, D. (2017). The assessment of developmental trauma in children and adolescents: A systematic review. Clinical Child Psychology and Psychiatry, 22, 260–287. https://doi.org/10.1177/1359104516631607 Elkjaer, H., Kristensen, E., Mortensen, E. L., Poulsen, S., & Lau, M. (2014). Analytic versus systemic group therapy for women with a history of child sexual abuse: 1-Year followup of a randomized controlled trial. Psychology and Psychotherapy: Theory, Research and Practice, 87(2), 191–208. https://doi.org/10.1111/papt.12011 Engel, R. J., & Schutt, R. K. (2017). The practice of research in social work (4th ed.). Thousand Oaks, CA: Sage. Franklin, C., Zhang, A., Froerer, A., & Johnson, S. (2017). Solution focused brief therapy: A systematic review and meta-summary of process research. Journal of Marital and Family Therapy, 43(1), 16–30. https://doi.org/10.1111/jmft.12193 Froerer, A. S., von Cziffra-Bergs, J., Kim, J. S., & Connie, E. E. (2018). Solution-focused brief therapy with clients managing trauma. New York, NY: Oxford University Press. Gillies, D., Taylor, F., Gray, C., O’Brien, L., & D’Abrew, N. (2012). Psychological therapies for the treatment of post-traumatic stress disorder in children and adolescents (Review). Cochrane Database of Systematic Reviews, (12), 1–110. https://doi.org/10.1002/14651858.CD006726.pub2. Gingerich, W. J., & Peterson, L. T. (2013). Effectiveness of solution-focused brief therapy: A systematic qualitative review of controlled outcome studies. Research on Social Work Practice, 23, 266–283. https://doi.org/10.1177/1049731512470859 Hiebert-Murphy, D., & Richert, M. (2000). A parenting group for women dealing with child sexual abuse and substance abuse. International Journal Of Group Psychotherapy, 50, 397–405. Herzog, J. I., & Schmahl, C. (2018). Adverse childhood experiences and the consequences on Journal of Solution Focused Brief Therapy – 63 37 Ray Eads and Mo Yee Lee Journal of Solution Focused Practices, Vol. 3 [2020], Iss. 1, Art. 12 neurobiological, psychosocial, and somatic conditions across the lifespan. Frontiers in Psychiatry, 9(September), 1–8. https://doi.org/10.3389/fpsyt.2018.00420 Imel, Z. E., Laska, K., Jakupcak, M., & Simpson, T. L. (2013). Meta-analysis of dropout in treatments for posttraumatic stress disorder. Journal of Consulting and Clinical Psychology, 81, 394–404. https://doi.org/10.1037/a0031474 Kessler, R. C., Aguilar-Gaxiola, S., Alonso, J., Benjet, C., Bromet, E. J., Cardoso, G., … Koenen, K. C. (2017). Trauma and PTSD in the WHO world mental health surveys. European Journal of Psychotraumatology, 8(sup5), 1353383. https://doi.org/10.1080/20008198.2017.1353383 Kim, J. S. (2008). Examining the effectiveness of solution-focused brief therapy: A metaanalysis. Research on Social Work Practice, 18, 107–116. https://doi.org/10.1177/1049731507307807 Kim, J. S., Brook, J., & Akin, B. A. (2018). Solution-focused brief therapy with substanceusing individuals: A randomized controlled trial study. Research on Social Work Practice, 28, 452–462. https://doi.org/10.1177/1049731516650517 Koob, J. J., & Love, S. M. (2010). The implementation of solution-focused therapy to increase foster care placement stability. Children and Youth Services Review, 32, 1346–1350. https://doi.org/10.1016/j.childyouth.2010.06.001 Kruczek, T., & Vitanza, S. (1999). Treatment effects with an adolescent abuse survivor’s group. Child Abuse and Neglect, 23, 477–485. https://doi.org/10.1016/S01452134(99)00023-X Lau, M., & Kristensen, E. (2007). Outcome of systemic and analytic group psychotherapy for adult women with history of intrafamilial childhood sexual abuse: A randomized controlled study. Acta Psychiatrica Scandinavica, 116, 96–104. https://doi.org/10.1111/j.1600-0447.2006.00977.x Liu, C. (2017). Examining the effectiveness of solution-focused art therapy (SF-AT) for sleep problems of children with traumatic experience (Doctoral dissertation, The Ohio State University). Retrieved from http://rave.ohiolink.edu/etdc /view?acc_num=osu1500567615448267 Magruder, K. M., McLaughlin, K. A., & Elmore Borbon, D. L. (2017). Trauma is a public health issue. European Journal of Psychotraumatology, 8(1), 1375338. https://doi.org/10.1080/20008198.2017.1375338 Mørkved, N., Winje, D., Dovran, A., Arefjord, K., Johnsen, E., Kroken, R. A., et al. (2018). Childhood trauma in schizophrenia spectrum disorders as compared to substance abuse disorders. Psychiatry Research, 261(October 2017), 481–487. https://doi.org/10.1016/j.psychres.2018.01.011 Newgent, R. A., Fender-Scarr, L. K., & Bromley, J. L. (2002). The retraumization of child sexual abuse: The second insult. Trauma & Loss: Research & Interventions, 2(2), 2–17. Pai, A., Suris, A., & North, C. (2017). Posttraumatic stress disorder in the DSM-5: Controversy, 64 – Journal of Solution Focused Brief Therapy https://digitalscholarship.unlv.edu/journalsfp/vol3/iss1/12 Solution Focused Therapy for Trauma Survivors change, and conceptual considerations. Behavioral Sciences, 7(1), 7. https://doi.org/10.3390/bs7010007 Sabalauskas, K. L., Ortolani, C. L., & McCall, M. J. (2014). Moving from pathology to possibility: Integrating strengths-based interventions in child welfare provision. Child Care in Practice, 20(1), 120–134. https://doi.org/10.1080/13575279.2013.847053 Salazar, A. M., Keller, T. E., Gowen, L. K., & Courtney, M. E. (2013). Trauma exposure and PTSD among older adolescents in foster care. Social Psychiatry and Psychiatric Epidemiology, 48(4), 545–551. https://doi.org/10.1007/s00127-012-0563-0 Schottenbauer, M. A., Glass, C. R., Arnkoff, D. B., Tendick, V., & Hafter Gray, S. (2008). Nonresponse and dropout rates in outcome studies on PTSD: Review and methodological considerations. Psychiatry: Interpersonal and Biological Processes, 71(2), 134–168. https://doi.org/10.1521/psyc.2008.71.2.134 Sin, J., Spain, D., Furuta, M., Murrells, T., & Norman, I. (2017). Psychological interventions for post-traumatic stress disorder (PTSD) in people with severe mental illness (Review). Cochrane Database of Systematic Reviews, (1), 1–103. https://doi.org/10.1002/14651858.CD011464.pub2.www.cochranelibrary.com Sklarew, B. H., & Blum, H. P. (2006). Trauma and depression. The International Journal of Psychoanalysis, 87(3), 859–861. https://doi.org/10.1516/14P5-CYAN-MPL8-6RL4 Slack, K. S., Font, S. A., & Jones, J. (2017). The complex interplay of adverse childhood experiences, race, and income. Health and Social Work, 42(1), e24–e31. https://doi.org/10.1093/hsw/hlw059 Smock, S. A., Trepper, T. S., Wetchler, J. L., McCollum, E. E., Ray, R., & Pierce, K. (2008). Solution-focused group therapy for level 1 substance abusers. Journal of Marital and Family Therapy, 34, 107–120. https://doi.org/10.1111/j.1752-0606.2008.00056.x Solution Focused Brief Therapy Association. (2013). Solution focused therapy treatment manual for working with individuals (2nd version). Retrieved from https://irp-cdn .multiscreensite.com/f39d2222/files/uploaded/SFBT_Revised_Treatment_Manual_ 2013.pdf Wamser-Nanney, R., & Vandenberg, B. R. (2013). Empirical support for the definition of a complex trauma event in children and adolescents. Journal of Traumatic Stress, 26, 671–678. Zhang, W., Yan, T., Du, Y., & Liu, X. (2014). Brief report: effects of solution-focused brief therapy group-work on promoting post-traumatic growth of mothers who have a child with ASD. Journal Of Autism And Developmental Disorders, 44(8), 2052–2056. https://doi.org/10.1007/s10803-014-2051-8 Ray Eads and Mo Yee Lee Email: lee.355@osu.edu; eads.34@buckeyemail.osu.edu Journal of Solution Focused Brief Therapy – 65 38 et al.: Volume 3 Issue 1 2019, Complete • The 3.0 version of “Reflections on Mark’s Paper SFBT 2.0” important evolution of existing practice A couple of comparisons: The 3.0 version of “Reflections on Mark’s Paper SFBT 2.0 - The new generation of SFBT has already arrived” Harry Korman Private Practice - SIKT Malmö When I read Mark’s paper the first time, I thought that Mark had done a pretty decent job at pointing out some of the key differences between what I call the BRIEF-model1 and my more traditional way of doing and teaching SFBT. I felt a tiny bit of unease as I read the paper but couldn’t put my finger on why that was except for a couple of things where I did not agree with his descriptions of the BFTC2-model. I thought most of my unease was due to me just being and feeling old-fashioned. Then the other day one of the people on our diploma training said, “Why do we need to learn SFBT 1.0 when SFBT 2.0 is already here?” I felt some more unease, so I re-read Marks paper again, and more carefully. Towards the end of the paper in the last paragraph Mark writes, “This is not to say that SFBT 1.0 is wrong, or bad, or outdated, or anything like that”(McKergow, 2016, p. 15). This phrase contradicts most of what came before it in the paper. Mark not only describes SFBT 2.0 in positive terms, it describes my way of doing Solution Focused Brief Therapy (the old way) in negatives. Language is powerful. Some people even have the idea that meaning making happens in language. I think Mark is one of them. The way we describe our world is how we live and take part in it. So, I decided that in this comment on Mark’s paper I will not accept the names he proposes because the numbering system in itself denotes that one is better, more advanced, a major update, etc. Since I am not yet convinced, I will instead talk here about the BRIEF-model and the BFTC-model. A couple of examples Some of the words qualifying the BRIEF model (quotes from Marks’s paper in italics; my emphasis in bold): • • even simpler in form left behind many elements 66 – Journal of Solution Focused Brief Therapy Published by Digital Scholarship@UNLV, 2020 • • • • losing hangovers from family therapy the end of the session has lost many of the trappings even more elegant than the previous versions. an even clearer commitment to offering power to the client, A description of the BFTC-model around the utilization of the team and descriptions of the summary: • The idea of others watching, hidden from view, seems not only costly but also rather creepy. • compliments in a sustained barrage, as the prelude to selling some kind of intervention (McKergow, 2016). I think my student’s question now makes sense. Who would want to do creepy things, sell interventions, use outdated elements, have hangovers3, be trapped by thinking about how to finish the session, be clumsy (instead of elegant) etc.4? A short look on what I don’t agree with Mark then compares the BFTC-model with the BRIEF-model where one is not “…trying to deliberately prompt the client to action” (McKergow, 2016, p. 11). Maybe this is a misunderstanding. I think that our much esteemed friends at BRIEF are saying that if you stop thinking about what to do at the end of the session you get more time and space to develop the preferred future in the future; the present and the past and new things will start happening in the session that you haven’t conceptualized and seen before. If they are actually saying (or thinking) that they are not deliberately trying to prompt the client to action, they are falling into the trap described by Weakland: Influence is inherent in all human interaction. We are bound to influence our clients, and they are bound to influence us. The only choice is between doing so without reflection, or even with attempted denial and doing so deliberately and responsibly (Weakland as cited in Gilligan & Price, 1993, p. 136-145). My view on influence is that it happens in the negotiation of meaning that is continuously on-going in the therapeutic conversation. My contribution in the process lies in the choices I make when I echo, paraphrase, and build questions on only parts of what the client told me (De Jong, Bavelas, & Korman, 2013; Korman, Bavelas, & De Jong, 2013), and in the presuppositions of my questions (McGee, Del Vento, & Bavelas, 2005). Since most people don’t like to be told what to do, and since one of the reasons they have come to see me is that they don’t know what to do, it would be rather unproductive to ask them what they need to do. Journal of Solution Focused Brief Therapy – 67 39 Harry Korman Journal of Solution Focused Practices, Vol. 3 [2020], Iss. 1, Art. 12 The 3.0 version of “Reflections on Mark’s Paper SFBT 2.0” Instead, I ask them how they will notice when things get better, how they feel, what they think, how they behave, and how other people will behave differently. People can only answer these questions when they have imagined themselves noticing that things are better, and what actions would follow or precede. Pretending that this is not deliberately prompting the client to action is ignoring the power of language and the whole post-structural revision of SFBT that Mark refers to in his paper (de Shazer, 1992 p. 92; McKergow, 2016). This is the largest beef I have with Mark’s paper. In more or less subtle ways, through his description of the BRIEF-model, he minimizes the effect of seemingly simple questions like: “So, after the miracle – what is the first thing you, or someone else, notices that you feel/do/think?” And pretends that this is not prompting the client into action. Since the above is also a major part of Mark’s description pointing to the BRIEF-model being a major upgrade from the BFTC-model, this is also questionable by inference. But let’s suppose for the sake of the argument that the BRIEF-model represents a major shift. information created in the session and you need to make decisions on how to proceed in order to construct a useful therapeutic reality. Sometimes it’s more complicated and that’s where the BFTC-model may have some advantages (and may have some disadvantages). There are more pathways described, there are more options available. Among them, there is for instance, the possibility of finishing a “bad” session in a useful way with a summary that you have taken some time to reflect on. More importantly than the summary though, the BFTC-model opens up a variety of pathways in the session. If Scott Miller is right in that the best therapists are the ones with a wider register of available behaviors, then de Shazer may be right that the BRIEF-model makes it more difficult to learn the variety that one needs to fit with all kinds of people in therapy. So, I personally believe that Steve’s comment about the BRIEF-model being more difficult to learn is that BRIEF-mapping makes it more difficult to learn the many different pathways of SBFT that I believe are useful to master. Is the BRIEF model one step forward? Is the BRIEF-model better? When I first heard Chris, Harvey, and Evan present the BRIEF-model at a conference some 15 years ago or so - I was enthusiastic. Steve de Shazer was walking out just in front of me, so I caught up with him, and asked him what he thought of this great simplification of his work, particularly considering his own fondness of simplicity. He grunted in response, so I pressed on. “Don’t you think it’s great that they have reduced the number of questions to only 2”; The questions being “What do you want? And what of that is already happening?”. He answered: “If it’s only a question of reducing the number of questions there is only one that is important.” That caught my attention. Being a fervent admirer of Steve’s thinking I asked him what THE QUESTION was. He answered: “What’s better?” He then added that one problem that BRIEF’s description “makes it damned difficult to teach”. So – a question I have been asking myself since I listened to BRIEF presenting their model is: There are some strong arguments for the BRIEF-model. For instance, the elegant simplicity of its description and the fit with many of the basic assumptions extracted from the work at BFTC. The strongest argument though are the claims that it has the same results in fewer sessions and that because of the simplicity it might be easier to learn. I’m not sure that this is what the guys at BRIEF think, but it’s certainly what my student was thinking. And it also feels like the gist of Mark’s paper – despite him negating this. Thinking about this, I re-read the paper where Shennan and Iveson (2011) present the development of the BRIEF-model and the research they did. I read with a critical eye, deliberately avoiding being solution focused. In the paper (Shennan & Iveson, 2011), they describe the 5 studies that the team at BRIEF was involved in. Talking about the fifth study they have a headline: Study 5: Briefer and Still Effective? The dip in outcomes (60% improved) of the fourth study compared to the previous studies spurred us on to put systems in place to enable more regular and systematic evaluations of our work. Because we wanted to check our practice as quickly as possible after the fourth study, the fifth was undertaken about a year later. On average, eighty-four percent of the clients reported that they had made progress towards their "best hopes" from the work, a year after its completion, with an average of 1.8 sessions per client. The questions asked in this study differed from those in the previous studies. The measure being about achievement of hopes rather than resolution of problems. This reflected the significant practice developments that had been set in motion by the earlier studies, and it is to these that we now turn. It honors Shennan and Iveson (2011) that they put a question mark after the heading. Regardless of their motive for changing the measure it makes all comparisons between study 4 and study 5 nonsense and opens alternative interpretations. An unexpected finding of the fifth study was that the average number of sessions across the 25 clients was as low as 1.8. Is it easier to learn the BRIEF model? Mark didn’t say in his paper that the BRIEF-model is easier to learn. I think my student thinks it as easier though. And Steve suspected that it might be more difficult to learn SFBT with the BRIEF-model than with the BFTC-model. So, I decided to talk about it here as well. If Solution Focused Brief Therapy is only, and I mean only, about creating a preferred future, and describing instances of that future, the answer would undoubtedly be: Yes! Obviously, the BFTC-model is more complex. If you use the model with the end of session message, including an experimental thing to do, something to observe, or just compliments requires that you create particular information in the session and it requires you to make a certain number of decisions. Is the miracle picture vague or concrete? If you were not able to construct a preferred future in the session, what is the form of the problem? The pieces of better something the client can do deliberately? problem? etc.? You need to categorize the 68 – Journal of Solution Focused Brief Therapy https://digitalscholarship.unlv.edu/journalsfp/vol3/iss1/12 Journal of Solution Focused Brief Therapy – 69 40 et al.: Volume 3 Issue 1 2019, Complete Harry Korman This reminded me of a study on clients at BFTC on 168 clients made by Kiser (1988) and Kiser and Nunnaly (1990), referenced in de Shazer, Putting Difference to Work (1991; See Table 1). At follow-up, 80% of the clients reported either “Complete relief of the presenting problem” or “Clear and considerable improvement.” About half of these clients had 4 sessions or more, and about half had 3 sessions or less. The researchers looked at if there was a difference in outcome correlated with length of therapy. In the group that had 4 sessions or more, 91% of the clients were better. In the group who had three sessions or less 69% were better. That is a big difference. Trying to be a brief therapist, I did not like that more therapy correlated with better results. The clients were also asked if things had improved in other areas then the problem that they had talked about. Again, there is a difference. There with more clients from the 4 sessions or more group meeting secondary goals. Table 1. Kiser (1988) and Kiser and Nunnaly (1990) cited in de Shazer (1991). BFTC 3 sessions or less BFTC 4 sessions or more (51,8 % of the clients) (48,2% of the clients) The 3.0 version of “Reflections on Mark’s Paper SFBT 2.0” Though overall our intention is to be successful therapists by helping clients move forward in their lives, client by client, the team endeavors to remain neutral about what the client does. Our hunch is that if clients are confronted in any way by our ideas concerning possible actions to take, then they will need to take time to consider these ideas. Conversely, the more we are able to keep out of their way, by simply inviting clients to describe future possibilities and whatever aspects of these possibilities are already in place, the more quickly they will be able to get on with whatever they choose to do. (Shennan & Iveson, p. 295) It makes sense to see the reduction of the number of sessions as the result of something good happening in sessions at BRIEF, but only if the outcomes of their therapies are not worse. Due to the fact that they changed their outcome measure between study number 4 and 5 this cannot be claimed. So, using my critical posture, here is another alternative. The BRIEF-model does not, in a significant number of cases, lead to the same kind of fit as the wider, older model that has more options on how to conduct the session and end it. Thus, the BRIEF-model might have more early dropouts and worse results (as they were in study number 4). Total % improved 69% 91% Conclusion Met secondary goal 44% 61% I agree with BRIEF that in lots of cases even having the idea that the client needs to do something doesn’t fit. If the therapist has the idea that the client needs to do something it will be visible in the presuppositions, in the selection process, and it will lead to a bad fit with that client. With other clients, for instance, someone who misheard what I asked with the best hopes/common project question; and to have heard me ask: “What can I do for you?” or “What needs to happen in this session for you to feel that it’s been useful?” and answers with: “I need you to give me some tools”. This answer will be taken into account in my BFTC-way of doing SFBT. It will be one of the threads of the conversation. And it might very well be a part in my summary, proposal of an experiment or what to pay attention to. I try to understand and work inside the client’s worldview even knowing that we are co-constructing it in the session. And I hope that it helps me develop/maintain the fit that I believe is crucial in developing a useful therapeutic reality with my clients. Maybe, the BRIEF-model is the next generation of Solution Focused Brief Therapy or maybe it has taken too many important and useful things away from the BFTC-model and is a step backwards. I don’t know. I want to see more research on both process and effect to decide where I stand. And maybe, maybe all of us brief therapist should do what Steve de Shazer did several times in his career. When the data doesn’t fit the theory, change the theory. Maybe we should at least ask ourselves if our old adage “briefer is better” is a useful way to think. So, I took a look at De Jong and Hopwood (1996), and in their findings of the follow-up of 276 clients at BFTC from 1992-93 there is a tendency in the same direction: More sessions – better results. Some years later, Scott Miller, who used to work at BFTC and who has been a strong advocate for practice-based evidence, told me that there are many studies that show that longer therapy is more effective than shorter. One of the key elements of his “Feedback Informed Treatment-model - FIT” is that we need to locate our unsuccessful cases early in therapy. Using the Session Rating Scale, we can find the cases at risk of dropping out in the first and second session. When we do, we can apologize for the bad fit we had with them and a lot of them will then return to a next session. Giving us the opportunity to perform better. Doing this improves outcome significantly. Another thing that Scott talks about in his later, and now on-going work, is that the best therapists regardless of model seem to have a wider variety of behaviors at their disposal when things become difficult in a session. Shennan and Iveson (2011) continue in their article: Our best guess about the reason for the reduction in the number of sessions is that it is related to our attempt to become non instrumental in our conversations with clients. 70 – Journal of Solution Focused Brief Therapy Published by Digital Scholarship@UNLV, 2020 Journal of Solution Focused Brief Therapy – 71 41 Harry Korman Journal of Solution Focused Practices, Vol. 3 [2020], Iss. 1, Art. 12 1 What Mark calls SFBT 2.0, I call the BRIEF-model which is also in homage of the people who developed it. 2 What Marks calls SFBT 1.0 I call the BFTC-model. 3 I originally wrote: “If you want a hangover – use SFBT 1.0”. This is a classical rhetoric move. You use the words the other person used but you use it with another sense. I think it irritated me that Mark chose the word “hangover” because I use lots of stuff coming from Erikson (and thus the MRI). 4 The numbering system is interesting by what it implies. This is my third version on the reflections on Marks paper. Suppose I said: “This is not to say that versions 1 and 2 were wrong, or bad, or outdated, or anything like that.” Would you believe me? The 3.0 version of “Reflections on Mark’s Paper SFBT 2.0” Weakland, J. H. (1993). Conversation—But what kind? In S. G. Gilligan & R. Price (Eds.), Therapeutic conversations (pp. 136–145) Harry Korman Email: harry@sikt.nu References Bavelas, J. B., de Jong, P., Jordan, S. S., & Korman, H. (2014). The theoretical and research basis of co-constructing meaning in dialogue. Journal of Solution Focused Brief Therapy 1, 1-24. De Jong, P. & Hopwood, L. E. (1996). Outcome research on treatment conducted at the Brief Family Therapy Center, 1992-1993. In S. D. Miller, M. A. Hubble, & B. L. Duncan (Eds.), Handbook of solution-focused brief therapy (pp. 272-298), San Francisco: Jossey-Bass Publishers. De Jong, P., Bavelas, J. B., & Korman, H. (2013). An introduction to using microanalysis to observe co-construction in psychotherapy. Journal of Systemic Therapies, 32, 17-30. doi:10.1521/jsyt.2013.32.3.17 deShazer, S. (1991). Putting difference to work. New York: Norton. deShazer, S., & Berg, I. K. (1992). Doing therapy: A poststructial revision. Journal of Marriage and Family Therapy, 18, 71-81. Kiser, D. (1988). A follow-up study conducted at the Brief Family Therapy Center. Unpublished manuscript. Milwaukee, WI: Brief Family Therapy Center. Kiser, D., & Nunnally, E. (1990). The relationship between treatment length and goal achievement in solution-focused therapy. Unpublished manuscript. Korman, H., Bavelas, J. B., & De Jong, P. (2013). Microanalysis of formulations in solutionfocused brief therapy, cognitive behavioral therapy, and motivational interviewing. Journal of Systemic Therapies, 32, 31-45. doi:10.1521/jsyt.2013.32.3.31 McGee, D. Del Vento, A., Bavelas, J. B. (2005). An interactional model of questions as therapeutic interventions. Journal of Marital and Family Therapy 31, 371-384 Shennan, G., & Iveson, C. (2011). From solution to description: Practice and research in tandem. In C. Franklin, T. S. Trepper, E. E. McCollum, & W. J. Gingerich (Eds.), Solution-focused brief therapy: A handbook of evidence-based practice (pp. 281–298). Oxford: Oxford University Press. 72 – Journal of Solution Focused Brief Therapy https://digitalscholarship.unlv.edu/journalsfp/vol3/iss1/12 Journal of Solution Focused Brief Therapy – 73 42 Mark McKergow et al.: Volume 3 Issue 1 2019, Complete Response to Harry Korman paper under discussion.) Is that “better” as a clear conception of what we are seeking to do? Yes, I think so, hence the various comments about “leaving behind” elements of existing Deliberately prompting the client to action Response to Harry Korman’s Reflections on SFBT 2.0 Paper Mark McKergow The Centre for Solutions Focus at Work First of all, many thanks to Harry for giving such detailed attention to this paper. I am very honored that he thinks these developments are worth paying serious attention to. He raises some very interesting points. At the outset, let me say that I don’t accept that what I have termed “SFBT 2.0” solely as the creation of BRIEF, although their work is clearly very important to it. I see this as much wider trend in the way SFBT is going and has been going for a decade or more. For example, I myself proposed a greater focus on ‘chunks’ of conversation all the way back in 2002. I have attempted to assemble the various changes and shifts into a coherent picture – which itself then reveals even more about new directions for our work. This also includes theoretical developments which are not the topic of the current SFBT 2.0 paper but are somewhat presaged in the Brief Therapy: Focused Description Development paper by Chris Iveson and me. Although I can see the temptation to refer to what I have termed “SFBT 1.0” as the BFTC model, I don’t think that’s a wise move either. As Harry knows, Steve and Insoo’s work developed over their careers in ways that were not always well documented. Peter de Jong has said to me recently (and I am happy to agree with him) that Insoo’s late work might have been seen as something like “SFBT 1.6”. It’s useful to set up an idea of “SFBT 1.0” as a counter point but that doesn’t mean that most people are doing exactly that – in fact I think most SFBT practitioners I have seen are moving along this spectrum, albeit with different degrees of awareness and deliberation. So, in this paper I am both attempting to point to shifts which are already happening, and also to show how a renewed and even more sharply honed set of practices might be emerging; which offers practitioners both clearer focus and a new connection to the question “how does it work”. The question “how does it work” could reposition SFBT in a clearer space for newcomers and the wider world. (Again, I accept that this element is not yet present in the 74 – Journal of Solution Focused Brief Therapy Published by Digital Scholarship@UNLV, 2020 Of course, I take the point of Harry’s reference to John Weakland about the all-pervading influence of communication. I am not seeking to pretend that our questions such as “so, after the miracle, what is the first thing you notice…” are not in the bigger picture intended to promote some kind of action in the client. I am making a rather narrow point – that these questions are not explicitly couched in “action” terms (“what will you do”) as in descriptive terms (“what will you notice”). In the initial work from BFTC (for example in Clues), the discussion about a hypothetical miracle future was only deployed if suitable exceptions had not been found. The advantage of these exceptions was seen, at the time, as being that as they had already happened, they could be deconstructed in detail to examine what the clients had done already that had helped. Then, as the practice developed, the miracle question took a more central part and the work seemed (to me) to take on a hybrid quality of being partly about descriptions of the future and examinations of past exceptions. And all of this was at the service of an intervention design, to be done in a group and delivered at the end of the session. Over the past 10-15 years, we have seen a shift away from deconstructing exceptions and building detailed interventions (in real-life action terms), and moving towards a much more creative and expressive way of working with clients. We want clients to feel encouraged to speak about themselves in the here-and-now and explore events in the future and past. Our goal is not to see what “really” happened but to connect with them in different ways. And of course, this is, in the end, about helping clients live their lives differently, in ways which give them less pain and more satisfaction. So, my point about prompting the client to action is about moving away from the blunt explorations of doing and towards the even more interactional and latent power of noticing. Is SFBT 2.0 one step forward? As I said above, I am seeing current practice as being at various points along an imaginary scale from SFBT 1.0 to SFBT 2.0. At the moment many people (including me) are doing something which allows for a mix of possibilities, and I get the sense from your document that you like the option to give tasks and get people to do things. I am reminded here, of the story Steve used to tell about the teenaged boy who came to therapy with his parents, watched what was going on for a while and then asked “So, are you asking these questions for you to get the answers, or for us to hear the answers?”. I think Steve’s response was a “hrmph” or something, because in the current practice it is ambiguous. It could well be both. What I am proposing here is that with SFBT 2.0 the answer is basically “so the client can hear the answers”. The practitioner hears them too – and their role is to construct more Journal of Solution Focused Brief Therapy – 75 43 Journal of Solution Focused Practices, Vol. 3 [2020], Iss. 1, Art. 12 Mark McKergow Response to Harry Korman questions to expand the description of the client. This is a different kind of endeavour from SFBT 1.0 where the practitioner is going to construct a task. I think / hope that in leaving that behind the practitioner is able to stay even better with the client in expanding their descriptions and their world, without having to worry about what to do with the information. That’s the proposal anyway, it clearly needs exploring and testing. By the way, I don’t think that BRIEF has only two questions – in order to make sense of that, we need a whole raft of skills, sub-questions, and tactics. They are (as ever) being modest about their skills. stuff (put together in a way which seemed to fit the situation) produced some useful ways forward. I think there are plenty of options in SFBT 2.0 – assuming a platform/project can be agreed, one can go to past, present or (more usually) future in various ways. The art gallery metaphor shows how what we’re after (a variety of different descriptions/pictures/scenes) can be approached in different ways. Also, if there is not a fit between client and therapist, it may be better to move on more quickly to an alternative therapist or treatment. Is SFBT 2.0 better? Distinguishing sessions of SFBT 1.0 and SFBT 2.0 Research – yes of course more is needed. As I say in the article, if we can name and define this as something to investigate, then investigation becomes likely. The research you mention about more sessions being better… it is of course, interesting and thought provoking. We could useful remember that most of the world is still not using a brief therapy mentality. I recently did a training for an agency here in Edinburgh where they give their clients 25 sessions routinely. (And guess what… the therapists report that there is a crisis around 20 sessions when the clients finally realizes they have to do something!) In this context, the difference between 4 and 5 sessions is less fundamental. Changing our emphasis in SFBT from “as many sessions as needed and not one more than necessary” would be a very big step. I guess we might, in situations (such as those here in the UK where therapist resources are limited in the NHS) discuss the relative benefit of one more session to an existing client who is well on the way to recovery, and giving a new client a first session. Yes of course we need more research. However, this can also act as a paralyser. I don’t think BFTC had a huge amount of weapons-grade research when they wrote Clues in 1988. They had some good ideas and some experience of them working, and look what that started. I also think there are some useful connections to come from SFBT 2.0 in terms of connection to things like enactive cognition, which is not included in the initial paper, but I hope to add soon from other work. You say that you’re not sure whether an observer could distinguish between sessions like this apart from the obvious lack of a break. It’s a very good question. Peter De Jong asked the same thing, so at his suggestion, we compared an Insoo session, (“Over The Hump”, chosen by him) and a Chris Iveson session, (“Mary and the cuddle”, chosen by me) by both looking at them to draw distinctions etc and then comparing notes. Of course, there are plenty of similarities – both sessions are clearly SFBT and not something else. However, there were also some clear distinctions. In what seemed as a complicated session due to the amount of children in the room, Insoo didn’t get a “project” agreement with the clients but rather assumed it. She may have been justified in doing that because of the situation. Then Insoo asks the miracle question and has to do a lot of clarifying because the family misunderstood it (unfortunate, but not very significant in this discussion). She then gets some “headline” answers but doesn’t expand much on them. Insoo tended to get a headline (in response to the miracle question, or a scale) and then repeat it, whereas Chris tended to dive into more detail from whatever starting point – he uses the question “what difference would that make?” six times (Insoo 0), and variations on “what might you notice?” over 20 times (Insoo 0). I will add my marked-up copy of the complete Over The Hump transcript with my comparison notes to this reply when I send it. (This includes the opening and closing, which were not examined for microanalysis purposes.) Peter De Jong concluded that these sessions show “very clear variations which could be very profitable to explore.” He also said that we were “reaffirming the spirit of SF” by looking directly at the work in action. Varieties of Pathways Yes, there are more possible pathways and options in SFBT 1.0. It’s not totally clear to me that it’s a useful thing – perhaps it means making decisions which takes one’s attention away from the client at hand. Perhaps there is enough variety within the options for SFBT 2.0 – we don’t know yet. What I do know is that I spent years recording open consultation sessions waiting for the perfect “difficult” one where the usual things didn’t work, resulting in doing something amazingly creative. And yes, I was frustrated that it never happened and the usual 76 – Journal of Solution Focused Brief Therapy https://digitalscholarship.unlv.edu/journalsfp/vol3/iss1/12 “Give me some tools” You ask about the kind of situation where the client, in answering questions about their hopes, say “I need you to give me some tools…” We can easily respond to this not by accepting it at totally face value but rather asking “and what difference would it make, if you had some tools?” We might get towards somewhere where the client knew what kind of tools to go look for, or they might realise that there are other ways to handle the situation – we just don’t know. Of course, defining a common project or platform is key in all of this, in all manner of SFBT variants. Thank you again Harry for taking all this trouble to respond to the paper. I am honoured and humbled that you felt it worth the time and energy. I hope this conversation will continue Journal of Solution Focused Brief Therapy – 77 44 Mark McKergow et al.: Volume 3 Issue 1 2019, Complete over many months and years to come – I value your experience, your knowledge, your commitment and your friendship. Cheers, Mark McKergow Edinburgh, February 2018 Mark McKergow Email: mark@sfwork.com 78 – Journal of Solution Focused Brief Therapy Published by Digital Scholarship@UNLV, 2020 Journal of Solution Focused Brief Therapy – 79 45
https://openalex.org/W4299633360	https://dash.harvard.edu/bitstream/1/13347564/1/4198701.pdf	English	null	The association of plasma biomarkers with computed tomography-assessed emphysema phenotypes	Carolina Digital Repository (University of North Carolina at Chapel Hill)	2,014	cc-by	9,452	Permanent link http://nrs.harvard.edu/urn-3:HUL.InstRepos:13347564 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of-use#LAA Citation Citation Carolan, B. J., G. Hughes, J. Morrow, C. P. Hersh, W. K. O’Neal, S. Rennard, S. G. Pillai, et al. 2014. “The association of plasma biomarkers with computed tomography-assessed emphysema phenotypes.” Respiratory Research 15 (1): 127. doi:10.1186/s12931-014-0127-9. http:// dx.doi.org/10.1186/s12931-014-0127-9. Published Version doi:10.1186/s12931-014-0127-9 Share Your Story The Harvard community has made this article openly available. Please share how this access benefits you. Submit a story . Accessibility Carolan et al. Respiratory Research 2014, 15:127 http://respiratory-research.com/content/15/1/127 The association of plasma biomarkers with computed tomography-assessed emphysema phenotypes Brendan J Carolan1,2, Grant Hughes3, Jarrett Morrow4, Craig P Hersh4, Wanda K O’Neal5, Stephen Rennard6, Sreekumar G Pillai7,12, Paula Belloni8,12, Debra A Cockayne9, Alejandro P Comellas10, Meilan Han11, Rachel L Zemans1,2, Katerina Kechris3 and Russell P Bowler1,2 RESEARCH Open Access Open Access Correspondence: CarolanB@NJHealth.org 1Department of Medicine, National Jewish Health, 1400 Jackson St, Denver, CO 80206, USA 2Department of Medicine, University of Colorado School of Medicine, Aurora, CO, USA Full list of author information is available at the end of the article © 2014 Carolan 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 credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Rationale: Chronic obstructive pulmonary disease (COPD) is a phenotypically heterogeneous disease. In COPD, the presence of emphysema is associated with increased mortality and risk of lung cancer. High resolution computed tomography (HRCT) scans are useful in quantifying emphysema but are associated with radiation exposure and high incidence of false positive findings (i.e., nodules). Using a comprehensive biomarker panel, we sought to determine if there was a peripheral blood biomarker signature of emphysema. Methods: 114 plasma biomarkers were measured using a custom assay in 588 individuals enrolled in the COPDGene study. Quantitative emphysema measurements included percent low lung attenuation (%LAA) ≤−950 HU, ≤−910 HU and mean lung attenuation at the 15th percentile on lung attenuation curve (LP15A). Multiple regression analysis was performed to determine plasma biomarkers associated with emphysema independent of covariates age, gender, smoking status, body mass index and FEV1. The findings were subsequently validated using baseline blood samples from a separate cohort of 388 subjects enrolled in the Treatment of Emphysema with a Selective Retinoid Agonist (TESRA) study. Results: Regression analysis identified multiple biomarkers associated with CT-assessed emphysema in COPDGene, including advanced glycosylation end-products receptor (AGER or RAGE, p < 0.001), intercellular adhesion molecule 1 (ICAM, p < 0.001), and chemokine ligand 20 (CCL20, p < 0.001). Validation in the TESRA cohort revealed significant associations with RAGE, ICAM1, and CCL20 with radiologic emphysema (p < 0.001 after meta-analysis). Other biomarkers that were associated with emphysema include CDH1, CDH 13 and SERPINA7, but were not available for validation in the TESRA study. Receiver operating characteristics analysis demonstrated a benefit of adding a biomarker panel to clinical covariates for detecting emphysema, especially in those without severe airflow limitation (AUC 0.85). Conclusions: Our findings, suggest that a panel of blood biomarkers including sRAGE, ICAM1 and CCL20 may serve as a useful surrogate measure of emphysema, and when combined with clinical covariates, may be useful clinically in predicting the presence of emphysema compared to just using covariates alone, especially in those with less severe COPD. Ultimately biomarkers may shed light on disease pathogenesis, providing targets for new treatments. Keywords: COPD, Biomarkers, RAGE, ICAM1, CCL20, Emphysema © 2014 Carolan 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 credited. Study population COPDGene is a multi-centered study of the genetic epi- demiology of COPD that enrolled 10,192 non-Hispanic White and African-American individuals, aged 45–80 years old with at least a 10 pack-year history of smoking, who had not had an exacerbation of COPD for at least the previous 30 days. Additional information on the COPDGene study and the collection of clinical data has been described previously [19]. 1839 COPDGene sub- jects (1599 non-Hispanic White (NHW) and 240 non- Hispanic Black) had fresh frozen plasma collected using a p100 tube (BD) at five COPDGene sites (National Jewish Health (N =916), University of Iowa (N =670), Harbor- UCLA Medical Center (N =202), Temple University (N =36), and Baylor Medical Center (N =15)). From this cohort a subset of 602 NHW subjects (no non-Hispanic Black subjects included due to limited numbers) were se- lected for a comprehensive biomarker study with an at- tempt to obtain a range of GOLD stages and match groups as closely as possible based on age, gender and smoking history. Of the 602 subjects, 588 subjects had quantitative HRCT measurements available. The institutional review boards of participating institutions approved the study (Additional file 1: Table S1). The best current non-invasive method of detecting em- physema is high-resolution computed tomography (HRCT) [8,9]. The drawbacks to HRCT include cost, radi- ation exposure, and a high rate of false positive clinical sig- nificant findings (e.g. benign nodules); however, HRCT can provide significant information relevant to lung pathology. For instance, lung attenuation area (LAA) at −950 or −910 Hounsfield units (HU) and the mean lung attenuation value at the 15th percentile (LP15A) on the lung attenu- ation curve are density-based measurements that correlate with emphysema [8,10,11]. Although the optimal method and normal values for describing radiologic emphysema have not been fully validated, it has been shown that con- trol smokers without COPD have percent LAA ≤−950 HU of <5% [9]. A separate validation cohort of 388 individuals (all former smokers with COPD) was obtained from the Treatment of Emphysema with a Selective Retinoid Agonist (TESRA) study. TESRA was a multi-centered randomized controlled trial assessing the safety and efficacy of palovarotene in ex- smokers with COPD. Only baseline samples before treat- ment were used for biomarker determination. Emphysema was quantitatively assessed by low dose spiral CT in the TESRA cohort. Additional information on the TESRA study has been described previously [20]. Introduction and with COPD, and relate the biomarker signature to dif- ferent methods of defining radiologic emphysema. Key findings were validated in an independent COPD cohort. Chronic obstructive pulmonary disease (COPD) is a phenotypically heterogeneous condition characterized by airflow limitation that is not fully reversible [1]. Some, but not all, COPD subjects have emphysema, i.e., airspace en- largement distal to the terminal bronchioles [2]. Deter- mining the presence of emphysema is important, as it has been independently associated with increased respiratory symptoms, more rapid decline in lung function, increased risk of lung cancer, higher rates of cardiovascular disease and increased mortality risk [3-6]. Surprisingly, there are some subjects with significant smoking history that have emphysema, but no airflow limitation [7]. Understanding the molecular signatures underlying emphysema may shed light on the pathogenesis of emphysema and its systemic complications. Abstract The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Carolan et al. Respiratory Research 2014, 15:127 http://respiratory-research.com/content/15/1/127 Carolan et al. Respiratory Research 2014, 15:127 http://respiratory-research.com/content/15/1/127 Page 2 of 10 Study population The first reported blood biomarker of emphysema was α1-antitrypsin (AAT); however, AAT deficiency accounts for only 1-2% of COPD [12]. Another recently reported in- dependent biomarker of emphysema is soluble RAGE or advanced glycosylation end product receptor (AGER) [13]. Peripheral blood adiponectin and bronchoalveolar lavage fluid eotaxin levels have also correlated with radiologic emphysema [14,15]. There are other reports of peripheral blood biomarkers of airflow limitation such as interleukin- 6, surfactant protein D and C-reactive protein [16,17]. Therefore, the presence of systemic biomarkers in periph- eral blood, which can be easily measured and offer infor- mation regarding COPD phenotypes, may provide another method of significant value in diagnosing and managing individuals with emphysema [18]. In addition, a biomarker signature of emphysematous phenotypes may provide insight to the pathogenesis of disease. Limitations of some previous emphysema biomarker studies include small sample size and lack of replication. With this in mind, using one of the largest studies to date, we sought to de- termine a peripheral blood biomarker signature of emphy- sema, independent of other clinical variables, in current and former cigarette smokers with normal lung function Clinical data and definitions COPD was defined as post bronchodilator ratio of forced expiratory volume in the first second (FEV1) to forced vital capacity (FVC) <0.70. Current or ex-smokers without spi- rometric evidence of airflow obstruction (FEV1/FVC ≥0.70) were classified as controls [1]. COPDGene study patients underwent whole lung volu- metric multi-detector computed tomography (CT) as pre- viously described [19,21]. Quantitative analysis of lung density was performed using the Slicer software package (http://www.slicer.org). Emphysema was primarily quanti- fied by the percent of lung voxels (%LAA) ≤−950 HU on the inspiratory images of CT scans for the whole lung. Emphysema was additionally quantified by percent of lung Carolan et al. Respiratory Research 2014, 15:127 http://respiratory-research.com/content/15/1/127 Page 3 of 10 (R > 0.6) was observed between proinsulin intact (INS in- tact) and proinsulin total (INS total) so INS intact was removed from the analysis. Also, brain derived neuro- tropic factor (BDNF) was removed, as it was collinear with angiopoietin 1, CCL5 (T cell specific protein RANTES), epithelial-derived neutrophil-activating protein 78, alpha-1 antitrypsin and latency associated peptide of transforming growth factor beta 1. For modeling of multiple biomarkers, stepwise regression, with a combination of backwards and forwards selection and a p-value threshold <0.15 for entry and exit from the model, was used to arrive at the final model. A p-value of <0.05 was taken as statisti- cally significant for association with the outcome em- physema variables. voxels (%LAA) ≤−910 HU on inspiratory CT scans and as mean lung attenuation at the 15th percentile on lung volume-adjusted attenuation curve (LP15A). In the TESRA cohort emphysema was quantified as %LAA ≤−910 HU and LP15A on HRCT scans [20]. Densiometric analyses of the HRCTs were completed in a central lab (BioClinica, Leiden, The Netherlands) using PulmoCMS software (Medis specials, Leiden, The Netherlands). The study design and clinical outcomes have been previously reported [13,20]. Statistical analysis Differences in demographic characteristics of study subjects were analyzed using a t-test for continuous variables and a Chi-squared test for categorical vari- ables. Emphysema severity was classified as none, mild, moderate and severe. For %LAA ≤−950 HU the cutoffs were <5%, 5- < 10%, 10- < 20% and ≥20%, respectively, while for %LAA ≤−910 HU the cutoffs were <35%, 35- < 45%, 45- < 55% and ≥55%, respectively. Cutoffs were based on mean values from COPDGene studies and balancing the sample size in each group [9]. Biomarker selection and measurement For the COPDGene cohort, 114 candidate biomarkers were selected based on a review of the literature and pre- viously reported pilot work from the BIOSPIR group [22]. Biomarker levels were determined using a custom 15-panel assay created by Myriad-RBM (Austin, TX) multiplex tech- nology. Blood samples were drawn from non-fasting indi- viduals. Approximately 8.5 mL of blood was withdrawn from the ante-cubital vein into a sterile 13 × 1000 mm P100 Blood Collection Tube (BD, New Jersey, USA). The sample was immediately centrifuged at 2500 × g, 20 minutes at room temperature. Aliquots in 500 μL tubes were stored at −80°C until analyzed. In the TESRA cohort, 111 similarly chosen protein biomarkers were measured in ethylenediamine-tetraacetic acid (EDTA) plasma in dupli- cate at Rules Based Medicine (Austin, TX) and Quest Diagnostics (Valencia, CA). A full list of biomarkers ana- lyzed in the TESRA study has been published [13] To perform the meta-analysis, a single variable model was fit for each of the significant biomarkers that were also identified in the TESRA study. Equivalent covariates were included for the two studies and an ordered logistic and lin- ear regression was fit respectively for the %LAA ≤−910 HU and LP15A outcomes. P-values from both studies were combined by calculating the average Z-score of the inverse normal quantiles of the two p-values to determine a com- bined p-value that accounted for consistent effects of the biomarker levels on emphysema severity in the two studies [23]. A Bonferroni adjustment was applied based on all tested markers. Receiver operating characteristic (ROC) curves were generated for covariates alone and covariates with bio- markers with the presence of emphysema compared to no emphysema as the outcome. Nominal logistic regres- sion was performed, with emphysema considered present if %LAA ≤−950 HU was ≥5% compared to no em- physema (%LAA ≤−950 HU <5%). Similarly, ROC curves were generated including different severities of airflow limi- tation based on FEV1 percent predicted. Statistical analyses were performed using JMP 9.0 (SAS Institute, Cary, NC) and R (version 3.0.2) statistical software packages [24]. Study population Demographics, physiology, quantitative HRCT measure- ments and patient-reported outcomes for COPDGene and TESRA cohorts are listed in Table 1. In the COPD- Gene biomarker study, there were 588 individuals with complete data available. Subjects with COPD were sig- nificantly older, had lower BMI, higher pack-year history of smoking and worse SGRQ scores compared to those without COPD (p < 0.01, all comparisons). The distribu- tion of gender and current smokers was similar between non-COPD and COPD groups. The following variables were associated with emphysema (LAA ≤−950 HU): lower FEV1 (p < 0.001), lower body mass index (p < 0.001), male gender (p = 0.002), older age at enrollment (p = 0.038) and current non-smoking status (p < 0.001); these variables Biomarkers (n = 17) with >10% and <95% of values below the lower limit of quantitation (LLOQ) for that par- ticular biomarker were transformed into binary variables (present or absent). Biomarkers (n = 16) with >95% values below LLOQ were excluded from the analysis. For regres- sion analysis, the remaining biomarker levels (n = 81) underwent an empirical normal quantile transformation projecting the ranks onto an inverse normal distribution so that they resemble a normal distribution and allow com- parison of biomarkers at different concentrations. Non- transformed biomarker levels are also presented (Additional file 1: Table S3). Collinearity among biomarkers and covari- ates was assessed using Pearson correlation. Collinearity Carolan et al. Study population FEV1 = Forced expiratory volume at one second; FVC = forced vital capacity; LAA = low area attenuation; N/A = data not available; LP15A = mean lung attenuation value at the 15th percentile on lung attenuation curve. MRC = Medical Research Council; SGRQ = St. George’s Respiratory Questionnaire. Presented are the means ± standard errors for COPDGene cohort and TESRA cohort. p values represent difference between no COPD and COPD groups for COPDGene. FEV1 = Forced expiratory volume at one second; FVC = forced vital capacity; LAA = low area attenuation; N/A = data not available; LP15A = mean attenuation value at the 15th percentile on lung attenuation curve. MRC = Medical Research Council; SGRQ = St. George’s Respiratory Questionnaire. were used as covariates for multiple regression (Additional file 1: Table S2). (CDH13, Figure 1D) and thyroxin-binding globulin (SER- PINA7) were positively correlated with emphysema se- verity (p < 0.001 for all comparisons). There were 3 biomarkers surfactant associated protein D (SFPD), FAS ligand receptor (FAS), and malondialdehyde-modified low-density lipoprotein (MDA LDL) associated with both %LAA ≤−910 HU and LP 15 emphysema out- comes (Table 2). Biomarkers associated with emphysema A full list of biomarkers analyzed in the COPDGene cohort is available (Additional file 1: Table S3). After adjusting for covariates, multiple regression analyses demonstrated a total of 24 biomarkers associated with radiologic emphy- sema including 15 biomarkers independently associated with %LAA ≤−950 HU (R2 = 0.4), 9 biomarkers associated with %LAA ≤−910 HU (R2 = 0.36) and 16 associated with LP15A (R2 = 0.64, Table 2). There were 6 biomarkers that were associated with all 3 radiologic emphysema outcome variables. Advanced glycosylation end-product receptor (RAGE) was negatively associated with more severe emphy- sema (Figure 1A). In addition, intercellular adhesion mol- ecule 1 (ICAM1, Figure 1B), macrophage inhibitory protein 3a (CCL20) and cadherin 1 (CDH1, Figure 1C) were nega- tively associated with emphysema severity. Cadherin 13 Study population Respiratory Research 2014, 15:127 http://respiratory-research.com/content/15/1/127 Page 4 of 10 Table 1 Demographics of individuals in COPDGene and TESRA studies COPDGene (n = 588) TESRA No COPD n = 247 COPD n = 341 p-value COPD (n = 388) Demographics Age (years) 61 ± 3 65 ± 0.5 p < 0.01 66.6 ± 0.4 Gender (male/female) 124/123 178/163 p = 0.63 267/121 Current smokers (%) 27 23 p = 0.23 0 Smoking history (pack-years) 38 ± 1 54 ± 2 p < 0.001 48 ± 1 Body mass index (kg/m2) 28.9 ± 2.3 27.8 ± 0.3 p = 0.009 26 ± 0.2 Physiology FEV1 post bronchodilator (% predicted) 98 ± 3.6 47 ± 1 p < 0.001 50 ± 0.5 FVC post bronchodilator (% predicted) 96 ± 3.6 79 ± 1 p < 0.001 93 ± 0.9 HRCT measurements Average % LAA ≤−950 HU 2.3 ± 1.6 15 ± 0.7 p < 0.001 N/A % Emphysema <5% 85 31 N/A % Emphysema 5- <10% 13 15 N/A % Emphysema 10- <20% 2 25 N/A % Emphysema ≥20% 0 29 N/A Average % LAA ≤−910 HU 22.6 ± 3.7 39 ± 0.7 p < 0.001 40.7 ± 0.8 % Emphysema <35% 79 35 % Emphysema 35- <45% 15 19 % Emphysema 45- <55% 5 19 % Emphysema ≥55% 1 27 Average LP15A −916 ± 4.3 −944 ± 1.3 p < 0.001 −945 ± 1.3 Patient-reported outcomes MRC dyspnea score 0.5 ± 0.1 2.2 ± 0.1 p < 0.001 2.0 ± 0.03 SGRQ 12 ± 3.9 39 ± 1.1 p < 0.001 46 ± 0.8 Presented are the means ± standard errors for COPDGene cohort and TESRA cohort. p values represent difference between no COPD and COPD groups for COPDGene. FEV1 = Forced expiratory volume at one second; FVC = forced vital capacity; LAA = low area attenuation; N/A = data not available; LP15A = mean lung attenuation value at the 15th percentile on lung attenuation curve. MRC = Medical Research Council; SGRQ = St. George’s Respiratory Questionnaire. Table 1 Demographics of individuals in COPDGene and TESRA studies cs of individuals in COPDGene and TESRA studies* Presented are the means ± standard errors for COPDGene cohort and TESRA cohort. p values represent difference between no COPD and COPD groups for COPDGene. Validation of emphysema biomarkers Using similar statistical methods (modeling, covariates, etc.), we attempted to validate the statistically significant biomarkers using an independent cohort from the TESRA study. Although %LAA ≤−910 HU and LP15A HRCT data were available in the TESRA cohort, % LAA ≤−950 HU measurements were not. Therefore, of the total 16 biomarkers statistically associated with the emphysema outcomes ≤−910 and LP15A in the COPD- Gene cohort, 9 biomarkers were available for validation Carolan et al. Respiratory Research 2014, 15:127 http://respiratory-research.com/content/15/1/127 Page 5 of 10 Page 5 of 10 in TESRA cohort After meta analysis and adjustment RAGE (p 2 5 × 10−10) ICAM1 (p 6 0 × 10−11) and Table 2 Biomarkers and covariates associated with radiologic emphysema in the COPDGene cohort (using multiple regression) %LAA ≤−950 HU %LAA ≤−910 HU LP15A# Covariate Beta coefficient p-value Beta coefficient p-value Beta coefficient p-value FEV1 (% predicted) −0.07 2.9 × 10−40 −0.05 6.4 × 10−29 0.42 2.1 × 10−47 Body mass index −0.15 3.2 × 10−10 −0.26 8.2 × 10−22 1.37 3.4 × 10−21 Current active smoking −1.16 9.1 × 10−5 −0.76 1.3 × 10−7 4.56 7.5 × 10−7 Male gender 0.35 0.002 0.71 7.3 × 10−9 −9.57 0.0001 Age at enrollment 0.04 0.039 0.04 0.006 −0.20 0.039 Biomarker RAGE −0.69 2.6 × 10−8 −1.10 0.005 10 0.0002 CCL20 (presence) −0.45 0.0006 −0.35 0.004 2.12 0.009 ICAM1 −0.42 0.001 −2.40 0.007 28.39 3.4 × 10−6 SERPINA7¶ 0.28 0.013 2.11 0.042 −13.69 0.038 CDH13¶ 0.29 0.025 2.62 0.005 −16.91 0.008 CDH1¶ −0.25 0.039 −2.04 0.006 13.09 0.006 TGFB1 LAP −0.54 0.0002 CCL13 0.35 0.013 TNFRSF11B 0.34 0.016 CCL8 −0.27 0.023 IgA −0.25 0.03 6.09 0.025 SORT1 −0.26 0.038 IL2RA 0.27 0.044 CCL2 0.25 0.045 IL12B (presence) 0.22 0.049 MDA LDL (absence)¶ 0.33 0.016 −2.07 0.025 FAS 1.16 0.016 −8.53 0.014 SFTPD −1.16 0.025 8.34 0.016 AXL 17.05 0.002 CXCL10 −11.80 0.002 ADIPOQ¶ −7.26 0.015 MB¶ −7.97 0.016 SOD1 11.08 0.009 NRCAM¶ −9.26 0.017 Presented are beta coefficients and p values for multiple regression models of biomarkers and covariates associated with emphysema outcomes. Validation of emphysema biomarkers %LAA = Percent low attenuation areas; LP15A = mean lung attenuation at 15th percentile on lung attenuation curve; HU = Hounsfield units; FEV1 = Forced expiratory volume in 1st second; RAGE = Receptor for advanced glycosylation end products; CCL20 = Macrophage Inflammatory Protein-3 alpha; ICAM1 = Intercellular Adhesion Molecule 1; SERPINA7 = Thyroxin-binding globulin; CDH 13 = Cadherin-13; CDH1 = Cadherin-1; TGFB1 LAP = Latency-Associated Peptide of Transforming Growth Factor beta 1; CCL13 = Monocyte Chemotactic Protein 4; TNFRSF11B = Osteoprotegerin; CCL8 = Monocyte Chemotactic Protein 2; IgA = Immunoglobulin A; SORT1 = Sortilin; IL2RA = Interleukin-2 receptor alpha; CCL2 = Monocyte Chemotactic Protein 1; IL-12B = Interleukin-12 Subunit p40; MDA LDL = Malondialdehyde-Modified Low-Density Lipoprotein; FAS = FASLG Receptor; SFTPD = Surfactant protein D; AXL = AXL Receptor Tyrosine Kinase; CXCL10 = Interferon gamma Induced Protein 10; ADIPOQ = Adiponectin; MB = Myoglobin; SOD1 = Superoxide dismutase 1; NRCAM = Neuronal Cell Adhesion Molecule. #Higher LP15A values indicate less severe emphysema, so positive coefficients are associated with less severe emphysema and negative coefficients are associated with more severe emphysema unlike higher %LAA which is associated with more severe emphysema. ¶Biomarkers not available for replication in TESRA. 2 Biomarkers and covariates associated with radiologic emphysema in the COPDGene cohort g multiple regression) Presented are beta coefficients and p values for multiple regression models of biomarkers and covariates associated with emphysema outcomes. %LAA = Percent low attenuation areas; LP15A = mean lung attenuation at 15th percentile on lung attenuation curve; HU = Hounsfield units; FEV1 = Forced expiratory volume in 1st second; RAGE = Receptor for advanced glycosylation end products; CCL20 = Macrophage Inflammatory Protein-3 alpha; ICAM1 = Intercellular Adhesion Molecule 1; SERPINA7 = Thyroxin-binding globulin; CDH 13 = Cadherin-13; CDH1 = Cadherin-1; TGFB1 LAP = Latency-Associated Peptide of Transforming Growth Factor beta 1; CCL13 = Monocyte Chemotactic Protein 4; TNFRSF11B = Osteoprotegerin; CCL8 = Monocyte Chemotactic Protein 2; IgA = Immunoglobulin A; SORT1 = Sortilin; IL2RA = Interleukin-2 receptor alpha; CCL2 = Monocyte Chemotactic Protein 1; IL-12B = Interleukin-12 Subunit p40; MDA LDL = Malondialdehyde-Modified Low-Density Lipoprotein; FAS = FASLG Receptor; SFTPD = Surfactant protein D; AXL = AXL Receptor Tyrosine Kinase; CXCL10 = Interferon gamma Induced Protein 10; ADIPOQ = Adiponectin; MB = Myoglobin; SOD1 = Superoxide dismutase 1; NRCAM = Neuronal Cell Adhesion Molecule. Validation of emphysema biomarkers #Higher LP15A values indicate less severe emphysema, so positive coefficients are associated with less severe emphysema and negative coefficients are associated with more severe emphysema unlike higher %LAA which is associated with more severe emphysema. ¶Biomarkers not available for replication in TESRA. Presented are beta coefficients and p values for multiple regression models of biomarkers and covariates associated with emphysema outcomes. %LAA = Percent low attenuation areas; LP15A = mean lung attenuation at 15th percentile on lung attenuation curve; HU = Hounsfield units; FEV1 = Forced expiratory volume in 1st second; RAGE = Receptor for advanced glycosylation end products; CCL20 = Macrophage Inflammatory Protein-3 alpha; ICAM1 = Intercellular Adhesion Molecule 1; SERPINA7 = Thyroxin-binding globulin; CDH 13 = Cadherin-13; CDH1 = Cadherin-1; TGFB1 LAP = Latency-Associated Peptide of Transforming Growth Factor beta 1; CCL13 = Monocyte Chemotactic Protein 4; TNFRSF11B = Osteoprotegerin; CCL8 = Monocyte Chemotactic Protein 2; IgA = Immunoglobulin A; SORT1 = Sortilin; IL2RA = Interleukin-2 receptor alpha; CCL2 = Monocyte Chemotactic Protein 1; IL-12B = Interleukin-12 Subunit p40; MDA LDL = Malondialdehyde-Modified Low-Density Lipoprotein; FAS = FASLG Receptor; SFTPD = Surfactant protein D; AXL = AXL Receptor Tyrosine Kinase; CXCL10 = Interferon gamma Induced Protein 10; ADIPOQ = Adiponectin; MB = Myoglobin; SOD1 = Superoxide dismutase 1; NRCAM = Neuronal Cell Adhesion Molecule. #Higher LP15A values indicate less severe emphysema, so positive coefficients are associated with less severe emphysema and negative coefficients are associated with more severe emphysema unlike higher %LAA which is associated with more severe emphysema. ¶Biomarkers not available for replication in TESRA. RAGE (p = 2.5 × 10−10), ICAM1 (p = 6.0 × 10−11), and AXL (p = 3.8 × 10−3) with radiologic emphysema inde- pendent of covariates (Table 3). CCL20 was signifi- cantly negatively associated with emphysema in both the TESRA and COPDGene cohorts; however, meta- analysis was not possible due to CCL20 being binary in in TESRA cohort. After meta-analysis and adjustment for multiple testing, biomarkers RAGE (p = 1.2 × 10−9) and ICAM1 (p = 1.5 × 10−7) were associated with % LAA < −910 HU (Table 3). Similarly, with regard to the LP15A emphysema outcome variable, meta-analysis with the TESRA cohort validated the association of Carolan et al. Respiratory Research 2014, 15:127 http://respiratory-research.com/content/15/1/127 Page 6 of 10 Figure 1 Biomarkers associated with CT-assessed emphysema in the COPDGene cohort. p values for COPDGene and TESRA are two sided p values. #CCL20 was a binary variable in COPDGene, therefore it is the presence CCL20 that is negatively associated with emphysema in COPDGene cohort, while CCL20 was a continuous variable in TESRA also associated negatively associated with more severe emphysema. Meta-analysis was not possible given difference in variables (N/A). Validation of emphysema biomarkers Biomarkers significant in the COPDGene study such as CDH1, CDH13, SERPINA7, MDA LDL, MB, NRCAM, and ADIPOQ were not measured in the TESRA study and therefore could not be included in the meta-analysis. COPDGene and continuous in TESRA. Biomarkers significant in the COPDGene study such as CDH1, CDH13, SERPINA7, MDA LDL, MB, NRCAM, and ADIPOQ were not measured in the TESRA study and therefore could not be included in the meta-analysis. using the TESRA data described in this study in combin- ation with the ECLIPSE investigators [13]. Some studies suggest that sRAGE is increased in the lungs of patients with COPD and high levels of sRAGE may be associated with progression of emphysema [33]. Interestingly, animal studies suggest RAGE/sRAGE plays a role in alveolar de- velopment and overexpression in mouse lung leads to the development of emphysema [34]. This suggests that sRAGE, by acting as a decoy molecule, may have a differ- ent role in the developing lung and the adult lung or low sRAGE levels in COPD may result in increased inflamma- tory signaling in the lung. ROC curves for covariates age, gender, BMI, current smoking status and FEV1 had an area under the curve (AUC) of 0.88 for the prediction of emphysema. ROC curves demonstrated a slight improvement in the AUC after adding 15 biomarkers to the model, raising the AUC to 0.92 (Additional file 1: Table S4 and Figure S1). However, when only considering those without severe airflow limitation (FEV1 ≥50%, n = 399), the AUC was 0.78 using covariates alone and the AUC increased to 0.85 when the biomarker panel was added to the model. In the present study, we found decreased ICAM1 levels correlate with increased severity of emphysema on CT scan, independent of smoking status, FEV1 and other covariates. ICAM1 is expressed on vascular endothelial and immune cells and mediates cell transmigration and adhesion [35]. ICAM1 plays a role in the recruitment of inflammatory cells to the lung. There is currently quite limited information about the association of ICAM1 to COPD and emphysema. Higher serum levels of soluble ICAM1 have been demonstrated in COPD, where it cor- related with the severity of airflow limitation, arterial hypoxemia and hypercarbia [36,37]. Discussion COPD is a phenotypically heterogeneous disease, with the presence of emphysema having implications for risk stratifi- cation and management [3-5,18]. In this study, we success- fully identified and replicated a panel of peripheral blood biomarkers that was associated with emphysema independ- ent of age, smoking status, body mass index, airflow limita- tion, and gender. These biomarkers (AGER, ICAM1 and CCL20) were associated with emphysema regardless of quantification technique (%LAA ≤−950 and ≤−910 HU and LP15A) and were replicated in an independent COPD cohort (TESRA), thus strengthening their poten- tial utility for defining clinically relevant emphysema. Our study reports lower RAGE levels in peripheral blood as a biomarker of increased emphysema percent- age in the lungs independent of gender, age, airflow limi- tation, body mass index and current smoking status. RAGE (advanced glycosylation end-product receptor or AGER) is an immunoglobulin family member that is highly expressed in human lung [25]. The RAGE path- way and soluble RAGE (sRAGE), a splice variant or pro- teolytic cleavage product of RAGE, have been associated with several inflammatory conditions such as diabetes mellitus, vascular disease and arthritis [26,27]. The sRAGE molecule binds damaged ligands preventing these from binding to cell surface receptors and activating cell signal- ing pathways [28]. RAGE is active in damage-related con- ditions such as hyperglycemia, hypoxia, inflammation and oxidative stress [29]. While fasting blood glucose measure- ments were not available, 66 individuals reported a history of diabetes mellitus in the COPDGene biomarker study and there was no association between RAGE levels and self-reported history of diabetes mellitus (p = 0.88). Lower levels of sRAGE have been described in individuals with airflow limitation [30,31]. Other studies have found lower sRAGE levels associated with CT-assessed emphysema se- verity and cor pulmonale [32] and with CT-assessed em- physema and lower diffusing capacity of carbon monoxide CCL20 or macrophage inhibitory protein 3a, a chemo- kine receptor ligand, is involved in the recruitment of in- flammatory cells through chemokine receptor 6 (CCR6), its only known receptor [40]. In both the COPDGene study and the TESRA study, CCL20 levels were inversely and significantly associated with emphysema although methodological considerations prevented a meta-analysis. Lower CCL20 levels have been described in bronchoalveo- lar lavage fluid of smokers [41]. Validation of emphysema biomarkers Other studies relate ICAM1 levels to active smoking [38] and preliminary analysis from The MESA Lung Study demonstrated that ICAM1 predicted 0.15%/year increase in CT-assessed emphysema, suggesting a role for this molecule as a bio- marker of emphysema and that it may play a role in em- physema pathogenesis [39]. Validation of emphysema biomarkers (A) Advanced glycosylation end-product receptor (RAGE); (B) Intracellular adhesion molecule 1 (ICAM1); (C) Cadherin 1 (CDH1); (D) Cadherin 13 (CDH13). Presented are normal quantile transformed biomarker levels on the ordinate and percent emphysema (% low attenuation ≤−950 HU) on CT scan on abscissa (p < 0.001 for all comparisons). Figure 1 Biomarkers associated with CT-assessed emphysema in the COPDGene cohort. (A) Advanced glycosylation end-product receptor (RAGE); (B) Intracellular adhesion molecule 1 (ICAM1); (C) Cadherin 1 (CDH1); (D) Cadherin 13 (CDH13). Presented are normal quantile transformed biomarker levels on the ordinate and percent emphysema (% low attenuation ≤−950 HU) on CT scan on abscissa (p < 0.001 for all comparisons). Table 3 Meta-analysis of biomarkers associated with emphysema in COPDGene and TESRA cohorts* COPDGene TESRA Adjusted meta-analysis p-value Variable Beta coefficient p-value° Beta coefficient p-value° Percent LAA ≤−910 HU RAGE −1.4 2.6 × 10−5 −0.52 9.2 × 10−7 1.2 × 10 −9 ICAM1 −3.2 9.2 × 10−6 −0.37 3.4 × 10−4 1.5 × 10−7 CCL20# −0.87 1.3 × 10−4 −0.29 2.2 × 10−3 N/A Mean lung attenuation at 15th percentile RAGE 10.78 1.3 × 10−5 7.08 3.0 × 10−8 2.5 × 10 −10 ICAM1 32.3 1.1 × 10−9 5.14 4.5 × 10−5 6.0 × 10−11 AXL 18.8 1.8 × 10−4 2.53 0.038 3.8 × 10−3 CCL20# 6.44 8.2 × 10−5 4.45 1.3 × 10−4 N/A Presented is the regression analysis for each biomarker with an adjusted meta-analysis p value. LAA = low attenuation area; RAGE = Receptor for advanced glycosylation end products; ICAM1 = Intercellular Adhesion Molecule 1; CCL20 = Macrophage Inflammatory Protein-3 alpha; AXL = AXL Receptor Tyrosine Kinase; °p values for COPDGene and TESRA are two-sided p values. #CCL20 was a binary variable in COPDGene, therefore it is the presence CCL20 that is negatively associated with emphysema in COPDGene cohort, while CCL20 was a continuous variable in TESRA also associated negatively associated with more severe emphysema. Meta-analysis was not possible given difference in variables (N/A). Meta-analysis of biomarkers associated with emphysema in COPDGene and TESRA cohorts Carolan et al. Respiratory Research 2014, 15:127 http://respiratory-research.com/content/15/1/127 Page 7 of 10 Page 7 of 10 COPDGene and continuous in TESRA. Biomarkers significant in the COPDGene study such as CDH1, CDH13, SERPINA7, MDA LDL, MB, NRCAM, and ADIPOQ were not measured in the TESRA study and therefore could not be included in the meta-analysis. COPDGene and continuous in TESRA. Conclusion f d With regard to the ability of biomarkers to predict the presence of any emphysema compared to no emphysema, ROC curves demonstrated a small contribution of plasma biomarkers separate to the covariates alone. This is likely because when individuals with more severe levels of em- physema are included, covariates alone, especially FEV1, are highly predictive of emphysema in their own right. How- ever, the biomarkers are more useful for predicting the presence of emphysema in those that do not already have severe airflow limitation, because the covariates alone were not as good at predicting emphysema in this group and biomarkers combined with covariates increased the area under the curve. This may be useful clinically since deter- mining the presence of underlying emphysema at this early stage in those that do not yet have severe airflow limitation may have outcome benefits for the individuals [3-6]. Our findings, particularly when combined with other studies of individual biomarkers, suggest that a panel of blood biomarkers including sRAGE, ICAM1 and CCL20 may serve as a useful surrogate measure of emphysema and may shed light on disease pathogenesis, providing targets for new treatments. Other biomarkers such as CHD1, CDH13 and SERPINA7 may also have a role in evaluating emphysema (especially milder emphysema), al- though require confirmation in other cohorts. Overall, these peripheral blood biomarkers could ultimately be used to diagnose emphysema at subclinical stages thereby reducing the need for CT, and perhaps may provide in- sights into disease prediction and progression. Discussion The CCR6/CCL20 com- plex is one of the most potent regulators of dendritic cell migration to the lung and CCR6 knockout mice may be partially protected against cigarette smoke-induced em- physema due to reduced recruitment of inflammatory cells to the lung [42]. These data suggest that increased activity of the CCL20/CCR6 pathway may increase the susceptibil- ity to emphysema. CDH1 was negatively correlated with radiologic emphy- sema across all emphysema outcome measurements. CDH1 or E cadherin is an epithelial cell adhesion molecule that regulates cell differentiation and morphogenesis, and is associated with lung fibrosis and cancer [43]. CDH1 may be a marker of epithelial cell injury and epithelial to mesen- chymal transition that is believed to play a role in small air- way remodeling in COPD [44]. Genetic polymorphisms in Carolan et al. Respiratory Research 2014, 15:127 http://respiratory-research.com/content/15/1/127 Page 8 of 10 CDH1 have been associated with development of COPD and decline in lung function [45]. CDH13 or H cadherin is another adhesion molecule that may influence surfactant protein D levels and serum adiponectin levels, both impli- cated in the pathogenesis of COPD; however, CDH13 itself has not been associated with quantitative emphysema to date [46,47]. We found higher levels of CDH13 to be asso- ciated with CT-assessed emphysema in the COPDGene cohort, but these were not available for validation in the TESRA cohort. Higher SERPINA7 levels were also associ- ated with more radiologic emphysema. SERPINA7 does not have protease inhibitor capabilities and is also known as thyroid binding globulin. This study represents a new association for SERPINA7 with COPD. populations remains unknown and emphysema measure- ments from both COPDGene and TESRA were cross sec- tional; therefore, the significance of these biomarkers for emphysema progression remains unknown. A final limita- tion of this study, an in many biomarker studies, is the magnitude of association between the change in biomarker levels and the change in emphysema severity. While the biomarker associations are highly statistically significant, and validation suggests the associations are real, further studies are needed to evaluate the role of these biomarkers in disease pathogenesis and as markers of disease presence and progression [48,49]. Additional file This COPDGene biomarker study is one of the largest emphysema biomarker studies to date on carefully pheno- typed individuals with COPD. The TESRA cohort pro- vides validation of a number of the findings. The study confirms a previously identified association between radio- logic emphysema and sRAGE, builds on data suggesting a role for ICAM1 as a biomarker, in addition to discovering previously not identified biomarkers associated with em- physema such as CCL20, cadherin 1, cadherin 13 and SERPINA7. The study also highlights the potential useful- ness of a panel of biomarkers to predict the presence of emphysema compared to using clinical data alone, espe- cially in those who do not yet have severe abnormalities in lung function. However there are limitations; the TESRA cohort was different from the COPDGene cohort in that its population was comprised of ex-smokers with at least mild COPD and did not include control subjects and only 2 of the 3 quantitative emphysema measurements were made (−910 HU and LP15A). Since emphysema can occur in smokers without COPD and emphysema measurements are highly co-linear, these limitations may be of minor im- portance. Other limitations include the fact that the ma- jority of subjects in both cohorts were non-Hispanic white, thus, the generalizability of these findings to other Additional file 1: Table S1. Institutional Review Boards of Participating Institutions. Table S2. Demographics of COPDGene cohort. Table S3. Biomarkers in COPDGene biomarker study. Table S4. Area under curve (AUC) for receiver operating characteristic (ROC) curves derived for emphysema (%LAA< -950 HU ≥5%) vs. no emphysema (%LAA< -950 HU <5%) as outcome. Figure S1. *Receiver operating characteristic (ROC) curve with emphysema (%LAA< -950 HU ≥5%) vs. no emphysema (%LAA< -950 HU <5%) as outcome for (A) covariates age, gender, body mass index, smoking status and FEV1 (all ranges); (B) same covariates with 15 biomarkers; (C) covariates with FEV1 (≥50% predicted) and (D) covariates with FEV1 (≥50% predicted) and 15 biomarkers. Authors' contributions 4. Li Y, Swensen SJ, Karabekmez LG, Marks RS, Stoddard SM, Jiang R, Worra JB, Zhang F, Midthun DE, de Andrade M, Song Y, Yang P: Effect of emphysema on lung cancer risk in smokers: a computed tomography- based assessment. Cancer Prev Res (Phila) 2011, 4(1):43–50. 4. Li Y, Swensen SJ, Karabekmez LG, Marks RS, Stoddard SM, Jiang R, Worra JB, Zhang F, Midthun DE, de Andrade M, Song Y, Yang P: Effect of emphysema on lung cancer risk in smokers: a computed tomography- based assessment. Cancer Prev Res (Phila) 2011, 4(1):43–50. BJC: Contributed to the data acquisition, analysis, and interpretation, manuscript drafting and critical review for intellectual content and final approval of the manuscript. BJC had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. GH: Contributed to the data analysis and interpretation, critical review for important intellectual content and final approval of the manuscript. JM: Contributed to the TESRA data analysis and interpretation. CPH: Contributed to TESRA data acquisition, analysis and critical review for the important intellectual content, and final approval of the manuscript. WKO: Critical review for the important intellectual content and final approval of the manuscript. SR: Critical review for the important intellectual content and final approval of the manuscript. SGP: Contributed to TESRA data acquisition, analysis and critical review for the important intellectual content, and final approval of the manuscript. PB: Contributed to TESRA data acquisition, analysis and critical review for the important intellectual content, and final approval of the manuscript. DAC: Contributed to TESRA data acquisition, analysis and critical review for the important intellectual content, and final approval of the manuscript. APC: Contributed to data acquisition, analysis and critical review for the important intellectual content and final approval of the manuscript. MH: Critical review for the important intellectual content and final approval of the manuscript. RLZ: Critical review for important intellectual content and final approval of the manuscript KK: Contributed to the data analysis and interpretation and drafting, critical review for important intellectual content, and final approval of the manuscript. RPB: Contributed to the study conception and design; data analysis and interpretation; manuscript drafting, critical review for the important intellectual content, and final approval of the manuscript. All authors read and approved the final manuscript. 5. Abbreviations ADIPOQ Adi ADIPOQ: Adiponectin; AGER/RAGE: Advanced glycosylation end products receptor; AXL: AXL Receptor Tyrosine Kinase; CCL13: Monocyte chemotactic protein 4; CCL2: Monocyte chemotactic protein 1; CCL20: Macrophage inflammatory protein-3 alpha; CCL8: Monocyte chemotactic protein 2; CDH 13: Cadherin-13; CDH1: Cadherin-1; COPD: Chronic obstructive pulmonary disease; CXCL10: Interferon gamma induced protein 10; FAS: FASLG receptor; FEV1: Forced expiratory volume in first second; FVC: Forced vital capacity; HRCT: High resolution computed tomography; HU: Hounsfield units; ICAM1: Intercellular adhesion molecule 1; IgA: Immunoglobulin A; IL-12B: Interleukin-12 Subunit p40; IL2RA: Interleukin-2 receptor alpha; LAA: Low attenuation area; LLOQ: Lower limit of quantitation; LP15A: Mean lung attenuation at the 15th percentile on lung attenuation curve; MB: Myoglobin; MDA LDL: Malondialdehyde-modified low-density lipoprotein; NRCAM: Neuronal cell adhesion molecule; SERPINA7: Thyroxin-binding globulin; SFTPD: Surfactant protein D; SOD1: Superoxide dismutase 1; SORT1: Sortilin; TGFB1 LAP: Latency-associated peptide of transforming growth factor beta 1; TNFRSF11B: Osteoprotegerin. Page 9 of 10 Page 9 of 10 Carolan et al. Respiratory Research 2014, 15:127 http://respiratory-research.com/content/15/1/127 Competing interests C l h h Dr. Carolan: The author has received lecture fees from Novartis. Dr. Hersh: The author is a consultant for CSL Behring and receives lecture fees from Novartis. Dr. Rennard: Entities with which SIR currently has relationships are as follows: GlaxoSmithKline, Boehringer Ingelheim, Forest, AstraZeneca, Chiesi, CME Incite, Takeda, Regeneron, Pearl, CIPLA, CSA, American Board of Internal Medicine, Merck, Medimmune, Synapse, Nycomed, Dalichi Sankyo, Novartis, Johnson and Johnson, Quadrant, Gerson Lehman, Able Associates, CSL Behring, CTS Carmel, Decision Resources, FirstWord, Gilead, Guidepoint Global, Pulmatrix, Saatchi and Saatchi, Schlesinger Associates, Cory Paeth, Frankel Group, Medical Knowledge, Pro Ed Communication, LEX Consulting. Dr. Belloni: The author is an employee of Genentech and receives stock or stock options from Genentech/Roche. Dr. Comellas: The author is a consultant for VIDA diagnostics. The other authors declare that they have no competing interests. Received: 29 May 2014 Accepted: 3 October 2014 Received: 29 May 2014 Accepted: 3 October 2014 Acknowledgements Th h ld lik The authors would like to thank Sarah Hawthorne for editorial assistance. The authors would like to thank Sarah Hawthorne for editorial assistance. 12. Stoller JK, Aboussouan LS: A review of alpha1-antitrypsin deficiency. Am J Respir Crit Care Med 2012, 185(3):246–259. 13. Cheng DT, Kim DK, Cockayne DA, Belousov A, Bitter H, Cho MH, Duvoix A, Edwards LD, Lomas DA, Miller BE, Reynaert N, Tal-Singer R, Wouters EF, Agusti A, Fabbri LM, Rames A, Visvanathan S, Rennard SI, Jones P, Parmar H, MacNee W, Wolff G, Silverman EK, Mayer RJ, Pillai SG: Systemic soluble receptor for advanced glycation endproducts is a biomarker of emphysema and associated with AGER genetic variants in patients with chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2013, 188(8):948–957. References b 1. Vestbo J, Hurd SS, Agusti AG, Jones PW, Vogelmeier C, Anzueto A, Barnes PJ, Fabbri LM, Martinez FJ, Nishimura M, Stockley RA, Sin DD, Rodriguez-Roisin R: Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease: GOLD executive summary. Am J Respir Crit Care Med 2013, 187(4):347–365. 2. Fletcher CM, Gilson JG, Hugh-Jones P, Scadding JG: Terminology, definitions, and classification of chronic pulmonary emphysema and related conditions Thorax 1959, 14:286–299. 3. Mohamed Hoesein FA, Zanen P, Boezen HM, Groen HJ, van Ginneken B, de Jong PA, Postma DS, Lammers JW: Lung function decline in male heavy smokers relates to baseline airflow obstruction severity. Chest 2012, 142(6):1530–1538. 3. Mohamed Hoesein FA, Zanen P, Boezen HM, Groen HJ, van Ginneken B, de Jong PA, Postma DS, Lammers JW: Lung function decline in male heavy smokers relates to baseline airflow obstruction severity. Chest 2012, 142(6):1530–1538. consultant for VIDA diagnostics. The other authors declare that they have no competing interests. Funding support h d This study was supported by National Heart, Lung and Blood Institute (NHLBI RO1HL 095432, U01 HL089856, U01 HL089897, P20 HL113445); UL1 RR025780 from NCRR/HIH and HHSN26820090020CP30 from NHLBI. Authors' contributions de Torres JP, Bastarrika G, Wisnivesky JP, Alcaide AB, Campo A, Seijo LM, Pueyo JC, Villanueva A, Lozano MD, Montes U, Montuenga L, Zulueta JJ: Assessing the relationship between lung cancer risk and emphysema detected on low-dose CT of the chest. Chest 2007, 132(6):1932–1938. 6. Haruna A, Muro S, Nakano Y, Ohara T, Hoshino Y, Ogawa E, Hirai T, Niimi A, Nishimura K, Chin K, Mishima M: CT scan findings of emphysema predict mortality in COPD. Chest 2010, 138(3):635–640. 7. Bastarrika G, Wisnivesky JP, Pueyo JC, Diaz L, Arraiza M, Villanueva A, Alcaide AB, Campo A, Seijo L, de Torres JP, Zulueta JJ: Low-dose volumetric computed tomography for quantification of emphysema in asymptomatic smokers participating in an early lung cancer detection trial. J Thorac Imaging 2009, 24(3):206–211. 8. Kirby M, Coxson HO: Computed tomography biomarkers of pulmonary emphysema. COPD 2013, 10(4):547–550. 9. Schroeder JD, McKenzie AS, Zach JA, Wilson CG, Curran-Everett D, Stinson DS, Newell JD Jr, Lynch DA: Relationships between airflow obstruction and quantitative CT measurements of emphysema, air trapping, and airways in subjects with and without chronic obstructive pulmonary disease. AJR Am J Roentgenol 2013, 201(3):W460–W470. 10. Mendoza CS, Washko GR, Ross JC, Diaz AA, Lynch DA, Crapo JD, Silverman EK, Acha B, Serrano C, Estepar RS: Emphysema Quantification in a Multi-Scanner Hrct Cohort Using Local Intensity Distributions. Proc IEEE Int Symp Biomed Imaging 2013: 474–477. 11. Parr DG, Dirksen A, Piitulainen E, Deng C, Wencker M, Stockley RA: Exploring the optimum approach to the use of CT densitometry in a randomised placebo-controlled study of augmentation therapy in alpha 1-antitrypsin deficiency. Respir Res 2009, 10:75. 17. Gaki E, Kontogianni K, Papaioannou AI, Bakakos P, Gourgoulianis KI, Kostikas K, Alchanatis M, Papiris S, Loukides S: Associations between BODE index and systemic inflammatory biomarkers in COPD. COPD 2011, 8(6):408–413. Author details 1D f 1Department of Medicine, National Jewish Health, 1400 Jackson St, Denver, CO 80206, USA. 2Department of Medicine, University of Colorado School of Medicine, Aurora, CO, USA. 3Department of Biostatistics and Informatics, Colorado School of Public Health, University of Colorado Denver, Aurora, CO, USA. 4Channing Division of Network Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA. 5Cystic Fibrosis/ Pulmonary Research and Treatment Center, Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA. 6Department of Internal Medicine, Pulmonary, Critical Care and Allergy Division, University of Nebraska Medical Center, Omaha, NE, USA. 7Hoffman La Roche, Nutley, USA. 8Genentech, Member of the Roche Group, South San Francisco, CA, USA. 9Department of Internal Medicine, University of Iowa, Iowa City, IA, USA. 10Department of Internal Medicine, Division of Pulmonary and Critical Care, University of Michigan Health System, Ann Arbor, MI, USA. 11Current address: Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN, USA. 12Current address: Boston Scientific, San Jose, CA, USA. 14. Carolan BJ, Kim YI, Williams AA, Kechris K, Lutz S, Reisdorph N, Bowler RP: The association of adiponectin with computed tomography phenotypes in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2013, 188(5):561–566. 15. Miller M, Ramsdell J, Friedman PJ, Cho JY, Renvall M, Broide DH: Computed tomographic scan-diagnosed chronic obstructive pulmonary disease- emphysema: eotaxin-1 is associated with bronchodilator response and extent of emphysema. J Allergy Clin Immunol 2007, 120(5):1118–1125. y gy 16. Thomsen M, Dahl M, Lange P, Vestbo J, Nordestgaard BG: Inflammatory biomarkers and comorbidities in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2012, 186(10):982–988. 17. Gaki E, Kontogianni K, Papaioannou AI, Bakakos P, Gourgoulianis KI, Kostikas K, Alchanatis M, Papiris S, Loukides S: Associations between BODE index and systemic inflammatory biomarkers in COPD. COPD 2011, 8(6):408–413. Page 10 of 10 Carolan et al. Respiratory Research 2014, 15:127 http://respiratory-research.com/content/15/1/127 37. Huang H, Jiang H, Kong X, Liu T, Tan Z, Chen P, Luo H: Association of intercellular adhesion molecule-1 gene K469E polymorphism with chronic obstructive pulmonary disease. Zhong Nan Da Xue Xue Bao Yi Xue Ban 2012, 37(1):78–83. 18. Rosenberg SR, Kalhan R: Biomarkers in chronic obstructive pulmonary disease. Transl Res 2012, 159(4):228–237. 19. Regan EA, Hokanson JE, Murphy JR, Make B, Lynch DA, Beaty TH, Curran-Everett D, Silverman EK, Crapo JD: Genetic epidemiology of copd (copedgene) study design. COPD 2010, 7:32–43. 38. Author details 1D f Gall TM, Frampton AE: Gene of the month: E-cadherin (CDH1). J Clin Pathol 2013, 66(11):928–932. 25. Buckley ST, Ehrhardt C: The receptor for advanced glycation end products (RAGE) and the lung. J Biomed Biotechnol 2010, 2010:917108. 44. Milara J, Peiro T, Serrano A, Cortijo J: Epithelial to mesenchymal transition is increased in patients with COPD and induced by cigarette smoke. Thorax 2013, 68(5):410–420. 26. Pullerits R, Bokarewa M, Dahlberg L, Tarkowski A: Decreased levels of soluble receptor for advanced glycation end products in patients with rheumatoid arthritis indicating deficient inflammatory control. Arthritis Res Ther 2005, 7(4):R817–R824. 45. Tsuduki KNH, Nakajima T, Tsujimura S, Yoshida S, Takahashi E, Nakamura M, Minematsu N, Tateno H, Ishizaka A: Genetic polymorphism of e-cadherin and COPD. Am J Respir Crit Care Med 2009, 179:A2999. 27. Falcone C, Bozzini S, Guasti L, D'Angelo A, Capettini AC, Paganini EM, Falcone R, Moia R, Gazzaruso C, Pelissero G: Soluble RAGE plasma levels in patients with coronary artery disease and peripheral artery disease. Scientific World Journal 2013, 2013:584504. 46. Kasahara DI, Williams AS, Benedito LA, Ranschr B, Kobzik L, Hug C, Shore SA: Role of the adiponectin binding protein, t-cadherin (cdh13), in pulmonaryt responses to subacute ozone. PLoS One 2013, 8:e65829. 28. Alexiou P, Chatzopoulou M, Pegklidou K, Demopoulos VJ: RAGE: a multi-ligand receptor unveiling novel insights in health and disease. Curr Med Chem 2010, 17(21):2232–2252. 47. Takeuchi T, Misaki A, Fujita J, Sonobe H, Ohtsuki Y: T-cadherin (cdh13, h-cadherin) expression downtregulated surfactant protein d in bronchioloalveolar cells. Virchows Arch 2001, 438:370–375. 29. Uchida T, Shirasawa M, Ware LB, Kojima K, Hata Y, Makita K, Mednick G, Matthay ZA, Matthay MA: Receptor for advanced glycation end-products is a marker of type I cell injury in acute lung injury. Am J Respir Crit Care Med 2006, 173(9):1008–1015. 48. Faner R, Tal-Singer R, Riley JH, Celli B, Vestbo J, MacNee W, Bakke P, Calverley PM, Coxson H, Crim C, Edwards LD, Locantore N, Lomas DA, Miller BE, Rennard SI, Wouters EF, Yates JC, Silverman EK, Agusti A: Lessons from ECLIPSE: a review of COPD biomarkers. Thorax 2013, 69(7):666–672. 48. Faner R, Tal-Singer R, Riley JH, Celli B, Vestbo J, MacNee W, Bakke P, Calverley PM, Coxson H, Crim C, Edwards LD, Locantore N, Lomas DA, Miller BE, Rennard SI, Wouters EF, Yates JC, Silverman EK, Agusti A: Lessons from ECLIPSE: a review of COPD biomarkers. Thorax 2013, 69(7):666–672. 30. Author details 1D f Lopez-Campos JL, Calero C, Arellano-Orden E, Marquez-Martin E, Cejudo-Ramos P, Ortega Ruiz F, Montes-Worboys A: Increased levels of soluble ICAM-1 in chronic obstructive pulmonary disease and resistant smokers are related to active smoking. Biomark Med 2012, 6(6):805–811. 20. Jones PW: Tesra (treatment of emphysema with selective retinoid agonist) study results. Am J Respir Crit Care Med 2011, 183:A6418. 21. Han MK, Kazerooni EA, Lynch DA, Liu LX, Murray S, Curtis JL, Criner GJ, Kim V, Bowler RP, Hanania NA, Anzueto AR, Make BJ, Hokanson JE, Crapo JD, Silverman EK, Martinez FJ, Washko GR: Chronic obstructive pulmonary disease exacerbations in the COPDGene study: associated radiologic phenotypes. Radiology 2011, 261(1):274–282. 39. Aaron CP, Schwartz JE, Tracy R, Hoffman EA, Austin JHM, Oelsner EC, Donohue KM, Kalhan R, Jacobs D, Barr RG: Intercellular Adhesion Molecule (icam)1 And Longitudinal Change In Percent Emphysema And Lung Function: The MESA Lung Study. Am J Respir Crit Care Med 2013, 187:A1523. y y 22. O'Neal WK, Anderson W, Basta PV, Carretta EE, Doerschuk CM, Barr RG, Bleecker ER, Christenson SA, Curtis JL, Han MK, Hansel NN, Kanner RE, Kleerup EC, Martinez FJ, Miller BE, Peters SP, Rennard SI, Scholand MB, Tal-Singer R, Woodruff PG, Couper DJ, Davis SM: Comparison of serum, EDTA plasma and P100 plasma for luminex-based biomarker multiplex assays in patients with chronic obstructive pulmonary disease in the SPIROMICS study. J Transl Med 2014, 12:9. 40. Dieu-Nosjean MC, Massacrier C, Homey B, Vanbervliet B, Pin JJ, Vicari A, Lebecque S, Dezutter-Dambuyant C, Schmitt D, Zlotnik A, Caux C: Macrophage inflammatory protein 3alpha is expressed at inflamed epithelial surfaces and is the most potent chemokine known in attracting Langerhans cell precursors. J Exp Med 2000, 192(5):705–718. 41. Meuronen A, Majuri ML, Alenius H, Mantyla T, Wolff H, Piirila P, Laitinen A: Decreased cytokine and chemokine mRNA expression in bronchoalveolar lavage in asymptomatic smoking subjects. Respiration 2008, 75(4):450–458. 23. Stouffer SA, Suchman EA, DeVinney LC, Star SA, Williams RM Jr: The American Soldier, Vol. 1: Adjustment during Army Life. Princeton: Princeton University Press; 1949. 42. Bracke KR, D'Hulst AI, Maes T, Moerloose KB, Demedts IK, Lebecque S, Joos GF, Brusselle GG: Cigarette smoke-induced pulmonary inflammation and emphysema are attenuated in CCR6-deficient mice. J Immunol 2006, 177(7):4350–4359. 24. Murdoch DR, O'Brien KL, Scott JA, Karron RA, Bhat N, Driscoll AJ, Knoll MD, Levine OS: Breathing new life into pneumonia diagnostics. J Clin Microbiol 2009, 47(11):3405–3408. 43. Author details 1D f Smith DJ, Yerkovich ST, Towers MA, Carroll ML, Thomas R, Upham JW: Reduced soluble receptor for advanced glycation end-products in COPD. Eur Respir J 2011, 37(3):516–522. 49. Agusti A, Sin DD: Biomarkers in COPD. Clin Chest Med 2014, 35(1):131–141. 49. Agusti A, Sin DD: Biomarkers in COPD. Clin Chest Med 2014, 35(1):131–141 doi:10.1186/s12931-014-0127-9 Cite this article as: Carolan et al.: The association of plasma biomarkers with computed tomography-assessed emphysema phenotypes. Respiratory Research 2014 15:127. 31. Cockayne DA, Cheng DT, Waschki B, Sridhar S, Ravindran P, Hilton H, Kourteva G, Bitter H, Pillai SG, Visvanathan S, Muller KC, Holz O, Magnussen H, Watz H, Fine JS: Systemic biomarkers of neutrophilic inflammation, tissue injury and repair in COPD patients with differing levels of disease severity. PLoS One 2012, 7(6):e38629. 32. Miniati M, Monti S, Basta G, Cocci F, Fornai E, Bottai M: Soluble receptor for advanced glycation end products in COPD: relationship with emphysema and chronic cor pulmonale: a case–control study. Respir Res 2011, 12:37. 33. Wu L, Ma L, Nicholson LF, Black PN: Advanced glycation end products and its receptor (RAGE) are increased in patients with COPD. Respir Med 2011, 105(3):329–336. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: Submit your next manuscript to BioMed Central and take full advantage of: 34. Stogsdill MP, Stogsdill JA, Bodine BG, Fredrickson AC, Sefcik TL, Wood TT, Kasteler SD, Reynolds PR: Conditional overexpression of receptors for advanced glycation end-products in the adult murine lung causes airspace enlargement and induces inflammation. Am J Respir Cell Mol Biol 2013, 49(1):128–134. • Convenient online submission 35. Di Stefano A, Maestrelli P, Roggeri A, Turato G, Calabro S, Potena A, Mapp CE, Ciaccia A, Covacev L, Fabbri LM, Saetta M: Upregulation of adhesion molecules in the bronchial mucosa of subjects with chronic obstructive bronchitis. Am J Respir Crit Care Med 1994, 149(3 Pt 1):803–810. • Thorough peer review 36. El-Deek SE, Makhlouf HA, Saleem TH, Mandour MA, Mohamed NA: Surfactant protein D, soluble intercellular adhesion molecule-1 and high-sensitivity C-reactive protein as biomarkers of chronic obstructive pulmonary disease. Med Princ Pract 2013, 22(5):469–474.
https://openalex.org/W156730495	https://sjtrem.biomedcentral.com/track/pdf/10.1186/1757-7241-21-S1-S22	English	null	Is the policy of “no active re-warming in the emergency department” adequate for therapeutic hypothermia after cardiac arrest	Scandinavian journal of trauma, resuscitation and emergency medicine	2,013	cc-by	585	Keep et al. Scandinavian Journal of Trauma, Resuscitation and Emergency Medicine 2013, 21(Suppl 1):S22 http://www.sjtrem.com/content/21/S1/S22 Keep et al. Scandinavian Journal of Trauma, Resuscitation and Emergency Medicine 2013, 21(Suppl 1):S22 http://www.sjtrem.com/content/21/S1/S22 Objectives The National Institute for Health and Clinical Excellence (NICE) recommend that after out of hospital cardiac arrest (OHCA) in patients with return of spontaneous circulation (ROSC), therapeutic hypothermia is induced “as soon as possible” to maintain core body temperature at 32-34°C for 12-24 hours.[1] Surface or internal cooling techniques are technically challenging in the Emergency Department (ED), instead we have a policy of ‘No active re-warming’ in our department. This study aimed to assess the adequacy of this policy. Conclusions Our policy did not achieve hypothermia by the time of ICU admission. Transit to PCI after ROSC increases the mean time to arrival in ITU by approximately 1 hour and does not appear to affect ICU arrival temperature. Cooling techniques are required in the ED phase of the patient pathway to initiate therapeutic hypothermia as soon as possible after ROSC. Is the policy of “no active re-warming in the emergency department” adequate for therapeutic hypothermia after cardiac arrest JW Keep*, Q Adelasoye, JR Pallett, S Calvert From London Trauma Conference 2012 London, UK. 4-7 December 2012 temperature on arrival in the ED for all patients was 35.15°C. Mean temperature on arrival in ICU after direct transfer from the ED was 35.24°C and on arrival in ICU after PCI was 35.30°C. 13 patients had therapeutic hypothermia in ICU of which 69% survived to discharge. Mean time to initiation of cooling from arrival in the ED was 6hr 6mins (1hr 53mins – 12hr 27mins). Reference h . [http://www.nice.org.uk/nicemedia/live/12990/53610/53610.pdf], (Accessed 11 October 2012). © 2013 Keep 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. Methods A retrospective observational cohort study of OHCA patients achieving ROSC in the ED was performed. Patients < 18 years and those admitted for palliative care were excluded. Transit times from arrival in the ED to Intensive Care Unit (ICU) either directly or via per- cutaneous coronary intervention (PCI) were recorded. In addition, body temperature in the ED and on arrival in ICU with the time of active cooling was obtained. Results doi:10.1186/1757-7241-21-S1-S22 Cite this article as: Keep et al.: Is the policy of “no active re-warming in the emergency department” adequate for therapeutic hypothermia after cardiac arrest. Scandinavian Journal of Trauma, Resuscitation and Emergency Medicine 2013 21(Suppl 1):S22. Between Jan 2009 and June 2012, 258 patients with OHCA were identified. Notes were unavailable for 3 cases. 14 were excluded as < 18 years. Of the remaining 241, 71 achieved ROSC (29%) and 167 died in the ED. Of the sur- vivors: 62 were admitted to ICU, (44 directly from the ED and 18 via PCI) and 9 admitted for palliative care on the ward. 1 case of hyperpyrexia was excluded. Time from arrival at the ED to direct admission in ICU was 2hr 58mins (n=26). This time increased to a mean of 3hr 50mins when patients went via PCI (n=9). The mean Kings College Hospital, London, UK © 2013 Keep 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.
https://openalex.org/W2359715109	https://europepmc.org/articles/pmc4865999?pdf=render	English	null	Peroxisome proliferator-activated receptor-gamma dependent pathway reduces the phosphorylation of dynamin-related protein 1 and ameliorates hippocampal injury induced by global ischemia in rats	Journal of biomedical science	2,016	cc-by	10,946	Peroxisome proliferator-activated receptor-gamma dependent pathway reduces the phosphorylation of dynamin- related protein 1 and ameliorates hippocampal injury induced by global ischemia in rats g1,2,3†, Tsu-Kung Lin1†, Ding-I Yang4, Jenq-Lin Yang2, Chia-Wei Liou1 and Shang-Der Chen1,2* * Correspondence: chensd@adm.cgmh.org.tw †Equal contributors 1Department of Neurology, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Kaohsiung, Taiwan 2Center for Translational Research in Biomedical Sciences, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan Full list of author information is available at the end of the article RESEARCH Open Access Peroxisome proliferator-activated receptor-gamma dependent pathway reduces the phosphorylation of dynamin- related protein 1 and ameliorates hippocampal injury induced by global ischemia in rats Yao-Chung Chuang1,2,3†, Tsu-Kung Lin1†, Ding-I Yang4, Jenq-Lin Yang2, Chia-Wei Liou1 and Shang-Der Chen1,2* Chuang et al. Journal of Biomedical Science (2016) 23:44 DOI 10.1186/s12929-016-0262-3 RESEARCH Open Access Peroxisome proliferator-activated receptor-gamma dependent pathway reduces the phosphorylation of dynamin- related protein 1 and ameliorates hippocampal injury induced by global ischemia in rats Yao-Chung Chuang1,2,3†, Tsu-Kung Lin1†, Ding-I Yang4, Jenq-Lin Yang2, Chia-Wei Liou1 and Shang-Der Chen1,2* Chuang et al. Journal of Biomedical Science (2016) 23:44 DOI 10.1186/s12929-016-0262-3 Chuang et al. Journal of Biomedical Science (2016) 23:44 DOI 10.1186/s12929-016-0262-3 Chuang et al. Journal of Biomedical Science (2016) 23:44 DOI 10.1186/s12929-016-0262-3 Chuang et al. Journal of Biomedical Science (2016) 23:44 DOI 10.1186/s12929-016-0262-3 RESEARCH Open Access Peroxisome proliferator-activated receptor-gamma dependent pathway reduces the phosphorylation of dynamin- related protein 1 and ameliorates hippocampal injury induced by global ischemia in rats Yao-Chung Chuang1,2,3†, Tsu-Kung Lin1†, Ding-I Yang4, Jenq-Lin Yang2, Chia-Wei Liou1 and Shang-Der Chen1,2* RESEARCH Open Access Peroxisome proliferator-activated receptor-gamma dependent pathway reduces the phosphorylation of dynamin- related protein 1 and ameliorates hippocampal injury induced by global ischemia in rats Yao-Chung Chuang1,2,3†, Tsu-Kung Lin1†, Ding-I Yang4, Jenq-Lin Yang2, Chia-Wei Liou1 and Shang-Der Chen1,2* Animals and general preparations Animals and general preparations All the animal studies followed the Guide for the Care and Use of Laboratory Animals, National Research Council, USA and were approved by the Institutional Animal Care and Use Committee (IACUC) of Chang Gung Memorial Hospital (Permit Number: 2009121704). All efforts were made to minimize animal suffering and to reduce the number of animals sacrificed. Adult male Sprague-Dawley rats (250–325 g) were purchased from BioLASCO, Taiwan. They were housed in an Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC), an internationally accredited animal facility, under temperature control (24–25 °C) and 12-h light-dark cycle. Standard laboratory rat chow and tap water were available ad libitum. Animals were anesthe- tized with chloral hydrate (400 mg/kg, i.p.) to perform preparative surgery. An experimental model of TGI was performed as previously reported [29] with modifica- tions [19, 21]. Briefly, the animals were subjected to a 10-min period of global ischemia by clamping both com- mon carotid arteries and lowering blood pressure to 35– 40 mm-Hg by withdrawing blood from a femoral arterial catheter; blood pressure was restored by infusing the withdrawn blood afterwards. A PE-50 catheter was inserted to femoral artery to continuously record the arterial blood pressure and to keep blood pressure within 35–40 mm-Hg. After regaining consciousness, the animals were maintained in an air-conditioned room at 25 °C. A selective neuronal loss in hippocampal CA1 subfield is a histological hallmark of transient global ischemia (TGI) and reperfusion [12, 13]. This condition occurs in patients with anoxic-ischemic encephalopathy and car- diorespiratory arrest of various causes [14] The damage may cause long-term cognitive change as the hippocam- pus is an important player in memory consolidation and in conjunction with other cortical areas, is critical for the retrieval of remote episodic memories and antero- grade memory processes [15]. Emerging evidence sug- gests that the mitochondrion may play a major role in delayed neuronal death of the CA1 subfield after TGI [16–19]. Recent evidence implicates the regulatory pro- cesses of mitochondrial biogenesis as a protective effect in the pathogenesis of cerebral ischemia [18, 20]. Lately, mitochondrial dynamic has been recognized as a pivotal process in regulating cell survival and death; in particu- lar, mitochondrial fission occurs as an upstream and early event in neuronal death after cerebral ischemia [7–9]. Background following ischemic injury. Despite its ability of enhan- cing mitochondrial biogenesis [28], however, the potential correlation between PPARγ agonist and mitochondrial dynamics, in particular its effects on the expression of p-Drp1(Ser616), has never been investigated before. To resolve this question, we hypothesized that PPARγ- dependent mechanism may exert anti-oxidative and anti- apoptotic effects against TGI in rats which involves p-Drp1(Ser616) expression. Results derived from this work may further improve our understandings to- wards the molecular mechanisms underlying TGI- induced neuronal demise in the hippocampal CA1 subfield and provide novel insights for future develop- ment of therapeutic regimen. Mitochondria are the powerhouses of cells to produce ATP as well as to regulate signaling cascades, including apoptosis [1]. A recent progress towards the understand- ing of mitochondrial control over apoptosis is the discovery of a drastic morphological change of this or- ganelle under stressful conditions [2, 3]. Mitochondria are dynamic organelles that maintain their shape or morphology via two opposing processes: fission and fu- sion [4–6]. While the fission process involves the con- striction and cleavage of mitochondria, fusion process involves the lengthening of mitochondria by tethering and joining two adjacent mitochondria together [4–6]. It was shown that, just before the apoptotic processes, mitochondria fragment into multiple small units (fission) and blocking mitochondrial fission can inhibit cyto- chrome c release with delayed cell death [2]. Drp1, an important fission protein, plays a crucial role in focal cerebral ischemia and inhibition of Drp1 can reduce the infarct volumes [7–9]. Expression of the dominant- negative Drp1 mutant in cell lines decreases mitochon- drial fragmentation and blocks cell death in response to various apoptotic insults [10, 11]. Abstract Background: Dynamin-related protein 1 (Drp1) is a mitochondrial fission protein that, upon phosphorylation at serine 616 (p-Drp1(Ser616)), plays a pivotal role in neuronal death after ischemia. In the present study, we hypothesized that peroxisome proliferator-activated receptor-gamma (PPARγ)-dependent pathway can reduce the expression of p-Drp1(Ser616) and ameliorate hippocampal injury induced by global ischemia in rats. Results: We found that pretreatment of the rats with Mdivi-1, a selective Drp1 inhibitor, decreased the level of transient global ischemia (TGI)-induced p-Drp1(Ser616) and reduced cellular contents of oxidized proteins, activated caspase-3 expression as well as the extent of DNA fragmentation. Delivery of siRNA against Drp1 attenuated the expression of p-Drp1(Ser616) that was accompanied by alleviation of the TGI-induced protein oxidation, activated caspase-3 expression and DNA fragmentation in hippocampal proteins. Exogenous application of pioglitazone, a PPARγ agonist, reduced the p-Drp1(Ser616) expression, decreased TGI-induced oxidative stress and activated caspase-3 expression, lessened the extents of DNA fragmentation, and diminished the numbers of TUNEL-positive neuronal cells; all of these effects were reversed by GW9662, a PPARγ antagonist. Conclusions: Our findings thus indicated that inhibition of TGI-induced p-Drp1(Ser616) expression by Drp1 inhibitor and Drp1-siRNA can decrease protein oxidation, activated caspase-3 expression and neuronal damage in the hippocampal CA1 subfield. PPARγ agonist, through PPARγ-dependent mechanism and via decreasing p-Drp1(Ser616) expression, can exert anti-oxidative and anti-apoptotic effects against ischemic neuronal injury. Keywords: Apoptosis, Dynamin-related protein 1, Global ischemia, Hippocampus, Peroxisome proliferator-activated receptor-gamma, Pioglitazone * Correspondence: chensd@adm.cgmh.org.tw †Equal contributors 1Department of Neurology, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Kaohsiung, Taiwan 2Center for Translational Research in Biomedical Sciences, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan Full list of author information is available at the end of the article © 2016 Chuang 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. Chuang et al. Journal of Biomedical Science (2016) 23:44 Page 2 of 14 Animals and general preparations Recently, we have also shown that TGI induced a transient increase in the phosphorylation of Drp1 at serine 616 (p-Drp1(Ser616)), without significantly af- fecting the expression of total Drp1 proteins or its phosphorylation at serine 637, in the rat hippocampal CA1 proteins [21]. Together these findings support the crucial roles of mitochondrial dynamics in ische- mic neuronal death. Qualitative and quantitative analysis of DNA fragmentation Qualitative and quantitative analysis of DNA fragmentation Preparations of tissue samples from the hippocampal CA1 subfield for qualitative and quantitative analysis of DNA fragmentation was conducted as reported previously [18, 19]. With total DNA from the hippocampal tissues, nucleosomal DNA ladders were amplified using a DNA ladder assay kit (Maxim Biotech, San Francisco, CA, USA) as previously reported [19, 21]. Samples were sepa- rated by electrophoresis on 1 % agarose gels. A cell death enzyme-linked immunosorbent assay (Roche Molecular Biochemicals, Mannheim, Germany) was used to assess the level of histone-associated DNA fragments in the Pharmacological pretreatments In the experiments involving pharmacological pretreat- ments, the dosage for Mdivi-1 was tested as previously reported [8, 30], the dosage of GW9662 and pioglitazone were based on our previous studies [26, 31]. One group of rats were treated intraperitoneally with Drp1 inhibitor Mdivi-1 (2.4 mg/kg), which was purchased from Sigma- Aldrich Ltd (St. Louis, MO, USA), or the solvent Peroxisome proliferator-activated receptor gamma (PPARγ) agonist, such as pioglitazone or rosiglitazone has been shown to reduce inflammation [22, 23], de- crease oxidative damage [23–27], and reduce cell death Chuang et al. Journal of Biomedical Science (2016) 23:44 Page 3 of 14 Page 3 of 14 Page 3 of 14 Western blot analysis dimethyl sulfoxide (DMSO) 30 min before TGI. The other group of rats were microinjected into bilateral CA1 subfields with pioglitazone (Cayman Chemical, Ann Arbor, MI, USA; 20 nmol), GW9662 (Cayman Chemical, 500 ng) or DMSO as the vehicle and volume control 30 min before TGI. The test agents were micro- injected bilaterally in a volume of 100 nl on each side. Drug delivery into the hippocampal CA1 subfield was carried out as previously reported [18, 19]. The animals receiving chloral hydrate anesthesia and surgical prepa- rations without additional experimental manipulations served as sham-controls. dimethyl sulfoxide (DMSO) 30 min before TGI. The other group of rats were microinjected into bilateral CA1 subfields with pioglitazone (Cayman Chemical, Ann Arbor, MI, USA; 20 nmol), GW9662 (Cayman Chemical, 500 ng) or DMSO as the vehicle and volume control 30 min before TGI. The test agents were micro- injected bilaterally in a volume of 100 nl on each side. Drug delivery into the hippocampal CA1 subfield was carried out as previously reported [18, 19]. The animals receiving chloral hydrate anesthesia and surgical prepa- rations without additional experimental manipulations served as sham-controls. y Western blot analysis for Drp1 and α-tublin was car- ried out on proteins extracted from total lysates of hippocampal samples. The primary antibody were Drp1, p-Drp1(Ser616) and active cleaved fragment (17 and 19 kDa) of caspase-3 (Cell Signaling, Danvers, MA, USA), or mouse monoclonal antiserum against α-tubulin (Santa Cruz Biotechnology). The secondary antibody in- cluded a horseradish peroxidase-conjugated goat anti- rabbit (Chemicon) for Drp1 and p-Drp1(Ser616), donkey anti-rabbit IgG (Amersham Biosciences, Little Chalfont, U.K.) for activated caspase-3 and goat anti-mouse IgG (Chemicon) for α-tubulin. The specific antibody-antigen complex was detected and measured semiquantitatively as previously reported [19, 21, 32]. siRNA administration All siRNAs were injected into bilateral hippocampal CA1 subfield as previously described [19, 21, 32]. To evaluate transfection efficiency, we used fluorescein iso- thiocyanate (FITC)-conjugated siRNA as a non-targeting siRNA (sc-36869; Santa Cruz Biotechnology, Santa Cruz, CA, USA). As previously reported, animals were killed 24 h after administration of FITC-siRNA in sham- control and 4 h after TGI/reperfusion before observation under a fluorescence microscope [21]. To inhibit Drp1 expression, we used pre-designed Drp1-siRNA from MISSION® siRNA, (Sigma-Aldrich Ltd.). The sequences were as follows: sense, 5′CAGAGUAUUGUAACACU AU3′, antisense, 5′AUAGUGUUACAAUACUCUG3′. For negative control siRNA (NC), the sequences were as follows: 5′GAUCAUACGUGCGAUCAGA3′, antisense, 5′UCUGAUCGCACGUAUGAUC3′. The final concen- tration of siRNA was 0.05 nM in a total volume of 400 nl for injection into each side of hippocampal CA1 subfield 24 h before TGI. Immunofluorescence staining g Immunofluorescence staining was carried out in animals as reported previously [19]. Briefly, free-floating sections (thickness = 30 μm) of the hippocampus were incubated with a rabbit polyclonal antiserum against p-Drp1 (Ser616) (Cell Signaling) and a mouse monoclonal anti- serum, neuron-specific nuclear protein (NeuN, Chemicon). Two secondary antibodies were used that included a goat anti-rabbit IgG-conjugated with Alexa Fluor 488 for p-Drp1(Ser616) and a goat anti-mouse IgG conjugated with Alexa Fluor 568 for NeuN (Molecular Probes, Eugene, OR, USA). The merged images indicated the pres- ence of p-Drp1(Ser616) immunoreactivity in the cytosol and NeuN in the nucleus of neurons. For double immuno- fluorescence staining of p-Drp1(Ser616) and COXIV, the sections of the hippocampus were first incubated with a rabbit polyclonal antiserum against p-Drp1(Ser616) (Cell Signaling). The sections were subsequently incubated with a goat anti-rabbit IgG conjugated with Alexa Fluor 488 for p-Drp1(Ser616). After fixed with 4 % paraformaldehyde for 5 min, the same sections were incubated with a polyclonal rabbit antiserum against COXIV (Cell Signaling) and then with DyLight 405-conjugated AffiniPure goat anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA, USA) for labeling COX IV. Collection of tissue samples from the hippocampus p pp p At predetermined time intervals (1, 4, 24, or 48 h) after induction of TGI, rats were anesthetized and perfused intracardially with 50 ml of warm (37 °C) sa- line that contained heparin (100 U/ml). The tissues from bilateral hippocampal CA1 area were collected and concentration of proteins determined as previ- ously reported [19, 21]. Tem7poral changes of drp1 expressions in the hippocampal CA1 subfield after TGI 1 Transient induction of p-Drp1(Ser616) by TGI. Hippocampal CA1 samples were collected from the rats at indicated times after 10-min TGI or sham-operated controls followed by protein extraction and western analysis for detection of total Drp1 in (a) and p-Drp1(Ser616) in (b). The same blots were also probed with α-tubulin antibody to serve as an internal reference control for equal loading of proteins in each lane. The ratio change of p-Drp1(Ser616)/total Drp was shown in (c). Values are mean ± SEM from representative blots and quantitative analyses from 4-6 animals in each experimental group are shown. P < 0.05 versus sham control group in the Scheff′e multiple-range test Detection of protein oxidation The extent of protein oxidation was determined by a commercial kit (OxyBlot, Chemicon, Temecula, CA). Total proteins extracted from the hippocampal CA1 subfield at 24 h after ischemia/reperfusion were subjected to reactions with 2,4-initrophenylhydrazine and deriva- tized to 2,4-dinitrophenylhydrazone (DNP-hydrazone). Western blotting using a rabbit anti-DNP antibody and then incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary IgG antibody was performed according to manufacturer’s instruction. Chuang et al. Journal of Biomedical Science (2016) 23:44 Page 4 of 14 Fig. 1 Transient induction of p-Drp1(Ser616) by TGI. Hippocampal CA1 samples were collected from the rats at indicated times after 10-min TGI or sham-operated controls followed by protein extraction and western analysis for detection of total Drp1 in (a) and p-Drp1(Ser616) in (b). The same blots were also probed with α-tubulin antibody to serve as an internal reference control for equal loading of proteins in each lane. The ratio change of p-Drp1(Ser616)/total Drp was shown in (c). Values are mean ± SEM from representative blots and quantitative analyses from 4-6 animals in each experimental group are shown. P < 0.05 versus sham control group in the Scheff′e multiple-range test cytoplasm. The amount of nucleosomes in the cytoplasm was determined using 2,20-azino-di-[3-ethylbenzthiazoline] sulfonate as the substrate and the absorbance was mea- sured as previously reported [19, 21]. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining Animals were processed for TUNEL staining 48 h after the onset of reperfusion following a 10-min episode of TGI as previously reported [21]. In brief, the hippocampus was removed and fixed in 30 % sucrose in 10 % formaldehyde- saline solution for ≥72 h. Six-micrometer paraffin- embedded sections (thickness = 25 μm) of the hippocam- pus were processed for TUNEL staining with an apoptosis detection kit (ApopTag, Intergen Company, Purchase, NY, USA). The total numbers of TUNEL-positive cells in each section were counted using an Olympus AX70 microscope and expressed as the TUNEL indices [21]. Statistical analysis All values expressed as mean ± SEM. The one-way ana- lysis of variance (ANOVA) was used, as appropriate, to assess group means, followed by the Scheffe’ multiple- range test for post-hoc assessment of individual mean. P < 0.05 indicates statistical significance. Tem7poral changes of drp1 expressions in the hippocampal CA1 subfield after TGI We first examined whether Drp1 is induced by TGI in the hippocampal CA1 subfield. The division of the mito- chondria, which is required for apoptosis as well as nor- mal cell growth and development, is controlled in part by the phosphorylation of Drp1 at Ser616 by Cdk1/cyclin B [33, 34]. We used Drp1 and phosphorylation of Drp1 at Ser616 (p-Drp1(Ser616)) antibodies to examine the ex- pression in the hippocampal CA1 subfield after TGI at the designed time. Total Drp1 (Fig. 1a) revealed no significant change and p-Drp1(Ser616) significantly increased as early as 1 h after TGI and remained elevated until 24 and 48 h after TGI in the total protein extracted from the hippo- campal CA1 subfield (Fig. 1b, c). This finding may suggest the active form, phosphorylated Drp-1(Ser616), in stead of total Drp1 expression, plays the functional role on the fis- sion process of mitochondria in this ischemic condition. This notion was reported before that Drp1 phosphoryl- ation at serine 616 results in its activation and recruitment to mitochondria [35]. i d f ( ) b l Fig. 1 Transient induction of p-Drp1(Ser616) by TGI. Hippocampal CA1 samples were collected from the rats at indicated times after 10-min TGI or sham-operated controls followed by protein extraction and western analysis for detection of total Drp1 in (a) and p-Drp1(Ser616) in (b). The same blots were also probed with α-tubulin antibody to serve as an internal reference control for equal loading of proteins in each lane. The ratio change of p-Drp1(Ser616)/total Drp was shown in (c). Values are mean ± SEM from representative blots and quantitative analyses from 4-6 animals in each experimental group are shown. P < 0.05 versus sham control group in the Scheff′e multiple-range test Fig. 1 Transient induction of p-Drp1(Ser616) by TGI. Hippocampal CA1 samples were collected from the rats at indicated times after 10-min TGI or sham-operated controls followed by protein extraction and western analysis for detection of total Drp1 in (a) and p-Drp1(Ser616) in (b). The same blots were also probed with α-tubulin antibody to serve as an internal reference control for equal loading of proteins in each lane. The ratio change of p-Drp1(Ser616)/total Drp was shown in (c). Values are mean ± SEM from representative blots and quantitative analyses from 4-6 animals in each experimental group are shown. P < 0.05 versus sham control group in the Scheff′e multiple-range test Fig. Effect of Drp1 inhibitor over p-Drp1(Ser616) expression, oxidative stress and neuronal injury in the hippocampal CA1 subfield After TGI Mitochondrial biogenesis may function as an endogen- ous protective mechanism [19, 20] while mitochondrial Page 5 of 14 Chuang et al. Journal of Biomedical Science (2016) 23:44 fission may initiate the apoptotic process under ischemic insult [7, 8]. As Drp1 phosphorylation at Ser616 in rat hippocampal CA1 regions shows a significant change that peaked at 24 h after TGI, with no significant change in total Drp1 protein expression after 1–48 h of reperfu- sion, we then test if downregulation of p-Drp1(Ser616) Fig. 2 Mdivi-1 reduced Drp1 phosphorylation, protein oxidation, and DNA fragmentation after TGI. Rats were treated intraperitoneally with Drp1 inhibitor Mdivi-1 (2.4 mg/kg) or its solvent DMSO 30 min before TGI. Total proteins were extracted from the hippocampal CA1 subfield of sham- operated controls or treated animals 24 h after 10-min TGI for detection of p-Drp1(Ser616) in (a), protein oxidation in (b) and activated caspase-3 expression in (c). DNA was isolated from the hippocampal CA1 subfield of sham-operated controls, DMSO + I/R and Mdivi-1 + I/R 48 h after TGI for detection of DNA fragmentation by PCR assay (d), protein lysates from hippocampal CA1 tissues were collected 48 h after TGI for detection of DNA fragmentation by sandwich ELISA in (e). Values are mean ± SEM from representative blots and quantitative analysis from 5–6 animals in each experimental group (a, b and c). Values in (e) are fold changes with reference to sham-operated controls and are mean ± SEM of 4 animals in each experimental group. P < 0.05 versus sham control group, #P < 0.05 versus DMSO + I/R group in the Scheffé multiple-range test. I/R: ischemia/reperfusion Fig. 2 Mdivi-1 reduced Drp1 phosphorylation, protein oxidation, and DNA fragmentation after TGI. Rats were treated intraperitoneally with Drp1 inhibitor Mdivi-1 (2.4 mg/kg) or its solvent DMSO 30 min before TGI. Total proteins were extracted from the hippocampal CA1 subfield of sham- operated controls or treated animals 24 h after 10-min TGI for detection of p-Drp1(Ser616) in (a), protein oxidation in (b) and activated caspase-3 expression in (c). DNA was isolated from the hippocampal CA1 subfield of sham-operated controls, DMSO + I/R and Mdivi-1 + I/R 48 h after TGI for detection of DNA fragmentation by PCR assay (d), protein lysates from hippocampal CA1 tissues were collected 48 h after TGI for detection of DNA fragmentation by sandwich ELISA in (e). Effect of Drp1 inhibitor over p-Drp1(Ser616) expression, oxidative stress and neuronal injury in the hippocampal CA1 subfield After TGI Values are mean ± SEM from representative blots and quantitative analysis from 5–6 animals in each experimental group (a, b and c). Values in (e) are fold changes with reference to sham-operated controls and are mean ± SEM of 4 animals in each experimental group. P < 0.05 versus sham control group, #P < 0.05 versus DMSO + I/R group in the Scheffé multiple-range test. I/R: ischemia/reperfusion ig. 2 Mdivi-1 reduced Drp1 phosphorylation, protein oxidation, and DNA fragmentation after TGI. Rats were treated intraperitoneally with Drp1 nhibitor Mdivi-1 (2.4 mg/kg) or its solvent DMSO 30 min before TGI. Total proteins were extracted from the hippocampal CA1 subfield of sham- Fig. 2 Mdivi-1 reduced Drp1 phosphorylation, protein oxidation, and DNA fragmentation after TGI. Rats were treated intraperitoneally with Drp1 inhibitor Mdivi-1 (2.4 mg/kg) or its solvent DMSO 30 min before TGI. Total proteins were extracted from the hippocampal CA1 subfield of sham- operated controls or treated animals 24 h after 10-min TGI for detection of p-Drp1(Ser616) in (a), protein oxidation in (b) and activated caspase-3 expression in (c). DNA was isolated from the hippocampal CA1 subfield of sham-operated controls, DMSO + I/R and Mdivi-1 + I/R 48 h after TGI for detection of DNA fragmentation by PCR assay (d), protein lysates from hippocampal CA1 tissues were collected 48 h after TGI for detection of DNA fragmentation by sandwich ELISA in (e). Values are mean ± SEM from representative blots and quantitative analysis from 5–6 animals in each experimental group (a, b and c). Values in (e) are fold changes with reference to sham-operated controls and are mean ± SEM of 4 animals in each experimental group. P < 0.05 versus sham control group, #P < 0.05 versus DMSO + I/R group in the Scheffé multiple-range test. I/R: ischemia/reperfusion Chuang et al. Journal of Biomedical Science (2016) 23:44 Page 6 of 14 expression will exert beneficial effect over hippocampal CA1 subfield under TGI. Firstly, we tested the effects of Mdivi-1, a selective inhibitor of Drp1, over TGI-induced p-Drp1(Ser616) expression. Western blot analysis re- vealed a reduction of p-Drp1(Ser616) protein level in the hippocampal CA1 subfield 24 h after TGI in the Mdivi- 1-treated group as compared to the vehicle groups (Fig. 1a). An excessive production of ROS underlies neuronal cell death in the CA1 subfield of the hippo- campus after TGI [18, 19]. Effect of Drp1 inhibitor over p-Drp1(Ser616) expression, oxidative stress and neuronal injury in the hippocampal CA1 subfield After TGI We therefore investigated the effects of Mdivi-1, which is capable of reducing TGI- induced phosphorylation of Drp1 at Ser616 (Fig. 2a), over ischemia-dependent oxidative stress and apoptosis related molecule. We found that, at 24 h following TGI, protein oxidation as well as activated caspase-3 expression in the hippocampal CA1 subfield decreased significantly by pretreatment with Mdivi-1 (Fig. 2b, c). We have also shown that an excessive production of ROS underlies DNA fragmentation and neuronal damage in the hippo- campal CA1 subfield after TGI [18, 19]. Our results de- rived from both qualitative (Fig. 2d) and quantitative (Fig. 2e) analyses of DNA fragmentation indicated that, at 48 h following TGI, apoptosis-like cell death decreased significantly by pretreatment of Mdivi-1. Thus, Mdivi-1 capable of reducing the extent of phosphorylation Drp1 at Ser616 also attenuates the TGI-mediated oxidative dam- age and neuronal injury in rat hippocampal CA1 regions. approach by using Drp1-siRNA to knock down its expression. We first confirmed its efficient delivery by injecting FITC-conjugated non-targeting siRNA (FITC- siRNA) into the hippocampal CA1 subfields bilaterally. Immunofluorescence demonstrated the cytosolic distri- bution of FITC-siRNA, in both the sham (Fig. 3a) and TGI (Fig. 3b) groups, in the hippocampal CA1 subfield 24 h after injection. These results indicated the siRNA could be successfully delivered into neurons under both sham-operated and ischemia/reperfusion condition. We then silenced the Drp1 protein expres- sion by this approach. We found that Drp1-siRNA decreased p-Drp1(Ser616) protein level in hippocampal CA1 subfield 24 h after TGI (Fig. 4a). Again as expected, total Drp1 revealed no significant change between sham and negative control (NC) siRNA + TGI group but showed decreased expression in Drp1-siRNA + TGI group (Fig. 4b). In parallel with the findings from Western blotting, under a laser-scanning confocal microscope, more p-Drp1(Ser616)-positive neurons, as revealed by its colocalization to the NeuN-positive cells, were observed in the hippocampal CA1 subfield at the same time point (Fig. 5b) as compared to the sham control animals (Fig. 5a). Pictures of higher magnification were shown in 5D. Microinjection of Drp1-siRNA into the hippocampus markedly reduced the numbers of p-Drp1(Ser616)- positive neurons induced by TGI under the same ex- perimental conditions (Fig. 5c, f). Fluorescent double immunostaining of p-Drp1(Ser616) and COXIV at higher magnification revealed co-localization of these two proteins in the mitochondria of hippocampal CA1 neurons at 24 h following TGI (Fig. 5e). Effect of Drp1 inhibitor over p-Drp1(Ser616) expression, oxidative stress and neuronal injury in the hippocampal CA1 subfield After TGI Pretreatment with Drp1-siRNA significantly retarded the extent of protein Drp1-siRNA silences Drp1 expression, attenuates oxidative stress and lessens neuronal Injury in the hippocampal CA1 subfield after TGI To further clarify the pivotal role of Drp1 in this ische- mic paradigm of the brain, we also employed molecular Fig. 3 Successful delivery of siRNA into hippocampal CA1 subfield. Fluorescent double staining of FITC-siRNA (green) and DAPI (blue) were observed in the hippocampal CA1 subfield 24 h after injection of 400 nl FITC-siRNA (10 μM), which was distributed in the cytosol of hippocampal CA1 in both sham-control (a) and in the rats subjected to TGI-reperfusion for 4 h (b). Scale bar: 10 μm. I/R: ischemia/reperfusion Fig. 3 Successful delivery of siRNA into hippocampal CA1 subfield. Fluorescent double staining of FITC-siRNA (green) and DAPI (blue) were observed in the hippocampal CA1 subfield 24 h after injection of 400 nl FITC-siRNA (10 μM), which was distributed in the cytosol of hippocampal CA1 in both sham-control (a) and in the rats subjected to TGI-reperfusion for 4 h (b). Scale bar: 10 μm. I/R: ischemia/reperfusion Page 7 of 14 Chuang et al. Journal of Biomedical Science (2016) 23:44 Fig. 4 Western blotting of p-Drp1(Ser616) and total Drp1 expression after Drp1-siRNA in the hippocampal CA1 subfield after TGI. After microinjection with Drp1-siRNA (0.05 nM in a total volume of 400 nl) into the CA1 subfield 24 h before TGI, total proteins were isolated from hippocampal CA1 subfield of sham-operated controls, control siRNA with TGI, or Drp1-siRNA animals after 10 min of TGI with 24 h reperfusion for detection of p-Drp1(Ser616) in (a) and total Drp1 in (b). The same blots were also probed with a α-tubulin antibody to serve as an internal control for equal loading of proteins in each lane. Values are mean ± SEM from representative blots and quantitative analysis from 4-6 animals in each experimental group. I/R: ischemia/reperfusion, NC: negative control siRNA in the CA1 subfield of rat hippocampus. These findings illustrated that inhibition of p-Drp1(Ser616) expression using Drp1-siRNA may decrease oxidative neuronal dam- age and DNA fragmentation in the hippocampal CA1 sub- field after TGI. Effect of PPARγ agonist on p-Drp1(Ser616) expression, oxidative stress and neuronal injury in the hippocampal CA1 subfield after TGI The same blots were also probed with a α-tubulin antibody to serve as an internal control for equal loading of proteins in each lane. Values are mean ± SEM from representative blots and quantitative analysis from 4-6 animals in each experimental group. I/R: ischemia/reperfusion, NC: negative control siRNA Effect of PPARγ agonist on p-Drp1(Ser616) expression, oxidative stress and neuronal injury in the hippocampal CA1 subfield after TGI Although PPARγ agonist including rosiglitazone or pio- glitazone can reduce inflammation and oxidative damage as well as diminishes cell death caused by ischemic injury [23–27], whether PPARγ-dependent pathway can reduce the expression of p-Drp1(Ser616) and ameliorate hippocampal injury induced by global ischemia is currently unknown. To address this issue, pioglitazone (20 nM) was microinjected into the CA1 subfield 30 min before TGI with or without prior microinjection of GW9662, a PPARγ antagonist, (500 ng) 30 min before pioglitazone. Western blotting revealed an increased p-Drp1(Ser616) protein level in hippocampal CA1 subfield 24 h after TGI, which was reduced by pio- glitazone pretreatment; moreover, GW9662 reversed the pioglitazone effect over p-Drp1(Ser616) protein expression (Fig. 7a). In parallel with the p-Drp1(Ser616) protein expression, pretreatment with pioglitazone de- creased TGI-induced protein oxidation and activated caspase-3 expression, whereas GW9662 partially reversed this beneficial effect of pioglitazone (Fig. 7b, c). Both quali- tative (Fig. 7d) and quantitative (Fig. 7e) studies of DNA fragmentation revealed that pioglitazone in part lessened TGI-induced DNA fragmentation, which was reversed by GW9662 pretreatment. Consistently, the extent of hippo- campal neuronal apoptosis based on TUNEL staining, re- vealed the same tendency after pioglitazone and GW9662 treatments (Fig. 7f). Fig. 4 Western blotting of p-Drp1(Ser616) and total Drp1 expression after Drp1-siRNA in the hippocampal CA1 subfield after TGI. After microinjection with Drp1-siRNA (0.05 nM in a total volume of 400 nl) into the CA1 subfield 24 h before TGI, total proteins were isolated from hippocampal CA1 subfield of sham-operated controls, control siRNA with TGI, or Drp1-siRNA animals after 10 min of TGI with 24 h reperfusion for detection of p-Drp1(Ser616) in (a) and total Drp1 in (b). The same blots were also probed with a α-tubulin antibody to serve as an internal control for equal loading of proteins in each lane. Values are mean ± SEM from representative blots and quantitative analysis from 4-6 animals in each experimental group. I/R: ischemia/reperfusion, NC: negative control siRNA Fig. 4 Western blotting of p-Drp1(Ser616) and total Drp1 expression after Drp1-siRNA in the hippocampal CA1 subfield after TGI. After microinjection with Drp1-siRNA (0.05 nM in a total volume of 400 nl) into the CA1 subfield 24 h before TGI, total proteins were isolated from hippocampal CA1 subfield of sham-operated controls, control siRNA with TGI, or Drp1-siRNA animals after 10 min of TGI with 24 h reperfusion for detection of p-Drp1(Ser616) in (a) and total Drp1 in (b). Discussion The results demonstrate that TGI increases p-Drp1(Ser616) expression, a phosphorylation site important for in- creasing mitochondrial fission, in the hippocampal CA1 subfield after TGI. In contrast, no significant change for total Drp1 expression under TGI. Both the Drp1 inhibitor Mdivi-1 and the siRNA targeting Drp1 decreased p-Drp1(Ser616) expression, lessened protein oxidation, and attenuated neuronal damage in the hippocampal CA1 subfield. These findings suggested the pivotal role of p-Drp1(Ser616) in TGI-induced neuronal injury. PPARγ agonist, pioglitazone, reduced the p-Drp1(Ser616) expression, decreased TGI-induced oxidative stress, lessened the extents of DNA fragmenta- tion, and diminished the numbers of TUNEL-positive neuronal cells; all of these effects were reversed by GW9662, a PPARγ antagonist. These findings indicated oxidation and activated caspase-3 expression in the hippo- campal CA1 subfield 24 h after TGI (Fig. 6a, b). Both qualitative (Fig. 6c) and quantitative (Fig. 6d) analyses re- vealed that downregulation of p-Drp1(Ser616) by siRNA significantly attenuated TGI-induced DNA fragmentation Page 8 of 14 Chuang et al. Journal of Biomedical Science (2016) 23:44 Fig. 5 (See legend on next page.) Chuang et al. Journal of Biomedical Science (2016) 23:44 Page 9 of 14 (See figure on previous page.) Fig. 5 Drp1-siRNA downregulates p-Drp1(Ser616) expression in the hippocampal CA1 subfield after TGI. Fluorescent double staining of p-Drp1 (green) and NeuN (red) in the hippocampal CA1 subfield in a sham control group, b ischemia/reperfusion 24 h with negative control siRNA and c siRNA for Drp1 and ischemia/reperfusion 24 h. NeuN showed the nuclear distribution while p-Drp1 were dispersed in the cytoplasm. Scale bars, 50 μm Merged images with higher magnification demonstrate that p-Drp1(Ser616) and NeuN-positive cells localized separately in the nucleus and non-nuclear cytoplasm in neurons in (d). Scale bars, 2 μm. A semi-quantitative data about the change of p-Drp1(Ser616) expression after Drp1-siRNA for Fig. 5 a-c was shown in (f). Fluorescent double staining of p-Drp1(Ser616) (green) and COXIV (blue) in the neuron of the hippocampal CA1 subfield; merged image shows the co-localization in mitochondria in neurons under the condition of ischemia/reperfusion for 24 h (e). Scale bars, 2 μm. I/R: ischemia/reperfusion, NC: negative control siRNA. COXIV: cytochrome c oxidase subunit 4 neuronal death in the hippocampal CA1 subfield [18, 19]. In this work, concomitantly with heightened protein oxi- dation, p-Drp1(Ser616) expression was increased (Fig. 2). It was known that Drp1 mediates mitochondrial fission [41, 42]. Discussion Drp1 activity results from phosphorylation by cyclin B/cyclin-dependent kinase (CDK), which causes phosphorylation of Serine 616 and promotes Drp1 recruit- ment to mitochondria for subsequent fission [41, 42]. The roles of calcium cascade in the cerebral ischemic paradigm in terms of necrosis and apoptosis are well established [43–45]. It was reported that calcium influx across the plasma membrane was an upstream event governing mitochondrial fission and ROS generation that can be re- versed by calcium chelation [46]. Increased intracellular calcium may cause Drp1 activation in cardiac ischemia [40]. The identification of mitochondrial calcium unipor- ter holds important clinical perspective, which allows the rapid calcium accumulation across the inner mitochon- drial membrane [47]. It was demonstrated that under ischemia/reperfusion injury, mitochondria accumulate sig- nificant amounts of calcium from the cytosol via mito- chondrial calcium uniporter and blocking mitochondrial calcium uniporter was demonstrated to exert protective effects against ischemia/reperfusion injury [48]. In a recent study, it revealed that mitochondrial calcium uniporter regulates the process of mitochondrial fission by control- ling the calcium transport, directly upregulating mito- chondrial fission proteins Drp1 [49]. All these evidence denote the importance of calcium and mitochondrial cal- cium uniporter in mitochondrial dynamics under ischemic condition. that PPARγ-dependent pathway can reduce the expression of p-Drp1(Ser616) and ameliorate hippocampal injury in- duced by global ischemia. Mitochondrial fission that occurs as an early event of neuronal cell death plays a pivotal role in cerebral ische- mia [7]. Drp1 is a large GTPase that cycles between the cytosol and mitochondrial outer membrane to function as a key contributor in the mitochondrial dynamic process when the cells encounter various stressful stim- uli, whereas Drp1-mediated mitochondrial fission and downstream mitochondrial death pathways are critically involved in the observed cell death [7, 36]. Phosphoryl- ation of Drp1 is crucial to regulating mitochondrial dy- namics [37]. Multiple phosphorylation sites have been characterized for their functional importance [34]. Drp1 phosphorylation at serine 616 can result in its activation and recruitment to mitochondria [35]. On the contrary, fission is inhibited when Drp1 is phosphorylated at Ser637 [34, 38]. The role of Drp1 in cerebral ischemia is just beginning to emerge [8, 9, 39]. Discussion It has been well dem- onstrated that knockdown of the fission protein Drp1 or with Drp1 inhibitors can block toxicity in a glutamate- induced oxidative stress model in HT22 cells and Drp1 inhibitors-Mdivi a or Mdivi b can reduce infarct volume in a mouse model of transient focal ischemia [8]. How- ever, the protective mechanism involving inhibition of Drp1 or Drp1 phosphorylation is still awaited to clarify with in vivo cerebral ischemia study. After focal cerebral ischemia, Drp1 phosphorylation is related to the apop- totic process in peri-infarct regions [39]. In our recent study, cerebral ischemia increased p-Drp1(Ser616) ex- pression with no evident change over total Drp1 and p-Drp1(Ser637) expression; further, down-regulation of PINK1 increased p-Drp1(Ser616) expression, heightened DNA oxidation, and augmented neuronal damage in the hippocampal CA1 subfield [21]. These studies denoted a pivotal role of p-Drp1 in cerebral ischemia and attenu- ation of p-Drp1 (Ser616) levels may exert neuroprotective effects. In a recent report, preventing dephosphorylation of Drp1(Ser637) with Mdivi-1 or Drp1-siRNA can pre- serve mitochondrial networking and ultrastructure follow- ing heart ischemia/reperfusion model [40]. It was suggested that mitochondrial oxidative stress modulates Drp1 expression and causes an imbalance be- tween mitochondrial fission and fusion, resulting in mitochondrial fragmentation and thus contributing ultimately to cellular dysfunction [50]. Treatment of an- tioxidants such as vitamin E or MitoQ can lessen mito- chondrial fragmentation and Drp1 expression [51, 52]. On the contrary, it was shown that knockdown of Drp1 expression suppressed production of mitochondrial ROS [53]. Both inhibition of Drp1 expression with antisense oligonucleotide and a dominant-negative mutant of Drp1 decrease oxidative stress [54, 55]. In this study, we showed that Mdivi-1 decreased p-Drp1(Ser616) We have reported before that TGI-induced ROS generation results in heightened protein oxidation and Page 10 of 14 Chuang et al. Journal of Biomedical Science (2016) 23:44 Fig. 6 Drp1 siRNA attenuates oxidative stress and decreases DNA fragmentation in hippocampal CA1 subfield after TGI. After microinjection with Drp1 siRNA (0.05 nM in a total volume of 400 nl) into the CA1 subfield 24 h before TGI, Total proteins were isolated from the hippocampal CA1 subfield of sham-operated controls, control siRNA with TGI, or Drp1 siRNA with TGI for protein oxidation in (a) and activated caspase-3 expression in (b). Discussion DNA was isolated from collected hippocampal CA1 subfield of sham-operated controls, vehicle with negative control siRNA, and Drp1- siRNA 48 h after TGI for detection of DNA fragmentation by PCR assay in (c) Hippocampal CA1 tissues were collected 48 h after TGI for detection of DNA fragmentation by sandwich ELISA in (d). Values are mean ± SEM from representative blots and quantitative analysis from 5–6 animals in each experimental group (a and b). Values are fold changes in (d) with reference to sham-control; mean ± SEM of 5–7 animals in each experimental group. P < 0.05 vs. sham-control group and #P < 0.05 vs. negative control siRNA + I/R in the Scheff′e multiple-range test. I/R: ischemia/reperfusion, NC: negative control siRNA Fig. 6 Drp1 siRNA attenuates oxidative stress and decreases DNA fragmentation in hippocampal CA1 subfield after TGI. After microinjection with Drp1 siRNA (0.05 nM in a total volume of 400 nl) into the CA1 subfield 24 h before TGI, Total proteins were isolated from the hippocampal CA1 subfield of sham-operated controls, control siRNA with TGI, or Drp1 siRNA with TGI for protein oxidation in (a) and activated caspase-3 expression in (b). DNA was isolated from collected hippocampal CA1 subfield of sham-operated controls, vehicle with negative control siRNA, and Drp1- siRNA 48 h after TGI for detection of DNA fragmentation by PCR assay in (c) Hippocampal CA1 tissues were collected 48 h after TGI for detection of DNA fragmentation by sandwich ELISA in (d). Values are mean ± SEM from representative blots and quantitative analysis from 5–6 animals in each experimental group (a and b). Values are fold changes in (d) with reference to sham-control; mean ± SEM of 5–7 animals in each experimental group. P < 0.05 vs. sham-control group and #P < 0.05 vs. negative control siRNA + I/R in the Scheff′e multiple-range test. I/R: ischemia/reperfusion, NC: negative control siRNA Fig. 6 Drp1 siRNA attenuates oxidative stress and decreases DNA fragmentation in hippocampal CA1 subfield after TGI. After microinjection with Drp1 siRNA (0.05 nM in a total volume of 400 nl) into the CA1 subfield 24 h before TGI, Total proteins were isolated from the hippocampal CA1 subfield of sham-operated controls, control siRNA with TGI, or Drp1 siRNA with TGI for protein oxidation in (a) and activated caspase-3 expression in (b). Discussion DNA was isolated from collected hippocampal CA1 subfield of sham-operated controls, vehicle with negative control siRNA, and Drp1- siRNA 48 h after TGI for detection of DNA fragmentation by PCR assay in (c) Hippocampal CA1 tissues were collected 48 h after TGI for detection of DNA fragmentation by sandwich ELISA in (d). Values are mean ± SEM from representative blots and quantitative analysis from 5–6 animals in each experimental group (a and b). Values are fold changes in (d) with reference to sham-control; mean ± SEM of 5–7 animals in each experimental group. P < 0.05 vs. sham-control group and #P < 0.05 vs. negative control siRNA + I/R in the Scheff′e multiple-range test. I/R: ischemia/reperfusion, NC: negative control siRNA permeabilization and effectively hinders Bid-activated Bax/Bak-dependent cytochrome c release from mito- chondria [57]. expression, lessened protein oxidation as well as acti- vated caspases 3, a marker of oxidative stress and apoptosis respectively (Fig. 2b, c). Mdivi-1 affects total Drp1 expression as well as phosphorylation level though the underlying mechanism is not well under- stood [33, 56]. Mdivi-1 attenuates mitochondrial div- ision by blocking dynamin GTPase activity, impedes apoptosis by inhibiting mitochondrial outer membrane We then used siRNA strategy to confirm the crucial role of Drp1 in this ischemic paradigm. Based on im- munofluorescence studies, we verified the successful de- livery of siRNA and reducing p-Drp1(Ser616) expression (Figs. 3, 4 and 5). In supporting the regulatory role of Page 11 of 14 Chuang et al. Journal of Biomedical Science (2016) 23:44 Fig. 7 Pioglitazone regulates Drp1 phosphorylation, protein oxidation, DNA fragmentation, and neuronal apoptosis in a PPARγ-dependant pathway after TGI. The chemical compounds microinjected into bilateral CA1 subfields as following with DMSO, pioglitazone (20 nmol) 30 min before TGI, or GW9663 (500 ng) 30 min before pioglitazone and 60 min before TGI. Total proteins were isolated from the hippocampal CA1 subfield of sham-operated controls or treated animals 24 h after 10 min of TGI for detection of p-Drp1 (Ser616) in (a) and protein oxidation in (b) and activated caspase-3 expression in (c). DNA was isolated from collected hippocampal CA1 subfield of sham-operated controls, DMSO + I/R, pioglitazone + I/R and GW9662 + pioglitazone 48 h after TGI for detection of DNA fragmentation by PCR assay (d) and hippocampal CA1 tissues were collected 48 h after TGI for detection of DNA fragmentation by sandwich ELISA in (e). Discussion Hippocampal slices were subjected to TUNEL staining to determine the extents of apoptosis in (f) which showed sham control in (a), ischemia/reperfusion with vehicle control in (b), pioglitazone with ischemia/reperfusion in (c) and GW9662 + pioglitazone and ischemia/reperfusion in (d). Values are mean ± SEM from representative blots and quantitative analyses from 5–6 animals in each experimental group (a, b and c); values in (e) are fold changes with reference to sham-control; mean ± SEM of 5-6 animals in each experimental group. P < 0.05 vs. sham-control group, #P < 0.05 vs. DMSO + I/R and + P < 0.05 versus Piog + I/R group in the Scheff′e multiple-range test. I/R: ischemia/reperfusion. Piog: pioglitazone n, DNA fragmentation, and neuronal apoptosis in a PPARγ-dependant pathway ubfields as following with DMSO, pioglitazone (20 nmol) 30 min before TGI, or e TGI. Total proteins were isolated from the hippocampal CA1 subfield of TGI for detection of p-Drp1 (Ser616) in (a) and protein oxidation in (b) collected hippocampal CA1 subfield of sham-operated controls, DMSO + I/R, etection of DNA fragmentation by PCR assay (d) and hippocampal CA1 tissues sandwich ELISA in (e). Hippocampal slices were subjected to TUNEL staining to control in (a), ischemia/reperfusion with vehicle control in (b), pioglitazone ischemia/reperfusion in (d). Values are mean ± SEM from representative mental group (a, b and c); values in (e) are fold changes with reference Fig. 7 Pioglitazone regulates Drp1 phosphorylation, protein oxidation, DNA fragmentation, and neuronal apoptosis in a PPARγ-dependant pathway after TGI. The chemical compounds microinjected into bilateral CA1 subfields as following with DMSO, pioglitazone (20 nmol) 30 min before TGI, or GW9663 (500 ng) 30 min before pioglitazone and 60 min before TGI. Total proteins were isolated from the hippocampal CA1 subfield of sham-operated controls or treated animals 24 h after 10 min of TGI for detection of p-Drp1 (Ser616) in (a) and protein oxidation in (b) and activated caspase-3 expression in (c). DNA was isolated from collected hippocampal CA1 subfield of sham-operated controls, DMSO + I/R, pioglitazone + I/R and GW9662 + pioglitazone 48 h after TGI for detection of DNA fragmentation by PCR assay (d) and hippocampal CA1 tissues were collected 48 h after TGI for detection of DNA fragmentation by sandwich ELISA in (e). Received: 7 January 2016 Accepted: 6 May 2016 Received: 7 January 2016 Accepted: 6 May 2016 Received: 7 January 2016 Accepted: 6 May 2016 Authors’ contributions YCC, TKL and SDC researched data, contributed to the discussion, and wrote the manuscript. SDC designed the study. DIY, JLY, CWL contributed to discussion, reviewed and edited the manuscript. All authors read and approved the final manuscript. Competing interests The authors declare that they have no competing interest. y In this study, we found that PPARγ agonist- pioglita- zone decreased p-Drp1(Ser616) expression, lessened oxi- dative stress as well as apoptotic process, and attenuated neuronal damage under TGI/reperfusion in the hippo- campal CA1 subfield, all of which were reversed by GW9662 (Fig. 7). PPARγ is a ligand-activated transcrip- tional factor that belongs to the nuclear hormone recep- tor superfamily and influences the expression of genes under various physiological or pathological conditions, including redox balance and vascular integrity [58]. We and others have shown before that thiazolidinediones drugs, including rosiglitazone and pioglitazone, possess characters of PPARγ agonist exerting neuroprotective effects in various models, which were abrogated by GW9662, a PPARγ antagonist [31, 59, 60]. Several po- tential mechanisms related to PPARγ and mitochondrial dynamic were reported before. Pioglitazone demon- strates the ability to restoring mitochondrial defects and mitochondrial networking [61]. Another PPARγ agonist, ciglitazone, can prevents mitochondrial size reduction in hippocampal neurons induced by H2O2 [62]. The poten- tial mechanism may relate to the property of PPARγ be- ing able to directly induce the expression of several mitochondria-related proteins including Drp1. PPARγ agonists have been linked to calcium balance [63, 64], which may affect Drp1 phosphorylation [65] and offer the ability of PPARγ to control mitochondrial dynamics modulation. Another common point for the mitochon- drial dynamics modulation among these PPARγ agonist is the capability of activating PPARγ coactivator 1-α [66, 67], an important molecule that can counteract excessive oxidative stress and apoptosis in cerebral is- chemia [68]. Thus, pioglitazone has the potential to reduce neuronal injury after cerebral ischemia through mitochondrial dynamic-related proteins and PPARγ- dependent pathways. References Nii 1. Niizuma K, Endo H, Chan PH. Oxidative stress and mitochondrial dysfunction as determinants of ischemic neuronal death and survival. J Neurochem. 2009;109 Suppl 1:133–8. 2. Suen DF, Norris KL, Youle RJ. Mitochondrial dynamics and apoptosis. Genes Dev. 2008;22(12):1577–90. 3. Herzig S, Martinou JC. Mitochondrial dynamics: to be in good shape to survive. Curr Mol Med. 2008;8(2):131–7. 4. Chan DC. Mitochondrial fusion and fission in mammals. Annu Rev Cell Dev Biol. 2006;22:79–99. 5. Okamoto K, Shaw JM. Mitochondrial morphology and dynamics in yeast and multicellular eukaryotes. Annu Rev Genet. 2005;39:503–36. 6. Westermann B. Molecular machinery of mitochondrial fusion and fission. J Biol Chem. 2008;283(20):13501–5. 7. Barsoum MJ, Yuan H, Gerencser AA, Liot G, Kushnareva Y, Graber S, et al. Nitric oxide-induced mitochondrial fission is regulated by dynamin-related GTPases in neurons. EMBO J. 2006;25(16):3900–11. 8. Grohm J, Kim SW, Mamrak U, Tobaben S, Cassidy-Stone A, Nunnari J, et al. Inhibition of Drp1 provides neuroprotection in vitro and in vivo. Cell Death Differ. 2012;19(9):1446–58. 9. Zhao YX, Cui M, Chen SF, Dong Q, Liu XY. Amelioration of ischemic mitochondrial injury and bax-dependent outer membrane permeabilization by mdivi-1. CNS Neurosci Ther. 2014;20(6):528–38. 10. Frank S, Gaume B, Bergmann-Leitner ES, Leitner WW, Robert EG, Catez F, et al. The role of dynamin-related protein 1, a mediator of mitochondrial fission, in apoptosis. Dev Cell. 2001;1(4):515–25. 11. Karbowski M, Lee YJ, Gaume B, Jeong SY, Frank S, Nechushtan A, et al. Spatial and temporal association of Bax with mitochondrial fission sites, Drp1, and Mfn2 during apoptosis. J Cell Biol. 2002;159(6):931–8. 12. Pulsinelli WA, Brierley JB, Plum F. Temporal profile of neuronal damage in a model of transient forebrain ischemia. Ann Neurol. 1982;11(5):491–8. 1. Niizuma K, Endo H, Chan PH. Oxidative stress and mitochondrial dysfunction as determinants of ischemic neuronal death and survival. J Neurochem. 2009;109 Suppl 1:133–8. 2. Suen DF, Norris KL, Youle RJ. Mitochondrial dynamics and apoptosis. Genes Dev. 2008;22(12):1577–90. 3. Herzig S, Martinou JC. Mitochondrial dynamics: to be in good shape to survive. Curr Mol Med. 2008;8(2):131–7. 4. Chan DC. Mitochondrial fusion and fission in mammals. Annu Rev Cell Dev Biol. 2006;22:79–99. 5. Okamoto K, Shaw JM. Mitochondrial morphology and dynamics in yeast and multicellular eukaryotes. Annu Rev Genet. 2005;39:503–36. 6. Westermann B. Molecular machinery of mitochondrial fusion and fission. J Biol Chem. 2008;283(20):13501–5. 7. Barsoum MJ, Yuan H, Gerencser AA, Liot G, Kushnareva Y, Graber S, et al. Discussion Journal of Biomedical Science (2016) 23:44 Drp1 over oxidative stress in ischemic condition, Drp1- siRNA treatment decreased p-Drp1(Ser616) expression (Figs. 4 and 5), which was accompanied by attenuated protein oxidation, lessened apoptotic process and de- creased neuronal damage (Fig. 6). Based on these results, Drp1-dependent enhancement of ROS generation is a more favorable assumption than oxidative stress- mediated induction of Drp1, at least in our TGI para- digm. This observation may be vital for therapeutic purposes for ischemic stroke by impeding the activa- tion of p-Drp1(Ser616) expression rather than inhib- ition of oxidative stress only. hippocampal CA1 subfield. PPARγ-dependent pathway can decrease the expression of p-Drp1(Ser616) and protect hippocampal CA1 injury induced by global ischemia. Abbreviations ANOVA: one-way analysis of variance; CDK: cyclin-dependent kinase; COXIV: cytochrome c oxidase subunit 4; Drp1: dynamin-related protein 1; FITC: fluorescein isothiocyanate; p-Drp1(Ser616): phosphorylation at Drp1serine 616; PPARγ: peroxisome proliferator-activated receptor-gamma; TGI: transient global ischemia; TUNEL: terminal deoxynucleotidyl transferase- mediated dUTP-biotin nick end labeling. Acknowledgments This work was supported by research grants NSC 99-2314-B-182A-069- and 101-2314-B-182-081-MY2 from the National Science Council, Taiwan and BMRP555 from Chang Gung Medical Foundation, Taiwan to S.D. Chen. References Nii Nitric oxide-induced mitochondrial fission is regulated by dynamin-related GTPases in neurons. EMBO J. 2006;25(16):3900–11. 8. Grohm J, Kim SW, Mamrak U, Tobaben S, Cassidy-Stone A, Nunnari J, et al. Inhibition of Drp1 provides neuroprotection in vitro and in vivo. Cell Death Differ. 2012;19(9):1446–58. 9. Zhao YX, Cui M, Chen SF, Dong Q, Liu XY. Amelioration of ischemic mitochondrial injury and bax-dependent outer membrane permeabilization by mdivi-1. CNS Neurosci Ther. 2014;20(6):528–38. Author details 1 1Department of Neurology, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Kaohsiung, Taiwan. 2Center for Translational Research in Biomedical Sciences, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan. 3Department of Neurology, Faculty of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan. 4Institute of Brain Science, National Yang-Ming University, Taipei, Taiwan. Discussion Hippocampal slices were subjected to TUNEL staining to determine the extents of apoptosis in (f) which showed sham control in (a), ischemia/reperfusion with vehicle control in (b), pioglitazone with ischemia/reperfusion in (c) and GW9662 + pioglitazone and ischemia/reperfusion in (d). Values are mean ± SEM from representative blots and quantitative analyses from 5–6 animals in each experimental group (a, b and c); values in (e) are fold changes with reference to sham-control; mean ± SEM of 5-6 animals in each experimental group. P < 0.05 vs. sham-control group, #P < 0.05 vs. DMSO + I/R and + P < 0.05 versus Piog + I/R group in the Scheff′e multiple-range test. I/R: ischemia/reperfusion. Piog: pioglitazone Fig. 7 Pioglitazone regulates Drp1 phosphorylation, protein oxidation, DNA fragmentation, and neuronal apoptosis in a PPARγ-dependant pathway after TGI. The chemical compounds microinjected into bilateral CA1 subfields as following with DMSO, pioglitazone (20 nmol) 30 min before TGI, or GW9663 (500 ng) 30 min before pioglitazone and 60 min before TGI. Total proteins were isolated from the hippocampal CA1 subfield of sham-operated controls or treated animals 24 h after 10 min of TGI for detection of p-Drp1 (Ser616) in (a) and protein oxidation in (b) and activated caspase-3 expression in (c). DNA was isolated from collected hippocampal CA1 subfield of sham-operated controls, DMSO + I/R, pioglitazone + I/R and GW9662 + pioglitazone 48 h after TGI for detection of DNA fragmentation by PCR assay (d) and hippocampal CA1 tissues were collected 48 h after TGI for detection of DNA fragmentation by sandwich ELISA in (e). Hippocampal slices were subjected to TUNEL staining to determine the extents of apoptosis in (f) which showed sham control in (a), ischemia/reperfusion with vehicle control in (b), pioglitazone with ischemia/reperfusion in (c) and GW9662 + pioglitazone and ischemia/reperfusion in (d). Values are mean ± SEM from representative blots and quantitative analyses from 5–6 animals in each experimental group (a, b and c); values in (e) are fold changes with reference to sham-control; mean ± SEM of 5-6 animals in each experimental group. *P < 0.05 vs. sham-control group, #P < 0.05 vs. DMSO + I/R and + P < 0.05 versus Piog + I/R group in the Scheff′e multiple-range test. I/R: ischemia/reperfusion. Piog: pioglitazone Page 12 of 14 Chuang et al. Journal of Biomedical Science (2016) 23:44 Chuang et al. Journal of Biomedical Science (2016) 23:44 Chuang et al. 9. Zhao YX, Cui M, Chen SF, Dong Q, Liu XY. Amelioration of ischemic mitochondrial injury and bax-dependent outer membrane permeabilization by mdivi-1. CNS Neurosci Ther. 2014;20(6):528–38. 10. Frank S, Gaume B, Bergmann-Leitner ES, Leitner WW, Robert EG, Catez F, et al. The role of dynamin-related protein 1, a mediator of mitochondrial fission, in apoptosis. Dev Cell. 2001;1(4):515–25. Conclusion Cell signaling and mitochondrial dynamics: Implications for neuronal function and neurodegenerative disease. Neurobiol Dis. 2013;51:13–26. 18. Chen SD, Lin TK, Lin JW, Yang DI, Lee SY, Shaw FZ, et al. Activation of calcium/calmodulin-dependent protein kinase IV and peroxisome proliferator-activated receptor gamma coactivator-1alpha signaling pathway protects against neuronal injury and promotes mitochondrial biogenesis in the hippocampal CA1 subfield after transient global ischemia. J Neurosci Res. 2010;88(14):3144–54. 38. Giedt RJ, Yang C, Zweier JL, Matzavinos A, Alevriadou BR. Mitochondrial fission in endothelial cells after simulated ischemia/reperfusion: role of nitric oxide and reactive oxygen species. Free Radic Biol Med. 2012;52(2):348–56. 39. Liu W, Tian F, Kurata T, Morimoto N, Abe K. Dynamic changes of mitochondrial fission proteins after transient cerebral ischemia in mice. Brain Res. 2012;1456:94–9. 19. Chen SD, Lin TK, Yang DI, Lee SY, Shaw FZ, Liou CW, et al. Protective effects of peroxisome proliferator-activated receptors gamma coactivator-1alpha against neuronal cell death in the hippocampal CA1 subfield after transient global ischemia. J Neurosci Res. 2010;88(3):605–13. 40. Sharp WW, Fang YH, Han M, Zhang HJ, Hong Z, Banathy A, et al. Dynamin- related protein 1 (Drp1)-mediated diastolic dysfunction in myocardial ischemia-reperfusion injury: therapeutic benefits of Drp1 inhibition to reduce mitochondrial fission. FASEB J. 2014;28(1):316–26. 20. Yin W, Signore AP, Iwai M, Cao G, Gao Y, Chen J. Rapidly increased neuronal mitochondrial biogenesis after hypoxic-ischemic brain injury. Stroke. 2008;39(11):3057–63. 41. Westermann B. Mitochondrial fusion and fission in cell life and death. Nat Rev Mol Cell Biol. 2010;11(12):872–84. 21. Chen SD, Lin TK, Yang DI, Lee SY, Shaw FZ, Liou CW, et al. Roles of PTEN- induced putative kinase 1 and dynamin-related protein 1 in transient global ischemia-induced hippocampal neuronal injury. Biochem Biophys Res Commun. 2015;460(2):397–403. 42. Kashatus DF, Lim KH, Brady DC, Pershing NL, Cox AD, Counter CM. RALA and RALBP1 regulate mitochondrial fission at mitosis. Nat Cell Biol. 2011;13(9):1108–15. 22. Patzer A, Zhao Y, Stock I, Gohlke P, Herdegen T, Culman J. Peroxisome proliferator-activated receptorsgamma (PPARgamma) differently modulate the interleukin-6 expression in the peri-infarct cortical tissue in the acute and delayed phases of cerebral ischaemia. Eur J Neurosci. 2008;28(9):1786–94. 43. Chen SD, Lee JM, Yang DI, Nassief A, Hsu CY. Combination therapy for ischemic stroke: potential of neuroprotectants plus thrombolytics. Am J Cardiovasc Drugs. 2002;2(5):303–13. 44. Hofmeijer J, van Putten MJ. Ischemic cerebral damage: an appraisal of synaptic failure. Stroke. 2012;43(2):607–15. 23. Conclusion In summary, the present study demonstrated that inhib- ition of TGI-induced p-Drp1(Ser616) expression by Drp1 inhibitor and Drp1-siRNA can decrease protein oxidation, apoptotic process and neuronal damage in the 11. Karbowski M, Lee YJ, Gaume B, Jeong SY, Frank S, Nechushtan A, et al. Spatial and temporal association of Bax with mitochondrial fission sites, Drp1, and Mfn2 during apoptosis. J Cell Biol. 2002;159(6):931–8. 12. Pulsinelli WA, Brierley JB, Plum F. Temporal profile of neuronal damage in a model of transient forebrain ischemia. Ann Neurol. 1982;11(5):491–8. Page 13 of 14 Page 13 of 14 Page 13 of 14 Chuang et al. Journal of Biomedical Science (2016) 23:44 13. Smith ML, Auer RN, Siesjo BK. The density and distribution of ischemic brain injury in the rat following 2-10 min of forebrain ischemia. Acta Neuropathol. 1984;64(4):319–32. 32. Chuang YC, Yang JL, Yang DI, Lin TK, Liou CW, Chen SD. Roles of sestrin2 and ribosomal protein s6 in transient global ischemia-induced hippocampal neuronal injury. Int J Mol Sci. 2015;16(11):26406–16. 14. Petito CK, Feldmann E, Pulsinelli WA, Plum F. Delayed hippocampal damage in humans following cardiorespiratory arrest. Neurology. 1987;37(8):1281–6. 33. Taguchi N, Ishihara N, Jofuku A, Oka T, Mihara K. Mitotic phosphorylation of dynamin-related GTPase Drp1 participates in mitochondrial fission. J Biol Chem. 2007;282(15):11521–9. 15. Cipolotti L, Bird CM. Amnesia and the hippocampus. Curr Opin Neurol. 2006;19(6):593–8. 34. Knott AB, Perkins G, Schwarzenbacher R, Bossy-Wetzel E. Mitochondrial fragmentation in neurodegeneration. Nat Rev Neurosci. 2008;9(7):505–18. 35. Bossy B, Petrilli A, Klinglmayr E, Chen J, Lutz-Meindl U, Knott AB, et al. S-Nitrosylation of DRP1 does not affect enzymatic activity and is not specific to Alzheimer's disease. J Alzheimers Dis. 2010;20 Suppl 2:S513–26. 16. Endo H, Kamada H, Nito C, Nishi T, Chan PH. Mitochondrial translocation of p53 mediates release of cytochrome c and hippocampal CA1 neuronal death after transient global cerebral ischemia in rats. J Neurosci. 2006;26(30):7974–83. 36. Yuan H, Gerencser AA, Liot G, Lipton SA, Ellisman M, Perkins GA, et al. Mitochondrial fission is an upstream and required event for bax foci formation in response to nitric oxide in cortical neurons. Cell Death Differ. 2007;14(3):462–71. 17. Miyawaki T, Mashiko T, Ofengeim D, Flannery RJ, Noh KM, Fujisawa S, et al. Ischemic preconditioning blocks BAD translocation, Bcl-xL cleavage, and large channel activity in mitochondria of postischemic hippocampal neurons. Proc Natl Acad Sci U S A. 2008;105(12):4892–7. 37. Wilson TJ, Slupe AM, Strack S. Conclusion Gamboa J, Blankenship DA, Niemi JP, Landreth GE, Karl M, Hilow E, et al. Extension of the neuroprotective time window for thiazolidinediones in ischemic stroke is dependent on time of reperfusion. Neuroscience. 2010;170(3):846–57. 45. Pundik S, Xu K, Sundararajan S. Reperfusion brain injury: focus on cellular bioenergetics. Neurology. 2012;79(13 Suppl 1):S44–51. 46. Yu T, Jhun BS, Yoon Y. High-glucose stimulation increases reactive oxygen species production through the calcium and mitogen-activated protein kinase-mediated activation of mitochondrial fission. Antioxid Redox Signal. 2011;14(3):425–37. 24. Shimazu T, Inoue I, Araki N, Asano Y, Sawada M, Furuya D, et al. A peroxisome proliferator-activated receptor-gamma agonist reduces infarct size in transient but not in permanent ischemia. Stroke. 2005;36(2):353–9. 25. Zhao Y, Patzer A, Herdegen T, Gohlke P, Culman J. Activation of cerebral peroxisome proliferator-activated receptors gamma promotes neuroprotection by attenuation of neuronal cyclooxygenase-2 overexpression after focal cerebral schemia in rats. FASEB J. 2006;20(8):1162–75. 47. Patron M, Raffaello A, Granatiero V, Tosatto A, Merli G, De Stefani D, et al. The mitochondrial calcium uniporter (MCU): molecular identity and physiological roles. J Biol Chem. 2013;288(15):10750–8. 48. Zhao Q, Wang S, Li Y, Wang P, Li S, Guo Y, et al. The role of the mitochondrial calcium uniporter in cerebral ischemia/reperfusion injury in rats involves regulation of mitochondrial energy metabolism. Mol Med Rep. 2013;7(4):1073–80. 26. Chen SD, Wu HY, Yang DI, Lee SY, Shaw FZ, Lin TK, et al. Effects of rosiglitazone on global ischemia-induced hippocampal injury and expression of mitochondrial uncoupling protein 2. Biochem Biophys Res Commun. 2006;351(1):198–203. 49. Zhao L, Li S, Wang S, Yu N, Liu J. The effect of mitochondrial calcium uniporter on mitochondrial fission in hippocampus cells ischemia/ reperfusion injury. Biochem Biophys Res Commun. 2015;461(3):537–42. 27. Wu JS, Cheung WM, Tsai YS, Chen YT, Fong WH, Tsai HD, et al. Ligand- activated peroxisome proliferator-activated receptor-gamma protects against ischemic cerebral infarction and neuronal apoptosis by 14-3-3 epsilon upregulation. Circulation. 2009;119(8):1124–34. 50. Wu S, Zhou F, Zhang Z, Xing D. Mitochondrial oxidative stress causes mitochondrial fragmentation via differential modulation of mitochondrial fission-fusion proteins. FEBS J. 2011;278(6):941–54. 28. Miglio G, Rosa AC, Rattazzi L, Collino M, Lombardi G, Fantozzi R. PPARgamma stimulation promotes mitochondrial biogenesis and prevents glucose deprivation-induced neuronal cell loss. Neurochem Int. 2009;55(7):496–504. 51. de Arriba G, Calvino M, Benito S, Parra T. Cyclosporine A-induced apoptosis in renal tubular cells is related to oxidative damage and mitochondrial fission. Toxicol Lett. 2013;218(1):30–8. 29. Conclusion Sugawara T, Kawase M, Lewen A, Noshita N, Gasche Y, Fujimura M, et al. Effect of hypotension severity on hippocampal CA1 neurons in a rat global ischemia model. Brain Res. 2000;877(2):281–7. 52. Solesio ME, Prime TA, Logan A, Murphy MP, Del Mar A-JM, Jordan J, et al. The mitochondria-targeted anti-oxidant MitoQ reduces aspects of mitochondrial fission in the 6-OHDA cell model of Parkinson's disease. Biochim Biophys Acta. 2013;1832(1):174–82. 30. Zhang N, Wang S, Li Y, Che L, Zhao Q. A selective inhibitor of Drp1, mdivi-1, acts against cerebral ischemia/reperfusion injury via an anti-apoptotic pathway in rats. Neurosci Lett. 2013;535:104–9. 53. Kobashigawa S, Suzuki K, Yamashita S. Ionizing radiation accelerates Drp1- dependent mitochondrial fission, which involves delayed mitochondrial reactive oxygen species production in normal human fibroblast-like cells. Biochem Biophys Res Commun. 2011;414(4):795–800. 31. Chuang YC, Lin TK, Huang HY, Chang WN, Liou CW, Chen SD, et al. Peroxisome proliferator-activated receptors gamma/mitochondrial uncoupling protein 2 signaling protects against seizure-induced neuronal cell death in the hippocampus following experimental status epilepticus. J Neuroinflammation. 2012;9:184. 54. Ferrari LF, Chum A, Bogen O, Reichling DB, Levine JD. Role of Drp1, a key mitochondrial fission protein, in neuropathic pain. J Neurosci. 2011;31(31):11404–10. Page 14 of 14 55. Peng L, Men X, Zhang W, Wang H, Xu S, Xu M, et al. Dynamin-related protein 1 is implicated in endoplasmic reticulum stress-induced pancreatic beta-cell apoptosis. Int J Mol Med. 2011;28(2):161–9. 56. Mizumura K, Cloonan SM, Nakahira K, Bhashyam AR, Cervo M, Kitada T, et al. Mitophagy-dependent necroptosis contributes to the pathogenesis of COPD. J Clin Invest. 2014;124(9):3987–4003. 57. Cassidy-Stone A, Chipuk JE, Ingerman E, Song C, Yoo C, Kuwana T, et al. Chemical inhibition of the mitochondrial division dynamin reveals its role in Bax/Bak-dependent mitochondrial outer membrane permeabilization. Dev Cell. 2008;14(2):193–204. 58. Chen YC, Wu JS, Tsai HD, Huang CY, Chen JJ, Sun GY, et al. Peroxisome proliferator-activated receptor gamma (PPAR-gamma) and neurodegenerative disorders. Mol Neurobiol. 2012;46(1):114–24. 59. Wu JS, Lin TN, Wu KK. Rosiglitazone and PPAR-gamma overexpression protect mitochondrial membrane potential and prevent apoptosis by upregulating anti-apoptotic Bcl-2 family proteins. J Cell Physiol. 2009;220(1):58–71. 60. Stopponi S, Somaini L, Cippitelli A, Cannella N, Braconi S, Kallupi M, et al. Activation of nuclear PPARgamma receptors by the antidiabetic agent pioglitazone suppresses alcohol drinking and relapse to alcohol seeking. Biol Psychiatry. 2011;69(7):642–9. 61. Ding Y, Fang H, Shang W, Xiao Y, Sun T, Hou N, et al. Chuang et al. Journal of Biomedical Science (2016) 23:44 Conclusion Mitoflash altered by metabolic stress in insulin-resistant skeletal muscle. J Mol Med. 2015;93(10):1119–30. 62. Zolezzi JM, Silva-Alvarez C, Ordenes D, Godoy JA, Carvajal FJ, Santos MJ, et al. Peroxisome proliferator-activated receptor (PPAR) gamma and PPARalpha agonists modulate mitochondrial fusion-fission dynamics: relevance to reactive oxygen species (ROS)-related neurodegenerative disorders? PLoS One. 2013;8(5), e64019. 63. Khasabova IA, Xiong Y, Coicou LG, Piomelli D, Seybold V. Peroxisome proliferator-activated receptor alpha mediates acute effects of palmitoylethanolamide on sensory neurons. J Neurosci. 2012;32(37):12735–43. 64. Pancani T, Phelps JT, Searcy JL, Kilgore MW, Chen KC, Porter NM, et al. Distinct modulation of voltage-gated and ligand-gated Ca2+ currents by PPAR-gamma agonists in cultured hippocampal neurons. J Neurochem. 2009;109(6):1800–11. 65. Han XJ, Lu YF, Li SA, Kaitsuka T, Sato Y, Tomizawa K, et al. CaM kinase I alpha-induced phosphorylation of Drp1 regulates mitochondrial morphology. J Cell Biol. 2008;182(3):573–85. 66. Haemmerle G, Moustafa T, Woelkart G, Buttner S, Schmidt A, van de Weijer T, et al. ATGL-mediated fat catabolism regulates cardiac mitochondrial function via PPAR-alpha and PGC-1. Nat Med. 2011;17(9):1076–85. 67. Hondares E, Rosell M, Diaz-Delfin J, Olmos Y, Monsalve M, Iglesias R, et al. Peroxisome proliferator-activated receptor alpha (PPARalpha) induces PPARgamma coactivator 1alpha (PGC-1alpha) gene expression and contributes to thermogenic activation of brown fat: involvement of PRDM16. J Biol Chem. 2011;286(50):43112–22. 68. Chen SD, Yang DI, Lin TK, Shaw FZ, Liou CW, Chuang YC. Roles of oxidative stress, apoptosis, PGC-1alpha and mitochondrial biogenesis in cerebral ischemia. Int J Mol Sci. 2011;12(10):7199–215. 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 y p and we will help you at every step: • We accept pre-submission inquiries
https://openalex.org/W4301186485	https://www.degruyter.com/document/doi/10.1515/labmed-2022-0125/pdf	Latin	null	German Congress of Laboratory Medicine: 17th Annual Congress of the DGKL and 4th Symposium of the Biomedical Analytics of the DVTA e.V.	Journal of laboratory medicine	2,022	cc-by	51,986	Congress Abstracts German Congress of Laboratory Medicine: 17th Annual Congress of the DGKL and 4th Symposium of the Biomedical Analytics of the DVTA e.V. Mannheim, Germany, October 13–14, 2022 Under the auspices of International Federation of Clinical Chemistry and Laboratory Medicine European Federation of Clinical Chemistry and Laboratory Medicine Union Européenne des Médecins Spécialistes Congress Presidents Matthias Nauck (Greifswald, Germany) Christiane Maschek (Hannover, Germany) Sponsor of Abstract awards Dr. Neumann & Kindler Ltd. & Co. KG (Bochum, Germany) Scientiﬁc Committee Adler Jakob, Ahmad-Nejad Parviz, Bauer Matthias, Baum Hannsjörg, Bertsch Thomas, Biemann Ronald, Bietenbeck Andreas, Birschmann Ingvild, Brand Korbinian, Burkhardt Ralph, Chavakis Triantafyllos, Danckwardt Sven, Fiedler Georg Martin, Findeisen Peter, Fischer Andreas, Fraunberger Peter, Frey Oliver, Geisel Jürgen, Haferlach Thorsten, Holdenrieder Stefan, Holdt Lesca Miriam, Isermann Berend, Junker Ralf, Kessler Harald, Klawonn Frank, Klein Hans-Georg, Knabbe Cornelius, Kratzsch Jürgen, Lackner Karl, Ludwig- Kraus Beatrice, Luppa Peter B., Lämmle Bernhard, Maschek Christiane, Nauck Matthias, Neumaier Michael, Orth Matthias, Peter Andreas, Petersmann Astrid, Rauh Manfred, Renné Thomas, Rentsch-Savoca Katharina, Renz Harald, Ruland Jürgen, Sack Ulrich, Seger Christoph, Shipkova Maria, Spannagl Michael, Steimer Werner, Tiebel Oliver, Tolios Alexander, Vogeser Michael, von Eckardstein Arnold, von Meyer Alexander, Walter Michael, Wieland Eberhard https://doi.org/10.1515/labmed-2022-0125 — J Lab Med 2022; 46(5): eA1–eA87 https://doi.org/10.1515/labmed-2022-0125 — J Lab Med 2022; 46(5): eA1–eA87 Congress Abstracts These abstracts have been reproduced directly from the material supplied by the authors, without editorial alteration by the staff of this Journal. Insufficiencies of preparation, grammar, spelling, style, syntax, and usage are the authors. e author(s), published by De Gruyter. This work is licensed under the Creative Commons Attribution 4.0 International German Congress of Laboratory Medicine: 17th Annual Congress of the DGKL and 4th Symposium of the Biomedical Analytics of the DVTA e.V. Mannheim Germany October 13 14 2022 Mannheim, Germany, October 13–14, 2022 Serum ghrelin is positively associated with physiological anxiety but negatively associated with pathological anxiety in humans: Data from a large community-based study Dirk Wittekind Universitätsklinikum Leipzig, Institut für Laboratoriumsmedizin, Klinische Chemie und Molekulare Diagnostik, Leipzig, G Universitätsklinikum Leipzig, Institut für Laboratoriumsmedizin, Klinische Chemie und Molekulare Diagnostik, Leipzig, Germany The orexigenic hormone ghrelin is being increasingly recognized as a stress hormone being involved in anxiety regulation. In animals, ghrelin effects on, and responses to acute stress differed from those in chronic stress, an animal model for anxiety and depression. In humans, elevated ghrelin levels were reported in pathological anxiety (e.g. panic disorder). However, no reports exist on physiological anxiety in mentally healthy subjects. In addition, reports on generalized anxiety symptoms, both in mentally healthy subjects (e.g. worrying) or in adult patients, are lacking. Total serum ghrelin was determined in 1666 subjects of a population-based cross-sectional study (‘LIFE’). The 7-item Generalized Anxiety Disorder Scale (GAD-7), detecting also other anxiety disorders, was administered. For multiple linear regression analyses, 1091 subjects were finally included. Serum ghrelin and GAD-7 scores were positively but not significantly associated in the total group (ß=0.00025, standardized β = 0.039, 95% CI: −0.00006; 0.0006; p = 0.144), in subjects with no more than mild anxiety, there was a sig-niﬁcant positive association (GAD-7 ≤9: n = 1061, 97.25%, β = 0.00032; standardized β = 0.060; 95% CI: 0.000023; 0.00062; p = 0.036). In contrast, there was a negative association in subjects with anxiety symptoms above the GAD-7 cut-off (GAD-7 ≥10: n = 30, 2.75%, ß=−0.003, standardized β = −0.462; 95% CI: −0.006; 0.0001; p = 0.045). Ghrelin levels were only numerically (p = 0.23) higher in subjects with clinically relevant anxiety symptoms (963.5 ± 399.6 pg/ml; mean±SD) than in those without (901.0 ± 416.4 pg/ml). In conclusion, the positive association between ghrelin and no more than mild anxiety is an initial indication for a role for ghrelin in the regulation of physiological anxiety in humans. This association and the opposed associ- ation in pathological anxiety resemble ﬁndings in animals showing diverging ghrelin effects in acute and chronic stress. Scientiﬁc Committee Adler Jakob, Ahmad-Nejad Parviz, Bauer Matthias, Baum Hannsjörg, Bertsch Thomas, Biemann Ronald, Bietenbeck Andreas, Birschmann Ingvild, Brand Korbinian, Burkhardt Ralph, Chavakis Triantafyllos, Danckwardt Sven, Fiedler Georg Martin, Findeisen Peter, Fischer Andreas, Fraunberger Peter, Frey Oliver, Geisel Jürgen, Haferlach Thorsten, Holdenrieder Stefan, Holdt Lesca Miriam, Isermann Berend, Junker Ralf, Kessler Harald, Klawonn Frank, Klein Hans-Georg, Knabbe Cornelius, Kratzsch Jürgen, Lackner Karl, Ludwig- Kraus Beatrice, Luppa Peter B., Lämmle Bernhard, Maschek Christiane, Nauck Matthias, Neumaier Michael, Orth Matthias, Peter Andreas, Petersmann Astrid, Rauh Manfred, Renné Thomas, Rentsch-Savoca Katharina, Renz Harald, Ruland Jürgen, Sack Ulrich, Seger Christoph, Shipkova Maria, Spannagl Michael, Steimer Werner, Tiebel Oliver, Tolios Alexander, Vogeser Michael, von Eckardstein Arnold, von Meyer Alexander, Walter Michael, Wieland Eberhard Open Access. © 2022 the author(s), published by De Gruyter. This work is licensed under the Creative Commons Attribution 4.0 International License. German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 eA2 Association between self-rating depression scores and total ghrelin and adipokine serum levels in a large population-based sample Dirk Wittekind Dirk Wittekind Dirk Wittekind Universitätsklinikum Leipzig, Institut für Laboratoriumsmedizin, Klinische Chemie und Molekulare Diagnostik, Leipzig, Germany Background: Ghrelin and the adipokines leptin and adiponectin have been suggested to be involved in mood and anxiety regulation and to be altered in affective disorders. However, studies investigating the association between ghrelin, leptin and adiponectin and depressive symptomatology are scarce but might contribute to a better under- standing of their involvement in mood regulation. We thus aimed investigating the association between depressive symptomatology and total ghrelin as well as leptin and adiponectin serum levels in a large population-based sample. Methods: Total serum ghrelin, adiponectin and leptin levels were determined in 1666 subjects of a population-based cross-sectional study (‘LIFE’). The Center for Epidemiological Studies Depression Scale (CES-D) and the Inventory of Depressive Symptoms – Self Rating (IDS-SR) were administered. Multiple linear regression analyses were conducted to examine the association between total serum ghrelin, leptin and adiponectin and the intensity of depressive symptoms. German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 eA3 Results: In the total sample (n=1092), neither ghrelin nor leptin or adiponectin serum levels showed a signiﬁcant association with CES-D or IDS-SR sum scores in the total sample (N=1092) or in depressed/non-depressed subjects. Leptin serum levels showed a signiﬁcantly positive association with IDS-SR sum scores in elderly men (≥60 years; β = 0.122, 95% CI: 0.009; 0.236; p = 0.035). Results: In the total sample (n=1092), neither ghrelin nor leptin or adiponectin serum levels showed a signiﬁcant association with CES-D or IDS-SR sum scores in the total sample (N=1092) or in depressed/non-depressed subjects. Leptin serum levels showed a signiﬁcantly positive association with IDS-SR sum scores in elderly men (≥60 years; β = 0.122, 95% CI: 0.009; 0.236; p = 0.035). Conclusion: Our study suggests that peripheral levels of ghrelin and adipokines in a cross-sectional study design might not be sufﬁcient to measure their involvement in depression, suggesting that associations are more complex and multi- layered. Differences in biomarker profiles between heart failure patients with preserved versus reduced ejection fraction from the DIAST-CHF study Abass Eidizadeh1; Moritz Schnelle1; Andreas Leha2; Frank Edelmann³; Andreas Fischer1; Lutz Bind 1Universitätsmedizin Göttingen, Institut für Klinische Chemie, Göttingen, Germany; 2Universitätsmedizin Göttingen, Institut für Medizinische Statistik , Göttingen, Germany; ³Charité-Universitätsmedizin Berlin, Institut für Innere Medizin und Kardiologie, Berlin, Germany; ⁴Universitätsmedizin Göttingen, Klinik für Kardiologie und Pneumologie, Göttingen, Germany Introduction: Chronic heart failure (HF) is a common disease and one of the leading causes of death worldwide. Heart failure with preserved ejection fraction (HFpEF) and heart failure with reduced ejection fraction (HFrEF) are different diseases with distinct as well as comparable pathophysiologies and diverse responses to therapeutic agents. We aimed to identify possible pathobiochemical signaling pathways and biomarkers in HFpEF and HFrEF by using a broad proteomic approach. Methods: 180 biomarkers in the plasma of a representative subgroup of HFpEF and HFrEF patients (n = 127) from the DIAST-CHF trial were examined with a proximity extension assay and compared with a healthy control group (n = 40). Biostatic analyses were performed to identify possible differences in biomarker profiles and signalpathways between HFpEF and HFrEF, always compared to the control group. Results: Thus, we were able to identify 35 proteins that were expressed signiﬁcantly different in both HF groups compared to the control group. It was also able to determine 29 unique proteins expressed in HFpEF and 33 unique proteins in HFrEF. Additionally network analyzes showed a special role of platelet-derived growth factor subunit A (PDGF-A), Dickkopf-related protein 1 (Dkk-1) and tumor necrosis factor receptor superfamily member 6 (FAS) in HFpEF patients, while perlecan (PLC) and junctional adhesion molecule A (JAM-A) stood out in the HFrEF group. Overall, signaling pathways of metabolic processes, cellular stress and iron metabolism seemed to be important for HFrEF, while for HFpEF oxygen stress, hemostasis, cell renewal, cell migration and cell proliferation are in the foreground. Conclusion: The identiﬁed proteins and signaling pathways offer new therapeutic and diagnostic approaches for patients with chronic heart failure. Mechanistic insights into the progression of systolic heart failure in aortic stenosis patients Moritz Schnelle1; Dawid Lbik2; Miriam Puls2; Elisabeth Zeisberg2; Andre Fischer³; Andreas Fischer1; Gerd Hasenfuß2; Karl Toischer2 awid Lbik2; Miriam Puls2; Elisabeth Zeisberg2; Andre Fischer³; Andreas Fischer1; Gerd Hasenfuß2 German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 eA4 1Universitätsmedizin Göttingen, Institut für Klinische Chemie, Göttingen, Germany; 2Universitätsmedizin Göttingen, Abteilung für Kardiologie und Pneumologie, Göttingen, Germany; ³Deutsches Zentrum für Neurodegenerative Erkran- kungen, Abteilung für Epigenetik und Systemmedizin, Göttingen, Germany 1Universitätsmedizin Göttingen, Institut für Klinische Chemie, Göttingen, Germany; 2Universitätsmedizin Göttingen, Abteilung für Kardiologie und Pneumologie, Göttingen, Germany; ³Deutsches Zentrum für Neurodegenerative Erkran- kungen, Abteilung für Epigenetik und Systemmedizin, Göttingen, Germany The process of transition to heart failure (HF) in humans is poorly understood. This study was designed to identify key mechanistic features at different HF stages in severe human aortic stenosis (AS) in order to better understand the pathophysiological process of HF progression. The study included 57 AS patients presenting with different degrees of systolic dysfunction at the time of aortic valve implantation. Echocardiographic phenotyping, Next Generation Sequencing (NGS) as well as histological analyses were performed in left ventricular (LV) myocardial biopsies of respective patient subsets. Principal Component Analysis (PCA) of gene expression data combined with cardiac remodeling assessment identiﬁed three major stages of HF progression: i) AS with normal ejection fraction (ASnEF, EF ≥ 55%) and concentric LV hypertrophy due to increased myocyte size, ii) AS with mildly reduced EF (ASmrEF, EF 45-54%) and LV dilatation, and iii) AS with reduced EF (ASrEF, EF < 45%). NGS was used to characterize the three groups. On a cellular level, inﬂammatory mast cells dominated in ASmrEF, whereas monocytes and gene expression related to T-lymphocyte activity were found in ASrEF. At the level of epigenetic regulation, microRNAs and DNA methylation/ hydroxymethylation were dominant in ASrEF. Myocardial ﬁbrosis was signiﬁcantly increased only in ASrEF. This study identiﬁed LV dilatation as an early feature of HF in human AS that is followed by development of cardiac ﬁbrosis and further contractile impairment. Changes in the inﬂammatory response, epigenetic modiﬁcations and extracellular matrix remodeling appear to be critically involved in a stage-speciﬁc manner. Our results highlight the need for a more personalized concept of HF therapy that takes into account individual disease stages and distinct, underlying mechanisms. Hypercoagulability impairs plaque stability in diabetes-induced atherosclerosis Saira Ambreen1; Sameen Fatima1; Ahmed Elwakiel1; Rajiv Rana1; Kunal Singh1; Anubhuti Gupta1; Dheerendra Gupta1; Hamzah Khawaja1; Jayakumar Manoharan1; Christian Besler2; Ulrich Laufs³; Shrey Kohli1; Berend Isermann1; Khurrum Shahzad1 1University Hospital Leipzig, Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostic, leipzig, Germany; 2University Hospital Leipzig, Klinik und Poliklinik für Kardiologie, leipzig, Germany; ³Universität Leipzig, Cardiology, Leipzig Heart Center, leipzig, Germany Introduction: Diabetes mellitus, which is largely driven by nutritional and behavioral factors, is characterized by accel- erated atherosclerosis with impaired plaque stability. Atherosclerosis and associated complications is the major cause of mortality in diabetic patients. Efﬁcient therapeutic concepts for diabetes-associated atherosclerosis are lacking. Athero- sclerosis among diabetic patients is associated with reduced endothelial thrombomodulin (TM) expression and impaired activated protein C (aPC) generation. Here, we demonstrate that atheroscleroticplaque stability isreducedin hyperglycemic mice expressing dysfunctional TM (TMPro/Pro mice) which have a pro-coagulant phenotype due to impaired thrombin inhibition and markedly reduced aPC generation. Methods: Female ApoE-/- or TMPro/Pro ApoE-/- mice (age 6 to 8 weeks) were fed a normal chow diet and were made diabetic (DM) by injecting streptozotocin. After 22 weeks of age, the mice were sacriﬁced and analyzed for different blood parameters. Oil Red O, MOVAT, MOMA-2 or α-SMC actin stainings were conducted on thoracic aortae (opened longitu- dinally) or frozen sections of the brachiocephalic arteries. Results: The vessel lumen and plaque size of atherosclerotic lesions in the truncus brachiocephalic were decreased in diabetic TMPro/Pro ApoE-/- mice compared to diabetic ApoE-/- mice. While lipid accumulation in lesions of diabetic TMPro/Pro ApoE-/- mice was lower than that in diabetic ApoE-/- mice, morphometric analyses revealed more prominent signs of instable plaques, such as a larger necrotic core area and decreased ﬁbrous cap thickness in diabetic TMPro/Pro ApoE-/- mice. Congruently more macrophages and fewer smooth muscle cells were observed within lesions of diabetic TMPro/Pro ApoE-/- mice. German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 eA5 Conclusion: Thus, impaired TM function reduces plaque stability, a characteristic of hyperglycemia-associated plaques, thus suggesting the crucial role of impaired TM function in mediating diabetes-associated atherosclerosis. Conclusion: Thus, impaired TM function reduces plaque stability, a characteristic of hyperglycemia-associated plaques, thus suggesting the crucial role of impaired TM function in mediating diabetes-associated atherosclerosis. Association of Plasma Chemerin with All-Cause and Disease-Specific Mortality – Results from a Population-Based Study Stephanie Zylla1; Katharina Noppes1; Stefan Groß2; Marcello R.P. Markus2; Martin Bahls2; Henry Völzke³; Marcus Dörr2; Matthias Nauck1; Nele Friedrich1 1Universitätsmedizin Greifswald, Institute of Clinical Chemistry and Laboratory Medicine, Greifswald, Germany; 2Uni- versitätsmedizin Greifswald, Department of Internal Medicine B, Greifswald, Germany; ³Universitätsmedizin Greifswald, Institute for Community Medicine, Greifswald, Germany Background and objectives: Various cross-sectional studies have observed an association between high circulating chemerin concentrations and an unfavorable metabolic proﬁle. However, the actual prognostic value of chemerin for the risk of associated diseases and mortality was examined only in few studies mostly using small and speciﬁc patient populations. Therefore, this study aimed to analyze the association between plasma chemerin concentrations and all-cause as well as cause-speciﬁc mortality in a general population. Study design and methods: From the Study of Health in Pomerania (SHIP) 2903 SHIP-START-1 and 4111 SHIP-TREND- 0 participants were followed up for 15 and 9 years (median), respectively. The association between plasma chemerin and all-cause mortality was analyzed using Kaplan-Meier survival curves and multivariable Cox proportional hazard regression models. Additionally, cause-speciﬁc hazards for cardiovascular diseases (CVD) and cancer were modelled considering competing events. Results: A total number of 372 and 126 deaths occurred during follow-up in SHIP-START-1 and SHIP-TREND-0, respectively. Unadjusted Kaplan-Meier survival curves illustrated in both study cohorts that subjects with chemerin ≥ 109 ng/mL (66.67%-percentile) had a lower survival function than subjects with lower chemerin. Multivariable regression analyses revealed that this association was independent of major confounders. Each increase of chemerin per 30 ng/mL was associated with a 23% higher risk of all-cause mortality (95%-conﬁdence interval: 1.13 – 1.35). Cause- speciﬁc analyses have further shown that the effect estimates for cancer were greater than those for CVD. Conclusion: The present study detected a positive association between plasma chemerin concentrations and mortality from all-causes, cancer, and CVD in a large population-based study sample. In comparison, the highest effect estimates were found forcanceras cause of death suggesting that the association between chemerinandmortality ismainlyattributed to cancer related deaths. Comparison of multi-steroid LC-MS/MS assays used for routine operation in five laboratories in Switzerland and Germany for the simultaneous analysis of 9 steroids Valentin Braun1; Christoph Seger2; Manfred Rauh³; Matthias Weber⁴; Alexander Gaudl⁵; Uta Ceglarek⁵; Joana Gawinecka⁶; Daniel Müller⁶ Valentin Braun1; Christoph Seger2; Manfred Rauh³; Matthias Weber⁴; Alexander Gaudl⁵; Uta Ceglarek⁵; Joana Gawinecka⁶; Daniel Müller⁶ 1Dr Risch Ostschweiz AG, —, Buchs, Switzerland; 2Universität Innsbruck, Department of Pharmacy, Innsbruck, Austria; ³University Hospital Erlangen, Department of Pediatrics and Adolescent Medicine, Erlangen, Germany; ⁴Bioscientia MVZ Labor Karlsruhe GmbH, —, Karlsruhe, Germany; ⁵University Hospital Leipzig, Institute of Laboratory Medicine, Clinical German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 eA6 Chemistry and Molecular Diagnostics, Leipzig, Germany; ⁶University Hospital Zurich, Institute of Clinical Chemistry, Zürich, Switzerland Chemistry and Molecular Diagnostics, Leipzig, Germany; ⁶University Hospital Zurich, Institute of Clinical Chemistry, Zürich, Switzerland Introduction: Regarding selectivity and speciﬁcity, LC-MS/MS is the best technology available for the quantiﬁcation of steroids in human serum in a clinical setting. By simultaneous analysis of multiple steroids, it can provide the clinicians with more information for better diagnosis. For quality assurance, accuracy estimation and between-method stan- dardization laboratory (lab) comparisons are a valuable tool. We present results from an inter-lab comparison study for 9 steroid analytes with 5 participating labs. Methods: The sample set consisted of 40 pooled human serum samples generated by targeted mixing of pre-analyzed leftovers to get a well-balanced distribution across reference ranges of each steroid. All 5 labs measured a sample set once using own protocols and calibrators for their multi-steroid LC-MS/MS assay. 4 labs used in-house developed tests, from which 3 were using IVD-CE certiﬁed calibrators (Chromsystems, Munich, Germany), while the 5th lab was using the whole LC-MS kit of that manufacturer. All labs reported results for 17OH-progesterone (17P), androstenedione (A), cortisol (F) and testosterone (T) and 4 labs for 11-deoxycortisol (S), corticosterone (B), cortisone (E), DHEAS (DS) and progesterone (P). Results of each lab were compared against the mean of all labs using Bland-Altmann (BA) and Passing-Bablok (PB) statistics. Results: Mean differences in BA-analysis were within ±10% for 17P, A, DS, E, P, S and T and within ±15% for B and F. Slopes of individual labs in PB analysis were inside the 95% CI of the slope calculated from all values. Signiﬁcant deviations from the all-lab mean were found only in one site for 17P and A (mean differences 33.1% and -18.9%). Comparison of multi-steroid LC-MS/MS assays used for routine operation in five laboratories in Switzerland and Germany for the simultaneous analysis of 9 steroids BA 2 SD intervals, indicative of the variability of result differences, were < ±15 % for all analytes and labs except for one site (±17 to 30 %) and analytes P and T at all sites due to matrix effects from gel-barrier tubes and sensitivity and linearity issues at concentrations < 1 nM, respectively. Mean inter-lab CV was < 10% for E, F and S; < 15% for A, B, DS and T; 20.0% for 17P and 21.5% for P. Conclusions: Comparable results (concordance of PB slopes) were found for B, E, F, P, S, and T but also for 17P and A, if one lab was excluded. Reference method assigned target values are missing hence the study delivers no evidence on the absolute trueness of the assays. The number of samples, measurements and participating labs was limited, but study design would allow for upscaling and repetition, due to the use of pooled instead of single donor samples. Nevertheless, results of this study indicate good overall standardization for the LC-MS/MS measurement of 9 steroids between 5 labs and some sources of higher variance and bias like operator errors, non-supported matrix types and higher imprecision at lower ends of measuring intervals could be identiﬁed, thereby helping the participating laboratories enhancing their assays and delivering better results to clinicians. Analytical interference of different hemoglobin variants on HbA1c measurements comparing high-performance liquid chromatography with whole blood enzymatic assay. Bozena Zechmeister1; Tanja Erden2; Berit Kreutzig³; Matthias Weber⁴; Philippe Joly⁵; Jürgen Erdmann⁶; Christine Brockmann-Honig⁷; Andreas Fischer⁸; Abass Eidizadeh⁸ Bozena Zechmeister1; Tanja Erden2; Berit Kreutzig³; Matthias Weber⁴; Philippe Joly⁵; Jürgen Erdmann⁶; Christine Brockmann-Honig⁷; Andreas Fischer⁸; Abass Eidizadeh⁸ 1Universitätsmedizin Göttingen, Interdisziplinäres UMG-Labor, Göttingen, Germany; 2Tosoh Bioscience, Diagnostic Busi- ness Unit-Europe, Göttingen, Germany; ³Amedes MVZ wagnerstibbe , Laboratoriumsmed. med. Mikrobiologie und Immu- nologie, Göttingen, Germany; ⁴4Bioscientia MVZ Labor Karlsruhe GmbH, Bioscientia MVZ Labor Karlsruhe, Karslruhe, Germany; ⁵Laboratoire Interuniversitaire de Biologie de la Motricit´e (LIBM) EA7424, Team « Vascular Biology and Red Blood Cell », Lyon, France; ⁶Laborgemeinschaft Rottweil G.b.R., Laborgemeinschaft Rottweil G.b.R., Villingen- Schwenningen, Germany; ⁷Aesculabor Hamburg GmbH, Aesculabor Hamburg GmbH, Hamburg, Germany; ⁸University Medical Center Göttingen, Institute for Clinical Chemistry/Interdisciplinary UMG Laboratory, Göttingen, Germany German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 eA7 eA7 Introduction: The concentration of glycated hemoglobin (HbA1c) is an essential diagnostic and therapeutic biomarker in diabetes mellitus. However, it is known that Hb structural variants and synthesis disorders, can affect the HbA1c measurement in different assays. Although the analytical interference of various hemoglobinopathies on the chro- matographic measurement of HbA1c using HPLC has been well studied, data on the interference on the enzymatic assay are few. Introduction: The concentration of glycated hemoglobin (HbA1c) is an essential diagnostic and therapeutic biomarker in diabetes mellitus. However, it is known that Hb structural variants and synthesis disorders, can affect the HbA1c measurement in different assays. Although the analytical interference of various hemoglobinopathies on the chro- matographic measurement of HbA1c using HPLC has been well studied, data on the interference on the enzymatic assay are few. Methods: In this multi-center study, a large number (n = 104) of 33 different hemoglobin variants were collected over a period of one year and compared between an HPLC (Tosoh G8 and G11) and an enzymatic assay (Abbott Alinity c). Results: A good comparability between ion-exchange HPLC and the Alinity assay for most Hb variants was found. However, we were able to determine for the ﬁrst time that certain Hb variants (Hb Okayama, HbAE, Hb Lepore) can lead to clinically relevant discordant results. HbF (>5%) can already cause a relevant aberration. Conclusion: Overall, using the Abbott HbA1c assay in the presence of certain hemoglobin variants can induce clinically relevant interference that can affect diagnosis and therapy monitoring decisions, mainly because the enzymatic assay cannot provide any information about Hb variants. Sex-specific Comparison of Reference Intervals for 13 Serum Steroids Determined by Direct or Indirect Methods Sophie Anker1; Jakob Morgenstern1; Jakob Adler2; Maik Brune1; Sebastian Brings1; Thomas Fleming1; Elisabeth Kliemank1; Markus Zorn1; Andreas Fischer³; Julia Szendrödi1; Lars Kihm1; Johanna Zemva1 1University Hospital Heidelberg, Department of Internal Medicine I and Clinical Chemistry, Heidelberg, Germany; 2Medical Laboratory for Clinical Chemistry, Microbiology, Infectious Diseases and Genetics Prof. Schenk/Dr. Ansorge & Colleagues, Clinical Chemistry, Magdeburg, Germany; ³University Hospital Göttingen, Department of Clinical Chemistry, Göttingen, Germany Aims: For the quantiﬁcation of steroids, mass spectrometry offers a higher degree of speciﬁcity and sensitivity than con- ventional immunoassays. However, as reference intervals have not been adjusted accordingly [1], laboratories are required to determine their own values which is time- and cost-consuming due to the recruitment of a sufﬁcient number of healthy reference subjects [2]. In this study, we addressed the question whether reference intervals for serum steroids can be established by an indirect approach. Furthermore, we wanted to contribute to the methodological harmonization of reference intervals. Materials and Methods: We used data of 9801 individuals, of whom only age and gender were recorded, to establish post-hoc reference intervals for androstenedione, dehydroepiandrosterone, testosterone, progesterone, dihy- drotestosterone, 17α-hydroxyprogesterone/-pregnenolone, corticosterone, 11-deoxycorticosterone, 11- and 21-deoxycortisol, aldosterone, cortisol, and cortisone. Analyses were performed on a Waters® Acquity UPLC class I system coupled to a Waters® XEVO TQ-S LC-MS using a MassChrom® Steroids kit (Chromsystems). The indirect reference interval algorithm [3,4], includes three robust quantile-based steps which were executed in R (Version 4.1.1): 1. Bowley’s quartile skewness predicts whether a normal or a lognormal distribution should be assumed. 2. An iterative boxplot method is applied to remove obvious outliers. 3. A normal quantile-quantile plot provides the 2.5th and 97.5th percentiles, calculated from the intercept and slope of the linear regression line according to the formula RI = intercept±1.96∙slope [4]. Post-hoc reference intervals were compared to reference intervals derived from studies using a direct approach. Results: The majority of reference intervals generated by the indirect method showed a very good overlap with those derived from a direct approach. Signiﬁcant deviationswere seen for sexhormones such as progesterone,17α-hydroxypregnenolone/- progesterone, and testosterone. This was reﬂected by skewed quantile-quantile-plots, which indicated an inhomogeneous distribution of the underlying data. The indirect approach yielded complete reference intervals also in cases, in which the lower reference limit was not provided by the direct method, e. g. in the case of aldosterone, 17-OH-pregnenolon and 11-Deoxycorticosterone. Critical appraisal of the influence of anti-Thyroglobulin on the measurement of Thyroglobulin and Thyroglobulin recovery Robert Markewitz; Klaus-Peter Wandinger; Ralf Junker Robert Markewitz; Klaus-Peter Wandinger; Ralf Junker Universitätsklinikum Schleswig-Holstein, Institut für Klinische Chemie, Kiel, Germany Background: Antibodies against Thyroglobulin (Anti-Tg) are known to be able to cause interferences in the measure- ment of Thyroglobulin (Tg). Measuring Tg recovery after adding a known amount of Tg to the sample is a popular method to identify samples that are affected by this interference. Methods: Results of clinical samples in which Tg, anti-Tg and Tg recovery were measured in a high-throughput clinical laboratory via immunometric assays (by Roche, Switzerland) were retroactively anonymized and analyzed. Speciﬁcally, the associations between the analytes Tg, anti-Tg and Tg recovery with one another and with the variables age and sex were statistically evaluated. Results: 8871 samples were collected, of which only 60 (0.0068%) exhibited pathologically decreased Tg recovery, of which 47 contained quantiﬁable and 24 pathological levels of anti-Tg. Quantiﬁable levels of anti-Tg were associated with signiﬁ- cantly decreased overall levels of Tg recovery and Tg itself (both: p < 0.0001), with the strongest decreases being associated with pathological levels of anti-Tg. But even for pathological anti-Tg, median Tg-recovery is still well within the reference range at 94±10.4 %. Effect sizes of all detected differences or associations were small to very small. Conclusion: Tg recovery appears neither sensitive nor speciﬁc enough to detect interference of anti-Tg in the measurement of Tg onasingle-specimenbasis.Thisinterference canneverthelessbedetectedwithstatisticalmethodsbothforTgrecoveryand for Tg itself, albeit with small effect sizes. Methods other than Tg recovery are needed to reliably detect samples with impaired Tg measurement. Tg measurement. Sex-specific Comparison of Reference Intervals for 13 Serum Steroids Determined by Direct or Indirect Methods 8 German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 eA8 Discussion/Conclusion: Our results suggest that it is a valid approach to verify and establish reference intervals by an indirect method. It should, however, be noted that reference intervals for widely varying sex hormones may differ signiﬁ- cantly between direct and indirect methods due to missing background information on female cycle, menopause and stages of puberty when the indirect approach isapplied to routine laboratory results.Further, this studycontributesa substantial set of data to the methodological harmonization of steroid reference ranges using mass-spectrometry. German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 eA9 Aim of the Study: In this project we hypothesized that high glucose induces UPR causes cell senescence, results in endothelial dysfunction which leads to vulnerable plaques. Activated protein C or IRE1 alpha inhibitor prevent diabetes induced accelerated atherosclerosis by reducing maladaptive UPR induced endothelial cell senescence. Aim of the Study: In this project we hypothesized that high glucose induces UPR causes cell senescence, results in endothelial dysfunction which leads to vulnerable plaques. Activated protein C or IRE1 alpha inhibitor prevent diabetes induced accelerated atherosclerosis by reducing maladaptive UPR induced endothelial cell senescence. Methods: To gain insights into pathomechanisms of diabetes induced atherosclerotic plaque development we cultured human coronary artery endothelial cells (HCAECs) under hyperglycemic (HG) or hyperlipidaemic (HL) conditions. ApoE-/- mice (age 8 weeks) was made either diabetic by streptozotocin injections (A mouse model type 1 diabetes) or fed them HFD to induce hyperlipidemia. Mice were analyzed after 20 weeks of treatments. Results: HG conditions induced strong barrier disruption as compared to HL (TEER, FITC dextran leakage) and protein expression of the senescence markers (p21, p16, p53) and UPR markers (XBP1, IRE1α and ATF6). Activated protein C restored barrier integrity, reduced glucose induced expression of senescence and UPR markers in vitro. Targeting IRE1α RNAase activity prevented HG induced cellular senescence. Ex vivo, diabetic ApoE-/- mice revealed increased expression of senes- cence and UPR markers within atherosclerotic lesion as compared with non-diabetic ApoE-/- mice. Activated proetin C signiﬁcantly reduced expression of senescence and UPR markers within atherosclerotic lesions of diabetic ApoE-/- mice. Thus, senescence associated inﬂammation and UPR are associated with glucose-dependent endothelial cells dysfunction and loss of endothelial barrier integrity. Conclusion: These results demonstrate that diabetes-induced atherosclerosis is associated with cellular senescence and UPR. Targeting cellular senescence and UPR (with aPC or IRE1α inhibitor) may be a useful therapy of atherosclerosis in diabetic patients. Activated protein C reduces maladaptive unfolded protein response (UPR) to ameliorate diabetes-accelerated atherosclerosis Sameen Fatima; Saira Ambreen; Ihsan Gadi; Kunal Singh; Anubhuti Gupta; Ahmed Elwakiel; Shruthi Krishnan; Hamzah Khawaja; Akash Methew; Shrey Kohli; Berend Isermann; Khurrum Shahzad Institut für Laboratoriumsmedizin, Klinische Chemi, Department of molecular diagnostics, Leipzig, Germany Background: The mechanisms underlying the different atherosclerosis course in diabetic and non-diabetic patients remain unknown. Hyperglycemia causes endothelial cells dysfunction, a key disease driver. Atherosclerotic plaques display markers of senescence and unfolded protein response (UPR). UPR has been linked with the atherosclerosis. One of the outcome of UPR is to control the cell senescence hallmarks including cell cycle arrest, DNA repair capacity, morphological changes, metabolic changes, the secretory pathway, and changes in membrane lipid composition. Plasma levels of coagulation protease, activated protein C decline in diabetes and atherosclerosis. Results: Fasting glucose concentrations in immediately centrifuged LH samples were 94.0±5.9 mg/dl (mean±standard deviation), ranging from 80.0 to 105.2 mg/dl (n=42). For LAB1, relative differences in tubes with citrate/ﬂuoride additives were -5.9±1.2% (GE1, n=118), -6.1±1.3% (GE2, n=119), and -1.0±1.4% (VA, n=104). For LAB2a, differences of -1.5±0.5% (LH, n=41), -0.7±1.3% (GE1, n=123), and -1.8±1.4% (GE2, n=118) were found; differences for LAB2b were -0.1±0.5% (LH, n=41), +0.6±1.3% (GE1, n=123), and -0.4±1.4% (GE2, n=118). LH samples centrifuged after 20 min exhibited some glycolysis (-2.4±1.1% (LAB1, n=41), -3.1±1.1% (LAB2a, n=40), -1.8±1.2% (LAB2b, n=40)), although the effect was much more pro- nounced after a delay of 240 min (-17.8±3.6% (LAB1, n=41), -18.9±3.5% (LAB2a, n=41), -17.7±3.6% (LAB2b, n=41)). Results: Fasting glucose concentrations in immediately centrifuged LH samples were 94.0±5.9 mg/dl (mean±standard deviation), ranging from 80.0 to 105.2 mg/dl (n=42). For LAB1, relative differences in tubes with citrate/ﬂuoride additives were -5.9±1.2% (GE1, n=118), -6.1±1.3% (GE2, n=119), and -1.0±1.4% (VA, n=104). For LAB2a, differences of -1.5±0.5% (LH, n=41), -0.7±1.3% (GE1, n=123), and -1.8±1.4% (GE2, n=118) were found; differences for LAB2b were -0.1±0.5% (LH, n=41), +0.6±1.3% (GE1, n=123), and -0.4±1.4% (GE2, n=118). LH samples centrifuged after 20 min exhibited some glycolysis (-2.4±1.1% (LAB1, n=41), -3.1±1.1% (LAB2a, n=40), -1.8±1.2% (LAB2b, n=40)), although the effect was much more pro- nounced after a delay of 240 min (-17.8±3.6% (LAB1, n=41), -18.9±3.5% (LAB2a, n=41), -17.7±3.6% (LAB2b, n=41)). Conclusion: Systematic differences of about -6% were found for GE tubes compared to LH tubes on analyzer LAB1, whereas no relevant differences between GH and LH tubes were found on analyzers LAB2a and LAB2b. These results suggest that additives in GE tubes might affect glucose measurement with the hexokinase application used in analyzer LAB1. VA samples did not show relevant differences on analyzer LAB1. In LH samples, delayed centrifugation of 20 and 240 min led to systematic differences of up to -3% and -19%, respectively. Fasting plasma glucose concentrations in different sampling tubes measured on different glucose analyzers Stefan Pleus; Alexandra Beil; Annette Baumstark; Cornelia Haug; Guido Freckmann Institut für Diabetes-Technologie, Forschungs- und Entwicklungsgesellschaft mbH an der Universität Ulm, n.a., Ulm, Germany Introduction: The German Diabetes Association recommends use of sampling tubes with citrate and ﬂuoride additives for the diagnosis of diabetes by oral glucose tolerance test. In this study, venous blood was sampled in different tubes with and without citrate/ﬂuoride additives. Glucose concentrations were subsequently determined on different laboratory ana- lyzers in order to estimate the potential effect of these additives. Materials and Methods: Venous samples were obtained from 42 persons without anamnestically known diabetes mellitus in a fasting state. Samples were collected in lithium-heparinized tubes (LH) and in tubes with citrate/ﬂuoride additives: GlucoEXACT 66x11mm (GE1), GlucoEXACT 75x13mm (GE2), and Vacuette FC Mix (VA). For each participant, venous blood was sampled 3 times per tube type for centrifugation after different periods (immediately (≤5 min), 20 min, and 240 min). All measurements were performed in plasma. Measurements for LH and GE tubes were performed on 3 hexokinase-based laboratory analyzers: Cobas Integra 400 plus with GLUC2 Glucose HK application (LAB1; Roche) and two Cobas pro c503 with GLUC3 Glucose HK Gen.3 application (LAB2a, LAB2b; Roche). VA tubes were measured only on LAB1. Relative differences between glucose concentrations in immediately centrifuged LH tubes (LAB1) and glucose concen- trations in all other samples were calculated. Since glucose concentrations in GE1, GE2 and VA tubes were similar for the three incubation times when measured on the same analyzer, results were pooled. eA10 German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 eA10 Differences between venous, capillary and interstitial glucose concentrations during an oral glucose tolerance test Stefan Pleus; Alexandra Beil; Annette Baumstark; Sebastian Schauer; Manuela Link; Delia Waldenmaier; Cornelia Haug; Guido Freckmann Institut für Diabetes-Technologie, Forschungs- und Entwicklungsgesellschaft mbH an der Universität Ulm, n.a., Ulm, Germany Introduction: In this study, glucose concentrations in venous blood, capillary blood, and interstitial ﬂuid (ISF) obtained during an oral glucose tolerance test (oGTT) were compared. Capillary and venous blood glucose (BG) concentrations are known to be not necessarily comparable. This study aimed at quantifying potential differences between venous BG, capillary BG, and ISF glucose concentrations before and after glycemic load. Materials and Methods: An oGTT was performed on 41 persons without anamnestically known diabetes mellitus. Venous and capillary blood sampling was performed in a fasting state before consumption of a standardized 75-g glucose solution as well as 60 and 120 minutes afterwards. In parallel, ISF glucose was recorded with a FreeStyle Libre system for continuous glucose monitoring (CGM). Venous and capillary blood samples were collected in lithium-heparinized tubes for immediate centrifugation (median: 3 min) and subsequent plasma glucose (PG) measurement on a laboratory analyzer (LAB; Cobas Integra400 plus).Capillary BGmeasurementswerealso performedwith a BGmonitoring system(BGMS;ContourNextOne). Relative differences from venous PG (LAB) were calculated for capillary PG (LAB), plasma-equivalent capillary BG (BGMS) and ISF glucose (CGM). Results: Fasting glucose concentrations at the start of the oGTT showed only minimal differences from venous PG (LAB). Results are provided as mean ± standard deviation. Capillary PG (LAB) was +3.9% ± 3.6% higher than venous PG, capillary BG (BGMS) was +9.1% ± 5.2% higher, and ISF glucose (CGM) was +1.4% ± 12.3%. After 60 minutes, marked differences with increased variability were found: +34.9% ± 23.8% for capillary PG (LAB), +42.8% ± 25.5% for capillary BG (BGMS), and +30.8% ± 25.7% for ISF glucose (CGM). After 120 minutes, slightly smaller, but still marked differences with increased variability werefound: +27.0% ± 15.6% forcapillary PG (LAB), +33.2% ± 17.7% forcap BG(BGMS),and +17.6% ± 19.5% forISF glucose (CGM). Conclusion: After consumption of the oGTT glucose solution, capillary BG/PG and ISF glucose concentrations were substantially higher than venous PG concentrations. Fasting glucose concentrations showed minimal differences. The post-prandial differences exhibited marked inter-individual variability. eA11 German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 eA11 HbA1c-usage under intensified preanalytical scrutiny Norbert Ostendorf Norbert Ostendorf St. Franziskus-Hospital Münster, Institut für Laboratoriums- und Transfusionsmedizin, Münster, Germany Introduction: We established an algorithm to question the reliability of HbA1c-measurements in a tertiary care hospital by looking at concomitant data in the laboratory information system, especially full blood count and transfusion record. After one year, data obtained by this strategy were evaluated. Methods: Results of HbA1c testing were validated together with haemoglobin (Hb) concentration and reticulocyte counts using the following algorithm: Hb concentrations of 10 g/dl or more were considered unsuspicious. When Hb concentrations were below 10 g/dl but not lower than 8 g/dl, a comment was added to the HbA1c-result, stating that the value was questionable. When Hbconcentrationswerebelow8 g/dl,theHbA1cresultwasdeleted and replaced byacommentary stating that the result was questionable and reporting the HbA1c value within the comment only. Moreover, in these cases the patient record was checked for erythrocyte transfusions within the current hospital stay. Finally, in cases where the HbA1c value was implausibly low in the ﬁrst place, a reticulocyte count was performed to look for hyperregenerative states. HbA1c was considered implausibly low in patients above 80 years of age and a HbA1c result below 5%. Results: From 01.05.2021 to 30.04.2022, 2718 measurements were evaluated. Of these, 87 or 3.2% were replaced by a comment because a preanalytical impairment was very likely. In 29 cases, transfusions of erythrocytes were recorded prior to the requested HbA1c-measurement. From the remaining 2631 values, 165 or 6.3% were commented to highlight the possibility of preanalytical impairment. Conclusion: HbA1c-measurement has a special preanalytical issue: a normal erythrocyte turnover in the weeks before the investigation and avoidance of recent erythrocyte transfusions. Using a haemoglobin concentration of 10 g/dl as an indicator for bleeding or impaired erythropoesis, 9.5% of the investigations were suspicious for preanalytical errors. Erythrocyte transfusions interfered with 1% of HbA1c-measurement. Association of suPAR and cardiovascular risk factors in a young and healthy population Niklas Wohlwend1; Kirsten Grossmann1; Martin Risch2; Ornella C. Weideli1; Stefanie Aeschbacher³; David Conen⁴; Loren Risch2 Kirsten Grossmann1; Martin Risch2; Ornella C. Weideli1; Stefanie Aeschbacher³; David Conen⁴; Lorenz 1Labormedizinisches Zentrum Dr. Risch, Forschung, Vaduz, Liechtenstein; 2Labormedizinisches Zentrum Dr. Risch, Geschäftsleitung, Vaduz, Liechtenstein; ³University Hospital Basel, Cardiovascular Research Institute Basel and Division of Cardiology , Basel, Switzerland; ⁴McMaster University, Population Health Research Institute, Hamilton, Canada Aims: The increasing global burden of cardiovascular diseases led to further intensive research in its cause, its devel- opment, its therapy and more and more in possible preventive measures including individual risk stratiﬁcation strategies. The biomarker soluble urokinase plasminogen activator receptor (suPAR) is one candidate of potential biomarkers which could be used for the evaluation of the individual cardiovascular risk. Materials and Methods: The current analysis is based on data from the GAPP (Genetic and phenotypic determinants of blood pressure and other cardiovascular risk factors) study, a population-based cohort-study investigating young and healthy adults (aged 25-41 at baseline) living in the Principality of Liechtenstein. In this analysis we included the following cardiovascular risk factors and laboratory parameters: BMI, physical activity, alcohol consumption, smoking status, blood pressure parameters, glucose status, lipid levels, liver enzymes, and kidney function. Additionally, the Framingham Score and the Healthy Lifestyle Score as indicators of the overall cardiovascular risk were included. We compared these factors using sex-speciﬁc quartiles and multivariate regression analysis in relation to suPAR serum levels. Results: Comparing the baseline characteristics, it was shown that female participants have higher levels of serum suPAR levels than the male participants (1.73 vs 1.50, p-value < 0.001). An inverse correlation between serum suPAR levels and HDL-cholesterol in men (p-value < 0.001) and women (p-value < 0.001) was found. Furthermore, smoking participants showed higher levels of serum suPAR levels (p-value < 0.001). For male participants, a higher lifestyle score resulted in lower serum suPAR levels (p-value < 0.001). The HbA1c (p-value 0.008) and the Framingham Score (p-value < 0.001) correlated with serum suPAR levels for female participants. Discussion/Conclusion: The current analysis shows statistically signiﬁcant differences in average serum suPAR levels for male and female participants. The correlation of suPAR and cardiovascular risk factors differs among male and female participants. suPAR as a biomarker for future cardiovascular risk in the general population can support clinical pre- diagnostics. Plasma-glycerol determinations: comparison of an enzymatic assay versus NMR spectroscopy Mohammed Abdullah1; Kathrin Budde1; Ann-Kristin Henning1; Astrid Petersmann2; Matthias Nauck 1Uni.Med.Greifswald, Institut für Klinische Chemie und Laboratoriumsmedizin, Greifswald, Germany; 2Universitätsmedizin Oldenburg, Institut für Klinische Chemie und Laboratoriumsmedizin, Oldenburg, Germany Background: Glycerol is a trivalent alcohol (1,2,3-propanetriol) composing the backbone of the triglyceride molecule. The determination of plasma glycerol is not applied in general patient care, but it is the main reaction of the enzymatic determination of triglyceride concentrations after the hydrolysis is performed. In vivo, glycerol is metabolized quickly, so that the plasma concentrations are below 20 mg/dl in general. But the concentrations of glycerol represent the products of in vitro lipolysis. The hydrolysis of triglycerides can be reduced by storing samples refrigerated or even frozen in the biobanking context. The amount of glycerol is a marker for hydrolysis and therefore a biomarker for sample quality. Methods: In the presented study, we evaluated the performance of two different methods for glycerol concentration measurements: Classical enzymatic method and NMR spectroscopy. Fifty plasma samples from patients with normal and abnormal triglyceride concentrations were collected randomly. Glycerol concentration has been directly estimated (T0) and after two weeks of storage at room temperature (T1). The correlation and agreement between these methods have been analyzed according to laboratory standards. eA12 German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 eA12 Results: The glycerol concentration increased from 0.22 ± 0.14 mmol/l (T0) to 1.3 ± 0.45 mmol/l (T1), demonstrating the hydrolyses of triglycerides during this time period. Both glycerol measurements showed a strong correlation and agreement according to Passing-Bablok. Bland-Altman analysis conﬁrmed the good agreement between both methods, which was more pronounced at the higher concentration range. Conclusions: Due to its high reproducibility, robustness, sensitivity and good agreement with glycerol concentrations measured by enzymatic method, the NMR spectroscopy is a valuable analytical tool for glycerol determination in plasma samples. Keywords: Glycerol, Nuclear Magnetic Resonance (NMR), Triglyceride. 1University Hospital Bonn, Institute of Clinical Chemistry and Clinical Pharmacology, Bonn, Germany; 2University Hospital Bonn, Department of Internal Medicine I, Center for Cirrhosis and Portal Hypertension Bonn (CCB), Bonn, Germany; ³University Hospital Bonn, Department of Medicine III, Bonn, Germany; ⁴University Hospital Bonn, Depart- ment of Medicine III, Mildred Scheel School of Oncology, Bonn, Germany 1University Hospital Bonn, Institute of Clinical Chemistry and Clinical Pharmacology, Bonn, Germany; 2University Hospital Bonn, Department of Internal Medicine I, Center for Cirrhosis and Portal Hypertension Bonn (CCB), Bonn, Germany; ³University Hospital Bonn, Department of Medicine III, Bonn, Germany; ⁴University Hospital Bonn, Depart- ment of Medicine III, Mildred Scheel School of Oncology, Bonn, Germany Introduction: Cirrhosis is the common end-stage of liver disease. Established animal models of experimental cirrhosis have been widely used to study human pathogenesis of chronic liver disease and portal hypertension. Two of the most successful and reproducible models are a cholestatic model of bile-duct ligation (BDL) and a toxic model induced by exposure to carbon tetrachloride (CCL4). In the context of these models, some of the liver-related laboratory parameters have been investigated in the past. However, there are no standard laboratory parameter ranges for these models. Material and Methods: To induce cholestatic cirrhosis (BDL), in male Sprague-Dawley rats (Charles River, Sulzfeld, Germany) the bile duct was exposed after median laparotomy, ligated twice and dissected between the two ligatures. For toxic cirrhosis, the animals were exposed to CCL4 inhalation twice weekly in increasing intervals. Blood samples from these models and from healthy control animals were taken at deﬁned timepoints. Analyses were carried out at the central laboratory. Sodium and potassium were determined potentiometrically (cobas 8000 ISE, Roche Diagnostics). Total protein, alkaline phosphatase, alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma- glutamyltransferase, triglycerides, cholesterol, high and low density-lipoprotein cholesterol, direct and total bilirubin, creatinine, and urea nitrogen were determined with cobas c702 or c502 (Roche Diagnostics). Mean, median, standard deviation, minimum and maximum, 2.5-97.5, 5-95 or 10-90 percentiles (depending on group size) were calculated. Results: AST, ALT were slightly elevated in healthy controls compared to human reference ranges. Different stages ofcirrhosis inBDL and CCL4 models showeddistinctdifferences inliver-related parameters. InBDL rats, the development of clinical and laboratory features, such as weight changes, hypalbuminemia and hyperbilirubinemia, was similar to that of human cirrhosis. Kidney function worsened with progression of cirrhosis. In CCL4 rats, bilirubin was only slightly elevated, even in more advanced stages, compared to healthy controls. Over time, liver-related parameters worsened. In general, changes in laboratory parameters were more pronounced in the BDL model than in the CCL4 model. Interestingly, sodium levels did not differ between control animals and models of cirrhosis nor between compensated and more decompensated stages of cirrhosis. Conclusion: Standardizing laboratory values in experimental cirrhosis in rodents is of utmost importance. However, existing data mostly focus on few parameters in treated groups. With our data, we provide a much needed overview of standard laboratory values in experimental cirrhosis models and healthy controls in male Sprague Dawley rats. Our data show that some laboratory features behave in a similar manner to human cirrhosis (albumin, liver parameters), while others, such as sodium, do not. Standardizing laboratory parameters in different models and stages of experimental cirrhosis and in healthy control male Sprague-Dawley rats Ramona Christina Dolscheid-Pommerich1; Franziska Schneider2; Marc Hebest2; Lino Teichmann³; Ruth-Miriam Koerber⁴; Michael Praktiknjo2; Birgit Stoffel-Wagner1; Johannes Chang2 eA13 German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 Discussion: Our current results indicate that LIWL of more than 8% reduces the risk of prediabetes and recurrence of MetS up to 5 years. MetS up to 5 years. eA14 Methods: The study is embedded in a prospective, controlled, monocentric, 6-month LIWL intervention trial in in- dividuals with MetS (ICTRP Trial Number: U1111-1158-3672). Following LIWL, 61 participants were split based on the initial weight loss (WL) into four quartiles: Q1 (WL 2.0-8.1 %, n= 15), Q2 (WL 8.1- 13.0 %, n= 15), Q3 (WL 13.0- 16.6 %, n= 15), Q4 (WL 16.6- 27.5 %, n= 16) and followed up for 5 years. Changes of body weight and parameters reﬂecting MetS i.e. fasting plasma glucose (FPG), triglycerides (TG), HDL-cholesterol and blood pressure were analyzed before and after LIWL and at annual follow-up visits over 5 years. Methods: The study is embedded in a prospective, controlled, monocentric, 6-month LIWL intervention trial in in- dividuals with MetS (ICTRP Trial Number: U1111-1158-3672). Following LIWL, 61 participants were split based on the initial weight loss (WL) into four quartiles: Q1 (WL 2.0-8.1 %, n= 15), Q2 (WL 8.1- 13.0 %, n= 15), Q3 (WL 13.0- 16.6 %, n= 15), Q4 (WL 16.6- 27.5 %, n= 16) and followed up for 5 years. Changes of body weight and parameters reﬂecting MetS i.e. fasting plasma glucose (FPG), triglycerides (TG), HDL-cholesterol and blood pressure were analyzed before and after LIWL and at annual follow-up visits over 5 years. Results: The mean weight loss was 12% after completing the LIWL and 3.6% at 5 years follow-up. Weight gain after LIWL was comparable between the quartiles. A differentiation of the quartiles shows that participants with a higher initial weight loss (> Q1) were able to maintain their body weight below the initial weight after 5 years. In addition to weight gain, the magnitude of the FPG increase was more pronounced in Q1 compared to Q4 (54% versus 21% at 5-year follow-up). Accordingly, recurrence of prediabetes frequency (glycated hemoglobin 5.7 to 6.4 %) was increased during follow-up in Q1 as compared to quartiles with higher weight loss. The frequency of MetS was reduced after LIWL to 73 % (Q1), 43 % (Q2), 27 % (Q3) and 31 % (Q4). Whereas a signiﬁcant reduction of MetS frequency was maintained in Q3 and Q4 during the 5-year follow-up, the number of participants with MetS increased in Q1 and Q2 already after 1 (Q1) and 2 years (Q2) post LIWL. Silke Zimmermann; Akash Mathew; Berend Isermann; Ronald Biemann Silke Zimmermann; Akash Mathew; Berend Isermann; Ronald Biemann Universitätsklinikum Leipzig, Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, Leipzig, Germany Universitätsklinikum Leipzig, Institute of Laboratory Medicine, Clinical Chemistry and Molecular D Germany Aims: Lifestyle-induced weight loss (LIWL) is regarded an efﬁcient therapy to reverse or ameliorate metabolic syndrome (MetS). However, weight loss is difﬁcult to maintain. Studies have shown, that 4.5 years after a structured weight loss program the average sustained weight loss was only a -3.2 % of the initial weight. The aim of our study was to determine (i) whether the initial LIWL-induced weight loss predicts the sustained weight loss, (ii) the relation of weight initial loss changes to parameters characteristic of the MetS, and (iii) whether the extent of initial weight loss in a controlled LIWL predicts the risk of prediabetes and recurrence of the MetS at 5 years of follow-up. eA14 German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 Adjusted Calcium – It is the cutoff, not the equation! Felix Eckelt1; Maria Schmidt1; Anke Tönjes2; Berend Isermann1; Thorsten Kaiser1 1Universitätsklinikum Leipzig, Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, Leipzig, Germany; 2Universitätsklinikum Leipzig, Clinic and Polyclinic for Endocrinology, Nephrology, Rheumatology, Leipzig, Germany Introduction: Calcium (Ca) disorders are common and adverse events such as coma, cardiac arrest or tetany occur if left untreated. Severe calcium disorders can be treated, but are easy to miss due to multifaceted symptomatics. Diagnostic options available are: total calcium (tCa), free calcium (fCa) and adjusted calcium (aCa). fCa is the analytical gold standard, but blood sampling is laborious, prone to preanalytic error and expensive. tCa is the routine parameter at hospital admission for screening of calcium disorders, yet interpretability is hampered by its dependence on primarily albumin and secondarily anion concentrations. The relationship between tCa and albumin is known to physicians and incorporated into interpretations. aCa calculation is based on published equations (e.g. Payne, Smith), yet, it is consensus not to use published equations at other hospitals without validation. It is clinical practice nonetheless. Our objective at the University Medicine Leipzig (UML) was to develop our own equation for aCa, reevaluate its decision cutoffs based on the gold standard fCa and compare them to established tCa cutoffs. Methods: The ﬁrst German adjusted Ca (aCa) equation was developed using UML measurements of Ca in whole blood (fCa) and serum (tCa), and albumin (2014-2019). Set fCa cutoffs for severe hypocalcemia (< 0.9mmol/L) and severe hypercalcemia (> 1.6mmol/L) were translated to concordant aCa and tCa cutoffs. In a retrospective analysis of inpatient laboratory results and clinical data at UML (2018-19) the equation and cutoffs were applied and alerts for hypo- and hypercalcemia detected accordingly. Results: The presented optimized aCa cutoffs show best concordance with fCa and were similar for our equation and established ones by Payne et al. and Smith et al. Cutoffs differed considerably between tCa and aCa. Also, our optimized cutoffs do not match the recommendations of the European Society of Endocrinology for hypo- (1.9mmol/L) and hyper- calcemia (3.5mmol/L). Retrospective application to UML inpatients (598,592 Ca measurements in 59,209 cases with ≥5y of age) revealed severe hypocalcemia (tCa < 1.6 mmol/L, aCa < 1.9mmol/L) in 2,154 cases (61% female) and severe hyper- calcemia (tCa > 2.8 mmol/L, aCa > 3.1mmol/L) in 353 cases (47% female). Hospital length of stay was increased for severe hypocalcemia and hypercalcemia (8.3d and 17.2d vs. Adjusted Calcium – It is the cutoff, not the equation! 4.7d) as well as admission to ICU treatment (52% and 40% vs. 15%). Conclusions: The largest cohort is presented to evaluate aCa. Translating fCa cutoffs to aCa and tCa equivalents reveals the ﬁrst evidence-based decision thresholds for severe Ca disorders. Optimized cutoffs have greater inﬂuence potential on interpretation quality than optimized equations. We suggest to calculate an aCa and, moreover, to critically assess it with cutoffs tailored to the used equation for best results. Results have been incorporated into the clinical decision support system AMPEL and our laboratory information system. Effects of PCSK9 inhibitors on apolipoproteins Anne Mieritz1; Julia Dittrich1; Ilijana Begcevic Brkovic1; Markus Scholz2; Ulrich Laufs³; Uta Ceglarek1 1University Hospital Leipzig, Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, Leipzig, Germany; 2Leipzig University, Institute for Medical Informatics, Statistics and Epidemiology (IMISE), Leipzig, Germany; ³Universitätsklinikum Leipzig, Klinik und Poliklinik für Kardiologie, Leipzig, Germany Introduction: Apolipoproteins are promising biomarkers for atherosclerotic cardiovascular disease (ASCVD), which repre- sents one of the main causes of death in western society. Hyperlipidemia, especially of LDL, plays a major role in the pathogenesis of ASCVD. Therefore, reduction of atherosclerotic lipoproteins in blood is an important option in the prevention of cardiovascular events. In addition to conventional lipid-lowering drugs, PCSK9 inhibitors represent a new therapeutic approach. These monoclonal antibodies decrease the degradation of the LDL-receptor resulting in an increased LDL intake and, conse- quently, in a reduction of LDL cholesterol levels by 50-60%. However, information on effects on other lipoprotein constituents like apolipoproteins are missing, so far. Therefore, we studied apo proﬁles before and after administration of PCSK9 inhibitors. Methods: In this observational study, 92 patients with diagnosed hyperlipidemia were included. Blood samples were drawn in fasted state prior and 4-8 weeks after administration of the PCSK9 inhibitors Evolocumab and Alirocumab. Two different dosages (75 mg vs. 140/150 mg) were applied. Simultaneous quantiﬁcation of the apos A-I, A-II, A-IV, B-100, C-I, C-II, C III, D, E, H, J, and M was performed from 3 μl serum by LC-MS/MS. Group comparisons were performed using paired sample t-test, Kruskal-Wallis H test, Mann-Whitney-U-test and multivariate regression. Results: PCSK9 administration reduced serum levels of the LDL- and triglyceride-rich lipoproteins-associated apos B-100, C-I, C-II, C-III, and E by 12% to 46%. Furthermore, concentrations of apos D, and M were also decreased by up to 15%. These effects were independent from the subjects’ potential lipid-lowering premedication, naming statins, ezeti- mibe or a combined administration, as well as from PCSK9 inhibitor-induced changes in LDL cholesterol. Interestingly, the effects of PCKS9 inhibitor administration on LDL cholesterol and apos were not dose-dependent. German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 eA15 Conclusion: Apolipoprotein proﬁles are altered by PCSK9 inhibitors independently from total and LDL-cholesterol levels and provide additional information of PCSK9 effects on lipoprotein metabolism. Conclusion: Apolipoprotein proﬁles are altered by PCSK9 inhibitors independently from total and LDL-cholesterol levels and provide additional information of PCSK9 effects on lipoprotein metabolism. eA16 Zentrum für klinische Transfusionsmedizin, Transfusionsmedizin, Tübingen, Germany Aims: Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare but serious complication of vector based COVID-19 vaccines. Similar due heparin-induced thrombocytopenia (HIT), antibodies reacting to platelet factor 4 (PF4) are responsible from platelet activation in VITT. The diagnosis of VITT includes the detection of anti-PF4 antibodies. Particle gel immunoassay is one of the rapid tests, that is commonly used in the diagnosis of HIT to detect anti-PF4 antibodies. The aim of the current study was to investigate the diagnostic performance of rapid immunoassay in patients suspected of VITT. Materials and Methods: In this retrospective, single-center study, the correlation between rapid immunoassay, enzyme- linked immunosorbent assay (ELISA) and modiﬁed heparin induced platelet aggregation assay (HIPA) in patients with ﬁndings suggestive of VITT was investigated. A commercially available PF4 rapid immunoassay (ID PaGIA H/PF4, Diamed) and an anti PF4/Heparin ELISA (Zymutest HIA IgG, Hyphen) were used according to manufacturer´s in- structions. A sample was considered reactive in ELISA if the optical density (OD) was ≥0.500. Modiﬁed HIPA was accepted as the gold standard test. Results: Between March 8th and May 20th, 21 samples from clinically well-characterized patients were analyzed with rapid immunoassay, ELISA and HIPA. Of these sera 8 revealed positive results and 13 tested negative in rapid immu- noassay. Sensitivity and speciﬁcity of rapid immunoassay were 50% and 69%, respectively. On the other hand, sensitivity and speciﬁcity of ELISA were 88% and 92%, respectively. Discussion: Particle gel immunoassay is not reliable in the detection of anti-PF4 antibodies in patients suspected of VITT. Commercially available PF4 rapid immunoassays are validated for the diagnosis of HIT. They should not be used to rule out VITT. The reliability of rapid immunoassay for the detection of anti-PF-4 antibodies in patients suspected of vaccine-induced immune thrombotic thrombocytopenia after COVID-19 vaccination Günalp Uzun; Karina Althaus; Stefanie Hammer; Yvonne Wanner; Stefanie Nowak-Harnau; Sigrid Enkel; Tamam Bakchoul eA16 German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 Results: CHE was positively associated with liver fat content, independent of sex, age and BMI (p < 0.0001). CHE activity was higher in participants fulﬁlling the IDF-criteria for the metabolic syndrome (p < 0.0001). Results: CHE was positively associated with liver fat content, independent of sex, age and BMI (p < 0.0001). CHE activity was higher in participants fulﬁlling the IDF-criteria for the metabolic syndrome (p < 0.0001). CHE was also positively associated with the fasting plasma glucose and glucose during the OGTT (AUCglucose), inde- pendent of sex, age and BMI (both p< 0.0001). While CHE was not associated with insulin secretion (p=0.7, adj. sex, age, insulin sensitivity), it was negatively associated with insulin sensitivity, independent of sex, age and BMI (p < 0.0001), as well as after additional adjustment for liver fat content (p < 0.0001). The reduction of liver fat content during lifestyle intervention was associated with a reduction in CHE, independent of body weight loss (p < 0.0001). The change in CHE was furthermore associated with the improvement in insulin sensitivity (p < 0.0001). This remained signiﬁcant even after adjustment for sex, age, BMI and liver fat (p=0.01). Conclusion: Our cross-sectional and longitudinal results using state-of-the-art approaches for metabolic phenotyping conﬁrm that higher CHE is a marker for liver fat accumulation and is present in patients with metabolic syndrome. We furthermore detected links to glucose tolerance and insulin sensitivity. Of note, these were independent of liver fat content. This suggests that CHE could be not just a marker for liver steatosis but could be indicative of processes in hepatocytes that contribute to metabolic health. Further investigations are needed to clarify the mechanistic contribution and potential diagnostic value of elevated CHE in hepatic steatosis and metabolic diseases. Elevated cholinesterase activity and the metabolic syndrome - dissecting fatty liver, insulin resistance and dysglycemia Martin Heni1; Julia Hummel2; Louise Fritsche2; Robert Wagner³; Lasse Relker1; Jürgen Machann⁴; Fritz Schick⁴; Andreas Birkenfeld³; Hans-Ulrich Häring³; Norbert Stefan³; Andreas Fritsche³; Andreas Peter1 1Universitätsklinikum Tübingen, Institut für Klinische Chemie und Pathobiochemie, Tübingen, Germany; 2Helmholtz Zentrum München, IDM, Tübingen, Germany; ³Universitätsklinikum Tübingen, Medizinische Klinik IV, Tübingen, Germany; ⁴Universitätsklinikum Tübingen, Diagnostische und Interventionelle Radiologie, Tübingen, Germany Aims: While low plasma cholinesterase activity (CHE) is a well-established marker of reduced hepatic synthesis capacity, the clinical signiﬁcance of elevated activity is not clear. High CHE was long suspected to be present in hepatic steatosis and metabolic syndrome, as reported by older and smaller studies. We aimed to clarify the relation between CHE and the metabolic syndrome as well as with precisely quantiﬁed liver fat content. Materials and Methods: CHE activity was measured photometrically (Butyrylthiocholin 5-Thio-2-nitrobenzoat) in lithium- heparin plasma in 844 humans (554 women) of the cross-sectional Tübingen Diabetes Family Study with a wide BMI range (17.6 – 55.1 kg/m2) and without severe diseases (including liver diseases). It was furthermore retrospectively measured in 108 participants of the Tübingen Lifestyle Intervention Program (TULIP) before and after a 9-month lifestyle intervention. Liver fat content was quantified with MR-spectroscopy. All participants underwent detailed metabolic phenotyping including a 2-h 75 g oGTT with glucose, insulin and C-peptide measurements at every 30 minutes. From that, insulin sensitivity was assessed using the Matsuda formula and insulin secretion was estimated as the AUC0-30 of insulin / AUC0- 30 of glucose. German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 eA17 Platelets and neutrophil extracellular traps promote thrombo-inflammation and glomerular endothelial dysfunction in diabetic kidney disease Anubhuti Gupta1; Kunal Singh2; Ruaa Younis2; Sameen Fatima2; Saira Ambreen2; Silke Zimmermann2; Shruthi Krishnan³; Ronald Biemann⁴; Khurrum Shahzad2; Berend Isermann2; Shrey Kohli2 1Universitätsklinikum Leipzig, Institute of laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, 04103, Germany; 2Universitätsklinikum Leipzig, Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, 04103, Germany; ³Otto-von-Guericke Universität Magdeburg, Institute of Experimental Internal Medicine, 39120, Ger- many; ⁴Universitätsmedizin Leipzig, Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, 04103, Germany Aim: Diabetic kidney disease (DKD) is a major cause of end-stage renal failure contributing to morbidity and mortality worldwide. Therapeutic options to prevent or reverse DKD progression are limited or lacking, respectively. Endothelial dysfunction, platelet-hyperactivity, immune cell inﬁltration and glomerular ﬁltration barrier (GFB) disruption are associated with DKD. We aim to scrutinize the mechanistic interplay between platelets and neutrophil extracellular traps (NETs) and ensuing renal thrombo-inﬂammation. Method: A type 1 diabetes mouse model (streptozotocin) was used to evaluate the role of platelet activation and NET formation in DKD. Therapeutic interventions (ASA, Anakinra, Solulin, GSK484) were performed in sub-groups of mice between week 16 to 24 of diabetes to study disease reversal. In vitro studies were performed using glomerular endothelial cells (GENC), platelets and neutrophils exposed to high glucose (HG) in static and ﬂow conditions. Result: Experimental DKD in C57Bl6 mice resulted in albuminuria, increased fractional mesangial area, activated platelets (CD62P) and neutrophil extracellular traps (NETs; H3Cit, NE, PAD4) within glomeruli. In parallel, increased expression of inﬂammasome markers (NLRP3, IL1β) and reduced expression of coagulation regulator thrombomodulin (TM) was observed. In vitro, platelets and NETs exacerbate inﬂammasome markers (IL1β, NLRP3), reduce endothelial function markers (p-eNOS, KLF2, KLF4 and TM) in GENC and disrupted the glomerular ﬁltration barrier (enhanced FITC-dextran leakage, disoriented VE cadherin) in HG conditions. Under ﬂow condition, platelets enhanced NET for- mation on GENC monolayers exposed to HG. Inhibition of platelet activation (ASA), amelioration of NETs by inhibition of histone citrullination PAD4 inhibition (GSK484), IL-1 receptor inhibition (anakinra) or restoring TM expression (solulin) ameliorated these effects in vitro and in vivo. Further experiments targeting P-selectin mediated platelet-neutrophil interactions and evaluating the clinical relevance in patient cohorts are under-progress. Conclusion: Taken together, hyperglycemia promotes platelet-neutrophil interactions resulting in intraglomerular NET formation,sterileinﬂammation,glomerularendothelialdysfunction,andbarrierdisruption.Thisresultsinaggravateddisease course and impaired renal health in DKD. Inhibition of platelets or NETs is a promising therapeutic strategy for DKD. Sera of Syphilis patients contain Antiphospholipid Antibodies with procoagulant properties Nadine Müller-Calleja1; Anne Hollerbach2; Karl J. Lackner2 1Universitätsmedizin der Johannes Gutenberg-Universität Mainz, Institut für klinische Chemie und Laborator- iumsmedizin, Mainz, Germany; 2Universitätsmedizin der Johannes Gutenberg-Universität Mainz, Institut für klinische Chemie & Laboratoriumsmedizin, Mainz, Germany Objectives: Antiphospholipid antibodies (aPL) are causally involved in the development of the antiphospholipid syn- drome (APS), an autoimmune disease characterized by recurrent thrombosis and fetal loss. Beside APS, many infections have been found to be associated with elevated aPL titers. Syphilis was the ﬁrst infection to be linked to the aPL. Since Syphilis patients show a higher risk for stroke, we want to analyze whether this was due to the procoagulant properties of anti-cardiolipin(aCL) aPL that are found in these patients. Methods: IgG fractions were prepared from those Syphilis patient’s sera with a positive aCL titer. The ability of these IgG fractions to activate Tissue factor, to induce endosomal ROS generation and to stimulate TNFα production was measured in human monocytes by a single stage clotting assay, by ﬂow cytometry or by qRT-PCR, respectively. Results: aPL isolated from Syphilis patients could be characterized as cofactor-independent aCL since they bind to cardiolipin in the absence of any cofactors as shown by a self-made ELISA. In contrast, these Syphilis aCL (s-aCL) could not bind to b2GPI. S-aCL were able to activate the same signaling pathway that has recently been reported for autoimmune-type lipid-reactive aPL: lysobisphosphatidic acid (LBPA) presented by the CD1d-like endothelial protein C receptor (EPCR) EPCR serves as the cell surface receptor for s-aCL. Binding of s-aCL to this LBPA-EPCR complex mediates Tissue factor activation on monocytes, s-aCL internalization and subsequent sensitization of immune cells to toll-like receptor 7 agonists. Moreover, this signaling complex speciﬁcally participates in interferon responses in monocytes and dendritic cells. However, compared to aCL isolated from APS patients, s-aCL must be used in a 3-4 fold higher concen- tration than classical aPL to achieve the same effects. Conclusion: s-aCL have procoagulalant and proinﬂammatory properties. Therefore they can increase the risk for stroke. However, compared to aPL isolated from APS patients, s-aCL have a lower afﬁnity for cardiolipin. This may be the reason why thromboembolic events do not occur more frequently in syphilis patients. eA18 German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 eA18 Pseudoxanthoma elasticum (PXE) is an autosomal recessive disorder which is manly caused by diverse mutations in the gene encoding the ATP-binding cassette sub-family C member 6 (ABCC6). Clinical, PXE shows some characteristics of the elderly, like arteriosclerosis, loss of skin elasticity and visual impairment. As described in further studies, there are similarities between PXE and premature aging which could be seen by high activity of senescence associated ß-galacto- sidase (SA ßGal) or expression of proinﬂammatory factors like interleukin-6 (IL6) or monocyte chemoattractant protein-1 (MCP1), in ABCC6-deﬁcient ﬁbroblasts. On molecular level PXE shares some features of premature aging diseases such as Hutchinson-Gilford progeria syndrome (HGPS). Both diseases are characterized by decreased levels of adenosine- triphosphate (ATP) and pyrophosphate (PPi). Further studies revealed a permanent activation of JAK-STAT3 signaling pathway in dermal ﬁbroblasts from HGPS patients. It was shown that treatment with JAK-inhibitor bariticinib reduces inﬂammation in HGPS ﬁbroblasts. Thus, the aim of this study was to evaluate the activation level of JAK-STAT3 and the effect of JAK-inhibitor bariticinib in dermal ﬁbroblasts of PXE patients. Normal human dermal fibroblasts (NHDF) (n=3) and ﬁbroblasts from PXE patients (n=3) were seeded with a ﬁnal density of 177 cells/mm2. Medium was changed after 24 h to medium with lipoprotein-deﬁcient serum (LPDS) and bariticinib. The level of activated pSTAT3 was measured after additional 72 h incubation, by immunoﬂuorescence and western blot. Also, quantitative real-time polymerase chain reaction for analyzing mRNA expression of IL6 and MCP1, as well as IL6 protein concentration from cell culture supernatants was analyzed. Immunofluorescence and western blot analysis showed increased pSTAT3 levels in untreated PXE fibroblasts, compared to the NHDF. Treatment with bariticinib reduced activated pSTAT3 in PXE fibroblasts to the level of NHDF. Expression of IL6, MCP-1, as well as IL6 concentration in supernatants were increased in untreated and treated PXE fibroblasts compared to NHDF. There were no significant changes in IL6 expression and IL6 concentration in treated PXE fibroblasts in contrast to untreated PXE fibroblasts. The expression of MCP-1 was significantly reduced in bariticinib treated PXE fibroblasts and NHDF in comparison to the respective untreated fibroblasts. Our data indicate that JAK-STAT3 signaling pathway is activated in PXE fibroblasts. Treatment with JAK-inhibitor bariticinib, only reduces MCP-1 gene expression but had no effect on IL6 gene expression and IL6 protein concentration in supernatant of PXE fibroblasts. In conclusion, JAK-STAT3 signaling pathway seems to induce MCP-1 gene expression but does not seem the dominant driving force for enhanced IL6 secretion in PXE. Further studies are needed to solve the question by which mechanisms IL6 is activated in PXE. Bariticinib reduces STAT3 activation and partly inflammatory processes in dermal fibroblasts from patients with Pseudoxanthoma elasticum Christopher Lindenkamp; Ricarda Plümers; Michel Robin Osterhage; Isabel Faust-Hinse; Cornelius Knabbe; Doris Hendig Herz- und Diabeteszentrum NRW, Institut für Laboratoriums- und Transfusionsmedizin , Bad Oeynhausen, Germany Christopher Lindenkamp; Ricarda Plümers; Michel Robin Osterhage; Isabel Faust-Hinse; Cornelius Kn Christopher Lindenkamp; Ricarda Plümers; Michel Robin Osterhage; Isabel Faust-Hinse; Cornelius Knabbe; Doris Hendig Herz- und Diabeteszentrum NRW, Institut für Laboratoriums- und Transfusionsmedizin , Bad Oeynhausen, Germany Herz- und Diabeteszentrum NRW, Institut für Laboratoriums- und Transfusionsmedizin , Bad Oeynhausen, Germany German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 eA19 Anti-DFS70: prevalence and diagnostic significance in antinuclear antibody (ANA)-positive patients Ivana Markovic; Andreas Fischer; Gry Helene Dihazi Universitätsmedizin Göttingen, Interdisziplinäres UMG-Labor, Göttingen, Germany Aims: Laboratory screening of anti-nuclear antibodies (ANAs) by indirect immunoﬂuorescence method (IIF) is often positive in patients without proven autoimmune pathologies. The most frequent pattern, which is detected is a « dense ﬁne speckled » (DFS) pattern (AC-2), characterized by the ﬁne-granular ﬂuorescence of the nuclei in the chromatin interphase and metaphase, and could occur in 2-22% of healthy individuals, infection, cancer and inﬂammatory con- ditions. However, there is still need for information about its clinical signiﬁcance. This study aimed to investigate the performance of available routine screening methods for detection of ANAs and anti-DFS70 antibodies, and the clinical signiﬁcance of anti-DFS70 autoantibodies using remaining patient samples, which were sent to laboratory for ANA detection. eA20 German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 eA20 Material and Methods: 31 serum samples routinely requested for ANAs screening were analyzed using IIF on HEp-2 cell substrates (Euroimmun, Germany). The semi-quantitative determination of the anti-histone autoantibodies in the pa- tient’s serum was carried out using an enzyme immunoassay (Euroimmun, Germany). For detecting autoantibodies against dsDNA, U1RNP, Sm, Ro/SSA, La/SSB, Scl-70, Pm-scl, Jo-1 and CENP a quantitative ﬂuorescence enzyme immunoassay for extractable nuclear antigen screen was performed (Thermo Fisher Scientiﬁc, Germany). Immunoblot (Euroimmun, Germany) enabled the detection of 14 autoantibodies against EJ, Jo1, Ku, MDA5, Mi-2α, Mi-2β, NXP2, OJ, PL-7, PL-12, PMScl100, PMScl75, Ro-52, SAE1, SRP and TIF1γ. The quantitative in-vitro measurement of antibodies of the IgG class against DFS70 in serum was performed using a quantitative ﬂuorescence enzyme immunoassay (Thermo Fisher Scientiﬁc, Germany). Demographic and clinical data were analyzed from the medical records. Results: Among the 31 samples, which were tested for ANA, 20 (64.5%) were ANA positive by IIF. The frequency of AC-2 immunoﬂuorescence pattern by ANA-IIF was 16.2% (5/31), of these only four samples contained antibodies against DFS70 in serum. No signiﬁcant differences were observed between anti-DFS70 positive and anti-DFS70 negative patients concerning age, gender, symptoms, clinical signs or other disease-speciﬁc antibodies. 75% of the patients with positive DFS70 antibody was without proven autoimmune pathologies. However, of the four anti-DFS70 positive patients, only one patient had accompanying autoantibodies (anti-histone and anti-dsDNA). Conclusion: Autoantibodies against DFS70 are less prevalent in patients with proven autoimmune pathologies. Monospeciﬁc anti-DFS70 antibodies are signiﬁcant in excluding ANA-associated rheumatic disease in patients presented with an AC-2 pattern. It has been observed that anti-DFS70 autoantibodies may be associated with non-ANA-associated rheumatic diseases and in many diseases related to other systems. Therefore, it is essential to evaluate these pathologies in patients positive for anti-DFS70 antibodies. 1 year follow up study after mild COVID-19 still shows decent t-cell response in patients with weak antibody formation Tobias Flieder; Monika Wolny; Katharina von Bargen; Bastian Fischer; Cornelius Knabbe; Ingvild B Herz- und Diabeteszentrum Nordrhein-Westfalen, Universitätsklink der Ruhr-Universität Bochum, Bad Oeynhausen, Germany, Institut für Laboratoriums- und Transfusionsmedizin, Bad Oeynhausen, Germany Introduction: More than 2 years ago, there were the ﬁrst reports from China of infections with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19). The two systems the body has available to protect itself against pathogens are the humoral and cellular immune responses. The humoral immune response provides for the formation of antibodies against the corresponding pathogen, whereas in the cellular immune response, immune cells such as T cells directly attack the virus or secrete interferons. It is already known that the humoral immune response after vaccination or infection with SARS-CoV-2 is maintained for only a few months. Over which period the cellular immune response remains active and whether there is a correlation between humoral and cellular immune response after SARS-CoV-2 vaccination/infection is less investigated. Methods: In our study, we included 50 individuals from a collective (426 individuals) that we screened for antibodies to SARS-CoV-2 between March 8 and April 8, 2020. These 50 individuals had mild infection with SARS-CoV-2, conﬁrmed by viral detectionbyRT-PCR,butdid notdevelop antibodiestoSARS-CoV-2ordid soforashortperiod oftime.Approximatelyoneyear after infection (291 - 380 days), we retested these individuals for SARS-CoV-2 antibodies using six different assays. We also tested the individuals for interferon-gamma release by T cells upon exposure to SARS-CoV-2 peptides. Results: The mean age of the participants was 49 years (interquartile range (IQR): 16.35) and 52% of participants were female. The mean duration of COVID-19 was 12.5 days (standard deviation (sd): 7.6). No participant requiring hospital- ization. The time between symptom onset and readmission for the 1-year follow-up was 321.5 days (IQR: 49.3). Mea- surement of IFN-γ release by T-cells induced with SARS-CoV-2 peptides showed increased IFN-γ release in 76% of patients. This cut-off value for this assay was determined from measurements of individuals who had no history of infection with SARS-CoV-2. In the determination of antibodies, we were able to show that there were very large differences between the assays used. The proportion of positive results varied between 8% and 66%. Conclusion: We were able to show in our study that the majority of individuals still have a cellular immune response approximately 1 year after SARS-CoV-2 infection, although they showed only a mild course and no/weak humoral immune response. Investigation of the presence of immature platelets in COVID-19 Monika Wolny; Tobias Flieder; Katharina von Bargen; Anne-Kathrin Vollmer; Cornelius Knabbe; Ingvild Birschmann Herz- und Diabeteszentrum Nordrhein-Westfalen, Universitätsklinik der Ruhr-Universität Bochum, Institut für Labo- ratoriums- und Transfusionsmedizin, Bad Oeynhausen, Germany SARS-CoV-2 (severe acute respiratory syndrome coronavirus type 2), which first emerged in late 2019, has infected about 520 ⋅10∧6 people worldwide to date and led to more than 6.2 ⋅10∧6 deaths. Infection can lead to a spectrum of COVID-19 (coronavirus disease 2019), ranging from mild cold-like symptoms to severe courses with acute respiratory distress syndrome and organ failure. The latter is often associated with pathological coagulation parameters and thromboembolic complications. In particular, immature platelets appear, which are hyperreactive and show prothrombotic activity. Immature or reticulated platelets are the platelets newly formed by megakaryocytes and released from the bone marrow into the bloodstream. IPF (immature platelet fraction) refers to the proportion of immature platelets among the total number of platelets. The fraction is between 0 and 6% in healthy individuals. The aim of this study was to determine the course of IPF during severe COVID-19. From December 2020 to July 2021, hospitalized patients with a predominantly severe COVID-19 course were included in this study. Daily determination of IPF was performed using the Sysmex XN-1000 hematology analyzer. In case of platelet transfusion, the readings of the following three days were not included in the analysis. Because of the ﬂuctuating number of readings, the ﬁrst 40 days after intubation or hospitalization were analyzed. A total of 83 patients were enrolled in this study. 47 patients with a severe COVID-19 course (intubation and ECMO), 21 patients with a moderate course (intubation, no ECMO), and 15 patients with a mild COVID-19 course (no intubation, no ECMO) were included. The patients with a mild course showed no thrombocytopenia and a short-term increase in median IPF above the upper norm 14 days after hospitalization (max. 8.7%). The moderately ill patients also showed no median thrombocytopenia but increased IPF (max. 9.0%) with few exceptions in the ﬁrst 20 days after intubation. The severely ill German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 eA21 patients had the lowest median platelet count (90 - 171 ∙10∧6/mL) and the highest median percentage of IPF (max. 14.1%, day 39) from the second day after intubation to day 40. Investigation of the presence of immature platelets in COVID-19 Comparison of IPF between surviving and deceased patients in the moderate and severe groups revealed higher than average IPF in the ﬁrst 40 days after intubation (days 0 and 1 not included) for both groups. However, there was a clear difference between deceased (median 10.2-18.4%) and surviving (5.4-11.6%) patients. In this study, it was observed that in a severe course of COVID-19, the number of platelets decreases and the number of newly formed immature platelets increases. This increase in IPF appears to be more pronounced in patients who die than in surviving patients. However, it should be explicitly noted that this is a purely descriptive observation, as the measured values vary widely. Further studies are needed to predict disease progression based on IPF. Comparison of two procalcitonin reagents in SARS COV 2 patients Borros Arneth1; Samr Mkhlof2; Janina Trauth³; Susanne Herold³; Thomas Voitz⁴; Chrysanthi Skevak 1Justus Liebig University Giessen, Institute for Laboratory Medicine, Giessen, Germany; 2Justus Liebig University Giessen, Institute of Laboratory Medicine, Giessen, Germany; ³Justus Liebig University Giessen, Internal Medicine Infectious Diseases, Giessen, Germany; ⁴Synlab Trier, Labor, Trier, Germany Borros Arneth1, Samr Mkhlof1, Janina Trauth2, Susanne Herold2, Thomas Voitz3, Chrysanthi Skevaki1 and Harald Renz Borros Arneth1, Samr Mkhlof1, Janina Trauth2, Susanne Herold2, Thomas Voitz3, Chrysanthi Skev 1Institute of Laboratory Medicine and Pathobiochemistry, Justus Liebig University Giessen; 2Clinic of Internal Medicine, Division for Infectious Diseases, University Hospital Giessen; 3Synlab MVZ Trier Introduction: Plasma procalcitonin is extensively used in clinical laboratories for the early detection of bacterial infections and sepsis. Differences in sensitivity and speciﬁcity between commercially available reagents and auto- mated platforms have been previously described. The aim of the current study was to determine and compare the diagnostic performance of reagents of Diazyme laboratories on the ADVIA (Siemens) and the Thermoﬁsher Scientiﬁc / Brahms reagent on the Centaur (Siemens) among COVID-19 patients with a clinical suspicion for bacterial superinfection. Method: PCT reagents from Diazyme Laboratories for the ADVIA (Siemens) and from Thermoﬁsher Scientiﬁc /Brahms for the Centaur (Siemens) have been compared using SARS COV2 positive patient samples. For the comparison to a gold standard the collected samples were additionally measured with Thermoﬁsher/ Brahms reagent on a Kryptor (Brahms) in Trier. Results: We were able to determine signiﬁcant differences between the two reagents in about every second patient sample. Thereby the level of the Brahms/Thermoﬁsher measurement always was signiﬁcantly lower compared to the PCT level measured using the Diazyme reagent. The level of the Diazyme values often did not match with the clinical picture of the patient. Conclusion: There are signiﬁcant differences between the two reagents from Brahms/Thermoﬁsher and Diazyme, not only with regard to the sensitivity of the reagents, but also with regard to the speciﬁcity of the two reagents. 1 year follow up study after mild COVID-19 still shows decent t-cell response in patients with weak antibody formation This is encouraging ﬁnding in terms of long-term immunity to SARS-CoV-2. eA22 German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 Myeloid Activation Marker by Flowcytometry Borros Arneth1; Samr Mkhlof2; Silva Herzog2; David Krieger³; Chrysanthi Skevaki2; Harald Renz2 1Justus Liebig University Giessen, Institute for Laboratory Medicine, Giessen, Germany; 2Justus Liebig University Giessen, Institute of Laboratory Medicine, Giessen, Germany; ³Justus Liebig University Giessen, Emergency Medicine, Giessen, Germany Samr Mkhlof1, Borros Arneth1, Silva Herzog1,2, David Krieger2, Chrysanthi Skevaki1 and Harald Renz 1Institute of Laboratory Medicine and Pathobiochemistry, Justus Liebig University Giessen; 2Department of Emergency Medicine, University Hospital Giessen Introduction: The expression of speciﬁc markers (CD64 on neutrophils and CD169 on monocytes) occurs on leukocytes due to viral and bacterial infections. We here want to use these novel markers to identify the cause (viral, bacterial) of an infectious disease in early stages and additionally we want to compare them to the established markers. German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 eA eA23 Methods: In addition to the usual laboratory routine, we took an EDTA whole blood sample from about 100 patients at admission to the emergency room with the new onset of fever. Exclusion criteria were patients with already known infections, myocardial infarction, lung emboli, and stroke. In this EDTA whole blood we examined the markers (CD64, CD169 and HLA-DR) on leukocytes by means of ﬂow cytometry additionally to conventional lab routine (Complete blood count, CRP, PCT, microbiology). Methods: In addition to the usual laboratory routine, we took an EDTA whole blood sample from about 100 patients at admission to the emergency room with the new onset of fever. Exclusion criteria were patients with already known infections, myocardial infarction, lung emboli, and stroke. In this EDTA whole blood we examined the markers (CD64, CD169 and HLA-DR) on leukocytes by means of ﬂow cytometry additionally to conventional lab routine (Complete blood count, CRP, PCT, microbiology). Results: Bacterial infections can reliably be detected by using the overexpression of the marker CD64 on neutrophils. Most patients we detected with viral expression type (CD169 positive on monocytes) had SARS COV2 disease. The expression of markers on leukocytes is compared to the classical diagnostic tools as leukocytes, differential blood count, CRP, PCT and microbiology. Conclusion: Flow cytometry is excellent for distinguishing bacterial from viral infectious diseases. In addition, if both marker proﬁles are present at the same time, it is also possible to identify patients with a viral disease and a bacterial superinfection. Early detection of life-threatening infections in the emergency department using metabolomics Sophie Neugebauer1; Johannes Winning2; Johannes Winning³; Thomas Lehmann⁴; Michael Bauer³; Michael Kiehntopf1 1Jena University Hospital, Department of Clinical Chemistry and Laboratory Diagnostics, Jena, Germany; 2University of Applied Sciences, Ernst-Abbe-Hochschule, Jena, Germany; ³Jena University Hospital, Department of Anesthesiology and Intensive Care, Jena, Germany; ⁴Jena University Hospital, Institute of Medical Statistics, Computer Sciences and Data Sciences, Jena, Germany Aims: Despite intensive research, sepsis remains a life-threatening organ dysfunction with high mortality (25-50%) [1, 2]. Early sepsis diagnosis is complicated by primarily nonspeciﬁc symptoms. Up to 40% of patients with severe sepsis are admitted to the emergency department [3]. Early recognition in the emergency department and initiation of therapeutic interventions is necessary to increase and improve patient survival [2,4,5]. The appearance, accumulation, or persistence of metabolites during the infection-related host response could serve as a surrogate for impaired metabolic control and for monitoring disease severity. Thus, the study aims to identify the most appropriate metabolites or combinations of metabolites for early sepsis diagnosis, identiﬁcation of sepsis-related organ dysfunction and risk stratiﬁcation of patients with suspected sepsis. Materials and Methods: A total of 188 metabolites comprising six analyte classes were measured using LC-MS/MS in lithium heparin plasma samples from 400 patients on admission to the emergency department of the university hospital Jena. 160 and 24 patients developed sepsis and septic shock within 96 hours, respectively. Procalcitonin was used as reference laboratory parameter. Results: The primary study endpoint was to deﬁne metabolites that can early identify patients with sepsis or septic shock. 44 and 30 metabolites were altered after correction for multiple testing in patients developing sepsis or septic shock, respectively. Mainly three metabolite classes: amino acids, lysophosphatidylcholines and phosphatidylcholines were associated with sepsis or septic shock and mostly decreased in their concentration during infection. Promising metabolites were selected and combined with a LASSO-regression with a 20-fold cross-validation. ROC-analysis of these models showed a sensitivity of 80.6% or 91.7% and a speciﬁcity of 79.6% or 90.2% for early detection of sepsis or septic shock, respectively. Furthermore, amino acids, biogenic amines and lysophosphatidylcholines showed high potential for providing infor- mation of organ dysfunction or poor patients’ outcome. Significant altered metabolites were combined for prognosis of unfavorable outcome of patients or the need of interventions at the intermediate care unit. eA24 Discussion/Conclusions: The data suggests a low concordance of anti-S and anti-N antibodies in children. This could be due to a quick waning of anti-N antibodies. Therefore, antibody assays results of patients should be interpreted accordingly and seroprevalence studies, the ones relying on anti-N antibodies in particular, can only display the infection rates of the recent past. Our ﬁndings indicate a rising seroprevalence in the pediatric population in Germany. Our results are in line with other seroprevalence studies and therefore show that using residual blood samples of patients is a resource-saving but accurate way of sampling and obtaining patient material for seroprevalence estimation. Our study design can be easily implemented into daily laboratory routines and is a useful tool for seroprevalence surveillance. Continuous monitoring of SARS-CoV-2 seroprevalence in children using residual blood samples from routine clinical chemistry Felix Wachter1; Adrian Regensburger2; Antonia Sophia Peter³; Ferdinand Knieling⁴; Alexandra Wagner⁵; David Simon⁶ André Hörning⁴; Joachim Wölﬂe⁴; Klaus Überla³; Antje Neubert⁴; Manfred Rauh⁴ 1Friedrich-Alexander-Universtität Erlangen-Nürnberg, Department of Pediatrics and Adolescent Medicine, Erlangen, Germany; 2FAU Erlangen-Nürnberg, Pädiatrie Erlangen, Erlangen, Germany; ³Friedrich-Alexander University (FAU) Erlangen- Nürnberg, Institute of Clinical and Molecular Virology, Erlangen, Germany; ⁴Friedrich-Alexander-University (FAU) Erlangen-Nürnberg, Department of Pediatrics and Adolescent Medicine, Erlangen, Germany; ⁵Friedrich-Alex- ander-University (FAU) Erlangen-Nürnberg, Department of Pediatrics and Adolescent Medicine, 91054, Germany; ⁶Friedrich-Alexander University (FAU) Erlangen-Nürnberg, Department of Internal Medicine 3, Erlangen, Germany Aims: The assessment of SARS-CoV-2 infections in children is still challenging, but essential for appropriate political decisions. The aim of this study was to investigate whether residual blood samples can be used for SARS-CoV-2 sero- prevalence monitoring in pediatrics and to calculate the seroprevalence and underreporting in the pediatric population. Infection rates of SARS-CoV-2 in children remain a widely discussed topic. In this study we aim to establish a study design to determine seroprevalence of SARS-CoV-2 antibodies in the pediatric population in a resource saving and cost effective manner. Materials and Methods: Residual blood samples from pediatric patients were collected in six time periods (Oct.–Nov. 2020, April 2021, and June–July 2021, November 2021, January 2022 and March 2022). They were analyzed for SARS-CoV-2 Spike protein (anti-S) and nucleocapsid (anti-N) antibodies using a commercial antibody assay by Roche diagnostics. 28 samples were further analyzed for neutralizing capability. The calculated seroprevalence was then compared to the number of ofﬁcially reported cases to calculate the extent of underreporting. While analysis of the data for the collection periods 4, 5 and 6 is still ongoing, results for the first three sample periods are already available and published by Wachter et al: Seroprevalence obtained from the antibody results of 2,626 patient samples increased from 1.38% to 9.16% and to 14.59% during the three time periods. Nucleocapsid seroprevalence was lower in all three sample periods (1.26%, 6.19%, 8.56%). Seroprevalence therefore was 3.93-5.66-fold higher than the number of cases reported by the health authorities. However, a good correlation between the reported cumulative incidence of individual provinces and the assigned seroprevalence was found (r=0.74, p=0.0151). Of the 14 samples reactive for anti-S and anti-N antibodies 11 showed neutralization capabilities, while of the 14 samples only reactive for anti-S antibodies 8 showed neutralization capabilities. Antibody levels did not differ between age groups or sexes (all p>0.05). Continuous monitoring of SARS-CoV-2 seroprevalence in children using residual blood samples from routine clinical chemistry eA24 German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 Abass Eidizadeh; Mechthild Wiederhold; Moritz Schnelle; Andreas Fischer; Lutz Binder Abass Eidizadeh; Mechthild Wiederhold; Moritz Schnelle; Andreas Fischer; Lutz Binder Universitätsmedizin Göttingen, Institut für Klinische Chemie, Göttingen, Germany Universitätsmedizin Göttingen, Institut für Klinische Chemie, Göttingen, Germany Introduction: Procalcitonin (PCT) is an important biomarker of sepsis and respiratory infections. Various automated immunoassays for measuring PCT in patient plasma are available in medical laboratories. However, due to a lack of international reference material for PCT, the assays are not always comparable. Methods: In this study, we compared a new turbidimetric immunoassay from DiaSys, measured on the Abbott Architect c16000 and Alinity c, with four BRAHMS-associated chemiluminescence immunoassays (Abbott Architect i2000SR, Alinity i, Roche Cobas e411 and DiaSorin Liaison XL) using 120 random patient plasma samples from the clinical laboratory routine at the University Medical Center Goettingen. Results: The DiaSys assay showed clear differences as compared to the BRAHMS-associated assays when measured on Architect c: i.e. 58% positive mean bias vs. Architect i, 67% vs. Cobas and 23% vs. Liaison. As a result, additional 19% our patients would have a suspected bacterial infection, when using PCT values from the DiaSys assay and commonly accepted decision limits. A crosscheck of the DiaSys calibrator on the BRAHMS-associated systems showed a low recovery of the calibrator material (approx. 50%). Conclusions: Overall, this study shows signiﬁcant differences between the DiaSys and BRAHMS-associated assays. This could be attributed to a potential DiaSys calibrator problem. This highlights the need for an international reference material for harmonization of the PCT assays. Control of neutrophil effector function by the actin-regulatory protein Coronin-1a Anton Shaverskyi; Lee Kyeong-Hee; Niko Föger Medizinische Hochschule Hannover, Institut für Klinische Chemie, Hannover, Germany Background: Neutrophils are innate immune cells that as key players of acute inﬂammation play fundamental roles in antimicrobial responses but can also contribute to inﬂammation-related tissue damage. To encounter pathogenic challenge, neutrophils have developed powerful defense mechanisms, such as the release of bioactive mediators from secretory granules and the generation of reactive oxygen species (ROS) to kill phagocytosed microbes. Neutrophil activation and execution of effector function involves dynamic reorganization of the actin cytoskeleton. The regulatory pathways and functional links between neutrophil activation/function and actin cytoskeletal regulation are, however, still only poorly understood. Early detection of life-threatening infections in the emergency department using metabolomics Conclusion: The present targeted metabolomics approach allowed to achieve the two study endpoints: a) to indicate metabolite patterns that are of early diagnostic value for sepsis or septic shock; b) to identify some metabolites that can early provide information about risk stratiﬁcation of patients. These ﬁndings will lead to the development of improved novel diagnostic tools for early diagnosis and prognosis of sepsis and septic shock. eA25 German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 Objective: We here explored the role of the evolutionary highly conserved actin-regulatory protein Coronin-1a (Coro1a) for neutrophil effector function. Objective: We here explored the role of the evolutionary highly conserved actin-regulatory protein Coronin-1a (Coro1a) for neutrophil effector function. Methods: Employing neutrophils from Coro1a-deﬁcient and wild type mice we analyzed different neutrophil-mediated defense reactions that were induced by various stimuli. Additional experiments were aimed at uncovering the mechanism underlying Coronin-1a function in neutrophils. eA26 German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 Results: Gene and protein expression analysis conﬁrmed high expression of Coronin-1a in neutrophils. Analysis of developing neutrophil subsets in the bone marrow indicated normal neutrophil development in Coro1a-deﬁcient mice and allowed us to examine the effects of Coro1a-deﬁciency oneffector function ofmature neutrophils. Consistent with a negative regulatory function of Coronin-1a on actin polymerization, Coro1a-deﬁcient neutrophils had increased F-actin levels, indicating altered actin cytoskeletal organization. Importantly, evaluation of ROS generation (also called oxidative burst reaction), a key antimicrobial defense mechanisms of neutrophils, revealed impaired oxidative burst formation in Coro1a- deﬁcient neutrophils in response to a wide range of different stimuli. Similarly, the release of prestored mediators from neutrophil granules, termed neutrophil degranulation, was also signiﬁcantly reduced in Coro1a-deﬁcient neutrophils, despite normal levels of intragranular compounds in resting cells. Initial mechanistic studies indicate an involvement of Coronin-1a in cellular signaling pathways during neutrophil activation. Conclusion: In summary, our data revealed alterations in actin cytoskeletal regulation in Coro1a-deﬁcient neutrophils that were associated with impaired oxidative burst formation and decreased neutrophil degranulation (mediator release), thus indicating a critical involvement of Coronin-1a in neutrophil-mediated defense mechanisms. Ongoing experiments are aimed at further identifying the underlying mechanism and its physiological relevance. The First Infection Wave: Clinical And Laboratory Characteristics Of COVID-19 Patients At The University Hospital Schleswig-Holstein In Kiel Viktoria Backes; Frank Leypoldt; Jeanette Franzenburg; Justina Dargvainiene; Daniela Esser; Klaus-Peter Wandinger; Robert Markewitz; Ralf Junker Universitätsklinikum Schleswig-Holstein, Institut für Klinische Chemie, Campus Kiel, Kiel, German Aims: After the outbreak of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Wuhan in December 2019, the novel virus spread quickly throughout the world, causing a pandemic. Although most people develop mild symptoms, especially old and multimorbid patients have an increased risk for an adverse outcome. The aim of this study was to analyse the cohort of coronavirus disease 2019 (COVID-19) patients at the University Hospital Schleswig-Holstein Campus Kiel during the ﬁrst infection wave and derive a laboratory-value-based prediction model for severe COVID-19 in hospitalized patients. Methods: This retrospective cohort study was conducted on 43 consecutive hospitalized patients with positive SARS-CoV-2 real-time reverse transcriptase-polymerase chain reaction, admitted to the University Hospital Schleswig- Holstein in Kiel from March to August 2020. Non-hospitalized patients were excluded. All patients were categorized according to the Ordinal Scale for Clinical Improvement (WHO) into two groups: mild to moderate disease versus severe disease. Clinical and laboratory parameters were acquired from patient ﬁles and compared between the groups. Results: 31 patients were categorized as mild to moderate disease and 12 patients as severe disease. Age over 60 years (p=0.0479), chronical heart failure (p=0.0321) and oxygen supplementation at the day of admission (p < 0.0001) were associated with severe disease. Main complications of COVID-19 were acute renal failure, cardiac arrhythmia, and septic shock. 18.6 % of patients died during the evaluation period. Most common cause of death was septic shock. C-reactive protein (day 0+1 of hospitalisation: p=0.0007, day 4+5 of hospitalisation: p=0.0035), interleukine-6 (day 0+1: p=0.002, day 4+5: p < 0.0001), neutrophil-to-lymphocyte ratio (day 0+1: p=0.0002, day 4+5: p=0.0105) and procalcitonin concentrations (day 0+1: p=0.0002, day 4+5: p < 0.0001) were signiﬁcantly higher in patients with severe disease. Furthermore, a low prothrombin time at the day of admission was associated with severe disease (p=0.0002). Sodium (day 0+1: p=0.1435, day 4+5: p=0.0006) and creatine kinase (day 0+1: p=0.0547, day 4+5: p < 0.0001) were signiﬁcantly higher in patients with severe COVID-19 than in mild-to-moderate disease during the course of hospitalization but not at admission. Conclusion: We could identify several inﬂammatory and acute-phase parameters which were signiﬁcantly associated with a severe course of COVID-19 in hospitalized patients. The First Infection Wave: Clinical And Laboratory Characteristics Of COVID-19 Patients At The University Hospital Schleswig-Holstein In Kiel Our results support the hypothesis that worse outcomes are mainly associated with hyperinﬂammation leading to multi-organ failure, including kidney damage and altered coag- ulation. A prediction model of risk factors for severe course of COVID-19 in hospitalized patients did not yield sufﬁcient power due to the low incidence of COVID-19 in Schleswig-Holstein during the ﬁrst wave resulting in small patient numbers. eA27 Regrettably, there have been no noticeable improvements in the standardization of methods during the last year. Assuming that the lessons learned from the current pandemic prove true, the number of cases, and thus the workload of laboratories will decrease signiﬁcantly during the summer season. This break should be used urgently to refocus on and improve the quality of diagnostics offered. Current deﬁciencies should be addressed and laboratories should be aware of their responsibility for reported results. 2 years external quality assessment for the detection of anti-SARS-CoV-2 antibodies - a critical retrospective Maximilian Kittel1; Anna Bode2; Romy Eichner1; Sihem Aida1; Volker Ast³; Michael Neumaier³; Verena Haselmann1 1Universitätsmedizin Mannheim, Institut für klinische Chemie, Mannheim, Germany; 2Referenzinstitut für Bioanalytik, RfB, Bonn, Germany; ³Medical Faculty Mannheim of Heidelberg University, Institut für klinische Chemie, Mannheim, Germany In the last year, a large number of assays for the serological detection of antibodies to the new SARS-CoV-2 virus have been brought to market and are being widely used in laboratories. These new developments have highlighted the importance of controlling the analytical methods currently in use to ensure patient safety. External quality assessment (EQA) is an important tool for both standardization of test results and their harmonization, and thus for ensuring high-quality diagnostic procedures. As these results are essential to estimate the prevalence of SARS-CoV-2 infections, the effect of immunizations, and post-infection immunity, this level of quality is mandatory. The Reference Institute for Bioanalytics (RfB) was the ﬁrst provider to offer a proﬁciency test, for the detection of anti-SARS CoV-2 antibodies. In the CoVimm EQA-schemes blinded panels of pre-characterized human serum samples with variable anti-SARS-CoV-2 antibody titers for detection of different anti-SARS-CoV-2-antibodies (IgG, IgA, IgM, total, nucleocapsid, and spike- protein-specific). In this study, the 4 rounds of the CoVimm EQA were evaluated and compared in an aggregated format with the goal to gain insight into the quality and development of diagnostics for the detection of anti-SARS-CoV-2 antibodies. In the four distribution rounds from 2020 to 2021, a total of 296 laboratories from 25 countries reported a total of 5,020 results for anti-SARS-CoV-2 antibody detection using more than 26 different assays. In terms of diagnostic sensitivity and specificity, significant differences were found between the various assays used and also between certified and lab- developed tests. Moreover, it could be observed that with the progress of the pandemic and the availability of vaccines, different requirements were imposed on the methods of antibody detection. The evaluation of the EQA-results also revealed, that there are still considerable deﬁcits in the application of the test procedures on the part of the users. In particular, the use of obviously unsuitable assays concerning their intended use illustrates these application errors. In summary, the EQA highlighted various aspects of the diagnostic situation. First, it should be emphasized that the testing landscape remains heterogeneous, but is increasingly concentrated among large providers. German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 eA28 1Medical Faculty Mannheim of the University of Heidelberg, Institute for Clinical Chemistry, Mannheim, Germany; 2Medical Faculty Mannheim of the University of Heidelberg, Department of Radiology and Nuclear Medicine, Mannheim, Germany; ³Medical Faculty Mannheim of the University of Heidelberg, Department of Medicine II, Mannheim, Germany; ⁴Medical Faculty Mannheim of the University of Heidelberg, Department of Anaesthesiology and Surgical Intensive Care Medicine, Mannheim, Germany 1Medical Faculty Mannheim of the University of Heidelberg, Institute for Clinical Chemistry, Mannheim, Germany; 2Medical Faculty Mannheim of the University of Heidelberg, Department of Radiology and Nuclear Medicine, Mannheim, Germany; ³Medical Faculty Mannheim of the University of Heidelberg, Department of Medicine II, Mannheim, Germany; ⁴Medical Faculty Mannheim of the University of Heidelberg, Department of Anaesthesiology and Surgical Intensive Care Medicine, Mannheim, Germany Introduction: The establishment of integrative diagnostic models combining quantitative imaging and laboratory ﬁndings may support the identiﬁcation of vulnerable COVID-19 patients and aid assessments regarding required intensive care unit (ICU) treatment. We have investigated laboratory biomarkers including cell-free deoxyribonucleic acid (cfDNA) and radiomics for their synergistic integrated diagnostics potential. Methods: Hospitalized SARS-CoV-2 infected patientes (n=52) were enrolled between May 2020 and September 2021. Retrospective image segmentation analyses of chest computed tomography (CT) and analysis of routine laboratory biomarkers together with prospectively obtained quantitative cfDNA concentrations were performed using separate feature selection and application of a minimal redundancy algorithm for both diagnostic modalities. The algorithm was established using cross-wise validation and subsequent veriﬁcation in subset of algorithm-naïve patients. The clinical decision endpoint “ICU stay likely/unlikely” was optimized based on the prediction by the algorithm. Results: The integrated model comprises six radiomics and seven laboratory biomarkers. Root mean square of the deviations between actual and predicted ICU-days was 5.3 days in cross-validation set and 12.3 days in test-cohort. Radiomic model accuracy was 0.54, cfDNA model accuracy was 0.47, routine laboratory model accuracy was 0.74 and combined model accuracy was 0.87 with an AUC of 0.91. The combined model performed superior to the individual radiological and laboratory models to predicting ICU requirement (adjusted R2 = 0.896). Conclusion: The integration of radiomics and laboratory data shows synergistic potential to improve clinical decision making of COVID-19 patients. Based on the results of our routine patient cohort, this model may contribute to stratiﬁ- cation of ICU capacities. Potential of integrative diagnostics predicting ICU demand in COVID-19 patients Catharina Gerhards1; Verena Haselmann1; Samuel Schaible2; Volker Ast1; Maximilian Kittel1; Matthias Ebert³; Andreas Teufel³; Manfred Thiel⁴; Alexander Hertel2; Stefan Schönberg2; Matthias Frölich2; Michael Neumaier1 eA28 German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 Evaluation of a laboratory-based high-throughput SARS-CoV-2 antigen assay Sebastian Hörber1; Christoph Drees1; Tina Ganzenmueller2; Dirk Biskup³; Andreas Peter1 1Universitätsklinikum Tübingen, Institut für Klinische Chemie und Pathobiochemie, Tübingen, Germany; 2Uni- versitätsklinikum Tübingen, Institut für Medizinische Virologie, Tübingen, Germany; ³CeGat GmbH, CeGat GmbH, Tübingen, Germany Objectives: Antigen tests are an essential part of SARS-CoV-2 testing strategies. Rapid antigen tests are easy to use but less sensitive compared to nucleic acid ampliﬁcation tests (NAT) and less suitable for large-scale testing. In contrast, laboratory-based antigen tests are suitable for high-throughput immunoanalyzers. Here we evaluated the diagnostic performance of the laboratory-based Siemens Healthineers SARS-CoV-2 Antigen (CoV2Ag) assay. Methods: In a public test center, from 447 individuals anterior nasal swab specimens as well as nasopharyngeal swab specimens were collected. The nasal swab specimens were collected in sample inactivation medium and measured using the CoV2Ag assay. The nasopharyngeal swab specimens were measured by RT-PCR. Additionally, 9046 swab specimens obtained for screening purposes in a tertiary care hospital were analyzed and positive CoV2Ag results conﬁrmed by NAT. Results: In total, 234/447 (52.3%) participants of the public test center were positive for SARS-CoV-2-RNA. Viral lineage B1.1.529 was dominant during the study. Sensitivity and speciﬁcity of the CoV2Ag assay were 88.5% (95%CI: 83.7%- 91.9%) and 99.5% (97.4%-99.9%), respectively. Sensitivity increased to 93.7% (97.4%-99.9%) and 98.7% (97.4%-99.9%) for swab specimens with cycle threshold values < 30 and < 25, respectively. Out of 9046 CoV2Ag screening tests from hospitalized patients, 21 (0.2%) swab specimens were determined as false-positive by conﬁrmatory NAT. Conclusions: Using sample tubes containing inactivation medium the laboratory-based high-throughput CoV2Ag assay is a very speciﬁc and highly sensitive assay for detection of SARS-CoV-2 antigen in nasal swab specimens including the B1.1.529 variant. In low prevalence settings conﬁrmation of positive CoV2Ag results by SARS-CoV-2-RNA testing is recommended. 8 induces maladaptive unfolded protein response in diabetic kidney disease Ronald Biemann1; Shruthi Krishnan2; Jayakumar Manoharan1; Dheerendra Gupta1; Akash Mathew1; Shrey Kohli1; Khurrum Shahzad1; Michael Naumann2; Berend Isermann1 1Universitätsklinikum Leipzig AöR, Institut für Laboratoriumsmedizin, Klinische Chemie und Molekulare Diagnostik, Leipzig, Germany; 2Otto-von-Guericke University Magdeburg, Institute of Experimental Internal Medicine, Magdeburg, Germany Aims: Dysfunction of mesangial cells plays a major role in the pathogenesis of diabetic kidney disease (DKD), the leading cause of end-stage renal disease. The underlying molecular mechanisms, however, are incompletely understood. Methods and Results: By unbiased gene expression analysis of glucose-exposed mesangial cells, we identiﬁed the transmembrane receptor CD248 as the most upregulated gene and maladaptive unfolded protein response (UPR) as one of the most upregulated pathways. Upregulation of CD248 was conﬁrmed in glucose-stressed mesangial cells in vitro, in renal glomeruli isolated from diabetic mice (STZ and db/db models, representing type 1 and type 2 diabetes mellitus, respectively) in vivo, and in glomerular kidney sections from patients with DKD. Time course analysis revealed that glomerular CD248 induction precedes the onset of albuminuria, mesangial matrix expansion and maladaptive UPR activation (hallmarked by C/EBP homologous protein, CHOP, induction) but is paralleled by loss of the adaptive UPR regulator spliced X box binding protein (sXBP1). Mechanistically, CD248 induces the assembly of a multiprotein UPRosome comprising heat shock protein 90 (HSP90), BH3 interacting domain death agonist (BID) and inositol requiring enzyme 1 (IRE1α), in which BID impedes IRE1α-mediated XBP1 splicing and sXBP1-dependent gene expression. Over- expression of HSP90 or BID in vitro or genetic reduction of XBP1 in vivo abrogates the protective effects of CD248-deﬁciency. German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 eA29 Conclusion: In the current study, we identiﬁed CD248 as a regulator of the adaptive UPR mediator XBP1 that induces maladaptive UPR signaling in renal glomeruli under diabetic conditions and in mesangial cells exposed to high glucose conditions in vitro. This research is expected to provide new mechanistic insights and identify the transmembrane receptor CD248 as a potential biomarker and a new druggable target in DKD. Conclusion: In the current study, we identiﬁed CD248 as a regulator of the adaptive UPR mediator XBP1 that induces maladaptive UPR signaling in renal glomeruli under diabetic conditions and in mesangial cells exposed to high glucose conditions in vitro. This research is expected to provide new mechanistic insights and identify the transmembrane receptor CD248 as a potential biomarker and a new druggable target in DKD. Methods: The existence and location of several poly-A-sites in the human NRP1 gene was conﬁrmed by 3’ RACE PCR. To measure the inﬂuence of different APA factors on the expression of NRP1, we transfected BE2C cells with silencing RNAs to knockdown central components that regulate APA (PCF11, CPSF6 and NUDT21). The knockdown efﬁciency was controlled by western blot. The transfected cells were harvested to isolate RNA and proteins. RT-qPCR was performed to measure expression changes of NRP1 isoforms on mRNA level. Results: We conﬁrmed different poly-A-site usage in the human NRP1 gene, which leads to the expression of different NRP1 RNA isoforms. We also demonstrate that CPSF6, a key determinant regulating APA, controls poly-A-site usage, with knockdown of CPSF6 resulting in upregulation of the soluble NRP1 isoform. Conclusion: We show that APA regulates the expression of NRP1, the neuronal SARS-CoV-2 entry (co)receptor. Down- regulation of CPSF6 results in the expression of a truncated NRP1 mRNA isoform, encoding a soluble NRP1 protein that lacks the transmembrane domain. Truncated NRP1 thereby functionally competes with full-length membrane bound NRP1 and acts as a soluble decoy receptor. Based on these ﬁndings is tempting to speculate that APA evolved as a regulatory mechanism controlling SARS-CoV-2 cell entry and infectivity. Alternative polyadenylation regulates the SARS-CoV-2 entry factor NRP1 Sophie Hartleb; Essak Khan; Jamie Nourse; Sven Danckwardt Universitätsmedizin Mainz, Center for Thrombosis and Hemostasis (CTH), Mainz, Germany Background: Neuropilin-1 (NRP1) has recently been identiﬁed as neuronal SARS-CoV-2 (co)receptor controlling host cell entry and infectivity. Apart from membrane bound NRP1 a truncated isoform lacking the transmembrane domain exists, which acts as functional antagonist to full-length NRP1. Recently, diversiﬁcation of the transcriptome at the 3’end by alternative polyadenylation (APA) has emerged as a pervasive and evolutionarily conserved layer of gene regulation. APA is, for example, involved in the IgM heavy chain class switch in activated B-cells resulting in the conversion from a membrane bound to a soluble IgM. Here we set out to explore if and how APA affects the expression of soluble and membrane bound NRP1. eA30 German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 eA30 y and Specificity of the Roche SARS-Cov-2 Antigen Assay in a hospital setting Boris Rolinski1; Julian Gebauer1; Anja Prantz1; Dusan Krnac1; Christoph Kutschker2 Boris Rolinski ; Julian Gebauer ; Anja Prantz ; Dusan Krnac ; Christoph Kutschker 1Elblandkliniken, Elblab, Meißen, Germany; 2Elblandkliniken, Krankenhaushygiene, Meißen, Germany blandkliniken, Elblab, Meißen, Germany; 2Elblandkliniken, Krankenhaushygiene, Meißen, Germany Aim: We evaluated the performance of the Roche SARS-CoV-2 Antigen Assay with respect to calibration stability and robustness as well as sensitivity and speciﬁcity. Methods: We run the Roche SARS-CoV-2 Antigen Assay in three 400 bed hospitals on every patient at admission in conjunction with a SARS-CoV-2 rt-PCR (Seegene). Antigen tests were performed on two Cobas 6000 and one Elecsys e411. Data were evaluated from 10.3.2021 to 23.04.2022. A total of 35090 data sets were available for analysis. Results: Antigen results ranged from 0,07 to 34149 U/ml and ct values ranged from 9 to 39,89. A total of 32642 samples were negative both by PCR and Antigen Assay, whereas 1421 samples where positive in both assay. 151 samples were false positive and 522 false negative resulting in a sensitivity of 0,7313 and a speciﬁcity of 0,9954. Methods: We run the Roche SARS-CoV-2 Antigen Assay in three 400 bed hospitals on every patient at admission in conjunction with a SARS-CoV-2 rt-PCR (Seegene). Antigen tests were performed on two Cobas 6000 and one Elecsys e411. Data were evaluated from 10.3.2021 to 23.04.2022. A total of 35090 data sets were available for analysis. Results: Antigen results ranged from 0,07 to 34149 U/ml and ct values ranged from 9 to 39,89. A total of 32642 samples were negative both by PCR and Antigen Assay, whereas 1421 samples where positive in both assay. 151 samples were false positive and 522 false negative resulting in a sensitivity of 0,7313 and a speciﬁcity of 0,9954. Conclusion: The Roche SARS-CoV-2 Antigen assay performed very stable over more than one year and on three different instruments. The assay proved to be sufﬁcient sensitive and highly speciﬁc for the detection of SARS-CoV-2 infection. Performance evaluation of the EUROArray assay for molecular detection of dermatomycosis Michal Krolik1; Susanna Bigler2; Thomas Bodmer³; Martin Risch⁴; Lorenz Risch⁴; Nadia Wohlwend1 1Dr. Risch, Mikrobiologie, Buchs, Switzerland; 2Dr. Risch, Klinische Chemie , Liebefeld, Switzerland; ³Dr. Risch, Mikro- biologie, Liebefeld, Switzerland; ⁴Dr. Risch, Allgemeine Innere Medizin, Buchs, Switzerland Introduction: Superﬁcial and cutaneous dermatomycosis is one of the most common fungal infection and remains a global concern. Increased mobility facilitates spreading and manifestation of originally rare dermatophyte species. While conventional culture is considered the gold standard for diagnosis of dermatomycosis, it is laborious, lengthy and requires a high level of expertise. In contrast, novel molecular approaches promise rapid yet sensitive and speciﬁc detection of dermatophytes. Here, we evaluate the performance of a novel PCR-based microarray compared to culture as reference. Materials & Methods: A total of 272 KOH positive clinical samples were prospectively analysed in parallel by culture and the EUROArray Dermatomycosis (EUROImmun, Kriens, Switzerland). For cultural analysis, sample material was plated and incubated on Sabouraud, Dermatophyte and Candida Agar (BioMérieux, Petit-Lancy Switzerland). Primary material was digested by Proteinase K followed by automated extraction via easyMAG (BioMérieux, Petit-Lancy Switzerland) and sub- sequently used for the identiﬁcation of 23 dermatophyte, 3 yeast and 3 mould species by the EUROArray. Results: Dermathophytes were detected in 233/272 (85%) of the KOH positive clinical samples by EUROArray with Trichophyton rubrum (n=194) and Trichophyton interdigitale (n=36) clearly dominating over Microsporum canis (n=2) and Nannizzia gypsea (n=1). Whereas culturally only 93/272 (34%) of the obligate pathogenic agents could be identiﬁed. Results from culture were available after a mean of 25 days, while results from the EUROArray were obtained on the same day that the samples were processed. Conclusion: Our data illustrate the increased sensitivity and shortened time to result compared to cultural analysis. We demonstrate that clinically relevant and human pathogenic dermatophytes, which did not yield a corresponding result in the time-consuming cultural rearing, are identiﬁed by molecular biological detection. Overgrowth by mould and inho- mogeneous distribution of dermatophytes in the primary material are major obstacles. These can be overcome by the EUROArray enabling targeted therapy within a short period of time. German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 eA31 Antimicrobial Activity of CLEC3A: Potential in the Prevention and Treatment of Septic Arthritis Dzemal Elezagic Universitätsklinikum Köln, Institut für klinische Chemie Introduction: The dramatic increase in antibiotic resistance has caused bacterial infections to once again become a serious global health threat. Moreover, the stagnating development of novel antibiotics urges for alternative antimi- crobial agents. Particularly promising alternatives to conventional antibiotics are antimicrobial peptides (AMPs), which are part of the innate immune system. The cartilage-speciﬁc C-type lectin domain family 3 member A (CLEC3A) exhibits structural similarities to AMPs, which prompted us to investigate its antimicrobial activity. Methods: We performed immunoblot to detect CLEC3A peptides in human cartilage extracts. To investigate their anti- microbial activity, we designed peptides and recombinantly expressed CLEC3A domains and used them to perform viable count assays using E.coli, P.aeruginosa and S.aureus. We investigated the mechanism of their antimicrobial activity by ﬂuorescence and scanning electron microscopy. In addition, we coated CLEC3A peptides on titanium, a commonly used prosthetic material, and performed ﬂuorescence microscopy to quantify bacterial adhesion. Moreover, we assessed the peptides’ cytotoxicity against murine ﬁbroblasts (NIH3T3) using MTT cell viability assays. To enhance the peptides´ performance, we altered the native peptides’ sequences, generating 6 modiﬁed peptides. Results: CLEC3A fragments were indeed detected in human cartilage extracts. Moreover, bacterial supernatants lead to fragmentation of recombinant and cartilage-derived CLEC3A. CLEC3A-derived peptides killed E.coli, P.aeruginosa and S.aureus. The modiﬁed peptides exhibited even more efﬁcient bacterial killing (including that of a Methicillin-resistant S.aureus strain). The antimicrobial activity of the native peptides occurs by permeabilizing bacterial membranes. Coating CLEC3A-derived AMPs on titanium lead to signiﬁcantly reduced bacterial adhesion to the material. Additionally, modifying the peptides considerably reduces cytotoxicity levels against NIH3T3 cells. Conclusions: We ﬁrst identify cartilage-speciﬁc AMPs originating from CLEC3A and resolve the mechanism of their antimicrobial activity. In a translational approach, through modifying peptides´ sequences, we pinpoint CLEC3A-derived AMPs with enhanced antimicrobial activity and reduced cytotoxicity. In addition, by coating prosthetic material with the peptides, we point to a novel approach in the prevention and treatment of septic arthritis. In vivo experiments involving mouse infection models are currently ongoing and are expected to shed light on the practical use of native and modiﬁed CLEC3A-derived AMPs in ﬁghting infection. Conclusion: We have identiﬁed the main innate immune sensor proteins that link uric acid crystals to complement activation. The unique ability of CRP to drive production of the more inﬂammatory C5a suggests elevated CRP may enhance the ability of the immune system to detect the crystals and therefore favor the initiation of gout ﬂares. CRP in gout: Not just a biomarker? Konstantin Neumann; Korbinian Brand; Anika Alberts; Anne Kathrin Wessig Medizinische Hochschule Hannover, Institut für Klinische Chemie - Zentrallabor, Hannover, Germany Introduction: Gout is a common form of arthritis caused by crystallization of uric acid. The uric acid crystals induce a strong inﬂammatory response that is hardly distinguishable from inﬂammation induced by bacterial infection and includes increases in serum levels of C-reactive protein (CRP). It is unclear, if the innate immune system recognizes the crystals or merely reacts to the damage they cause. Aim: We aimed to identify proteins of the immune system that interact with gout crystals. Methods: We puriﬁed proteins from different body ﬂuids including synovial ﬂuid and identiﬁed the proteins by liquid chromatography-coupled mass spectrometry. Recombinant proteins of the identiﬁed proteins were used to test their binding to uric acid crystals and their impact on inﬂammatory responses induced by the crystals. Results: Besides strong binding of apolipoprotein B, we found speciﬁc binding of several proteins of the immune system capable of inducing inﬂammatory responses. Strongest binding was found for CRP. Binding was strong enough to deplete CRP from human serum by addition of uric acid crystals. Binding of CRP induced complement activation on the surface of uric acid crystals. In addition to CRP, we found immunoglobulin M (IgM) to bind to the crystals, in all donors tested. Antibodies usually arise in response to foreign antigens. However, the antibodies recognizing uric acid crystals were also found in cord blood serum, which means they are formed before birth, suggesting they are natural/innate antibodies. Both CRP and IgM were capable of activating the complement system, which alerts the immune system by releasing inflammatory peptides, C3a and C5a. In the absence of CRP and IgM, no complement activation was induced by uric acid crystals, indication that we have found the main complement sensors for uric acid crystals. However, while IgM was only able to induce release of C3a but not C5a, CRP was able to propagate the complement cascade to the very end and induce release of the more inflammatory C5a. eA32 German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 eA32 Naturally occuring non-radioactive Calcium isotope ratios in the human body as a biomarker for bone metastasis of prostate cancer -a pilot study- Burkhard Brandt1; Axel Semjonow2; Katrin Schlack³; Alexander Heuser⁴; Ana Kolevica⁵; Anton E 1; Axel Semjonow2; Katrin Schlack³; Alexander Heuser⁴; Ana Kolevica⁵; Anton Eisenhauer⁶ eA33 German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 Aims: Prostate cancer (PCa) is the second most frequent malignancy in men worldwide. Although 5-year survival in patients with organ-conﬁned PCa is nearly 100%, metastasis to the bones still remains incurable. Therefore, there is an urgent need for markers able to predict bone metastasis (BM), in order to personalize patients’ (pts) treatment. The ratios of Calcium isotopes like 44Ca and 42Ca (δ44/42Ca), can be used to identify unbalanced bone mineral disorders, e.g. osteoporosis. This is based on the kinetic isotope fractionation factor between blood and bones being constant in humans. In this study we tested how sensitive δ44/42Ca changes in serum reﬂect PCa bone metastasis. Materials and Methods: Sera from 20 pts were measured by plasma-mass spectroscopy for δ44/42Ca-values. For QC all data were compared and calibrated to international standards. The long-term standard reproducibility was in the order of 0.06 ‰. Nine pts (6 with PCa, 3 without) had no signs of bone metastasis. Eleven pts had moderate to multiple BM. BM were evaluated by PSMA-PET/CT or bone scintigraphy. BM pts received various androgen deprivation therapies, and, 4 received also taxane therapy. For osteoprotection Denosumab , Zoledronic acid and Vit D3 was given in 2, 1 and 1 pt., resp. Results: Six of 9 pts w/o BM had normal age adjusted δ44/42Ca values, 2 showed Ca resorption and 1 Ca absorption ratios. Ca resorption occurred in 3 BM pts with moderate numbers of BM and low PSA levels (< 0.05 – 8 ng/ml). Eight pts with multiple BM and high PSA levels (31 – 1443 ng/ml) had Ca absorption. In this high δ44/42Ca subgroup 3 pts had Denosumab/ Zoledronic acid as bone protection. Unexpectedly, Vitamin D3 supplementation made no δ44/42Ca increase in 1 BM pt. Discussion (Conclusion): We applied for the ﬁrst time Ca isotope ratios as a biomarker for PCa bone metastasis. Overall, 2/3 of non-metastatic pts showed balanced δ44/42Ca, whereas BM pts were found in both groups representing Ca resorption and absorption as well. From published reports it might be hypothesized that Ca resorption show earlier phases of metastases which is in agreement with the lower number of BM and PSA values of the pts. In contrary, pts with multiple metastases and high PSA levels where found in the Ca absorption group indicating the ﬁnal osteoblastic/ sclerotic phase of PCa bone metastases. This hypothesis might be substantiated by additional δ44/42Ca measurements from pts only displaying BM in PET-PSMA scan. The δ44/42Ca values in the absorptive range for pts with osteoprotective treatment by Denosumab and Zoledronic acid might also support this assumption. Nevertheless, an extended study is needed to control preanalytical inﬂuencing factors which may cause Ca disequilibrium. Those are benign gastrointes- tinal, kidney, endocrine and bone diseases and Ca supplementation. Possible inﬂuencing factors might explain why some patients in the study are misclassiﬁed by their δ44/42Ca measurements. Burkhard Brandt1; Axel Semjonow2; Katrin Schlack³; Alexander Heuser⁴; Ana Kolevica⁵; Anton Eisenhauer⁶ 1UKSH, Institut für Klinische Chemie, Kiel, Germany; 2UKM, Prostatazentrum - Uroonkologie, Klinik für Urologie, Münster, Germany; ³UKM, Münster, Prostatazentrum - Uroonkologie, Klinik für Urologie , Münster, Germany; ⁴Geomar, Helmholtz-Zentrum für Ozeanforschung, Kiel, Germany; ⁵Geomar/OsteolabsGmbH, Helmholtz-Zentrum für Ozeanfor- schung und Osteolabs, Kiel, Germany; ⁶Geomar/Osteolabs GmbH, Helmholtz-Zentrum für Ozeanforschung und Osteo- labs, Kiel, Germany Using quantitative morphometry data, the impaired formation of lamellipodia in Glanzmann thrombasthenia patients could be evaluated Katharina von Bargen1; Max-Joseph Kraus2; Marcus Dittrich³; Christina Brandenburger1; Monika Wolny1; Tobias Flieder1; Johannes Rheinlaender⁴; Tilman E. Schäffer⁴; Günther Kappert⁵; Susan Halimeh⁵; Cornelius Knabbe1; Erwin Strasser⁶; Heiko Neeb2; Ingvild B 1Herz- und Diabeteszentrum Nordrhein-Westfalen, Institut für Laboratoriums- und Transfusionsmedizin, Bad Oeynhausen, Germany; 2Universität Koblenz-Landau, Institut für Medizintechnik und Informationsverarbeitung, Koblenz, Germany; ³ Universität Würzburg, Institut für Humangenetik und Bioinformatik, Würzburg, Germany; ⁴Universität Tübingen, Institute of Applied Physics, Tübingen, Germany; ⁵Gerinnungszentrum Rhein-Ruhr, GZRR, Duisburg, Germany; ⁶Klinikum der Universität München, LMU München, Abteilung für Transfusionsmedizin, Zellther- apeutika und Hämostaseologie, München, Germany Introduction: After vascular injury, platelet adhesion to the extracellular matrix leads to platelet activation, which in turn induces platelet shape change. Thereby the reorganization of the cytoskeleton is mediated by a variety of signaling pathways. Among other things, the ﬁbrinogen receptor (GPIIb/IIIa complex) is also involved. This complex plays a crucial role in primary hemostasis by mediating platelet aggregation. Therefore, the study aimed to investigate the shape change (spreading) in dependence of the GPIIb/IIIa complex. For this purpose, platelets from Glanzmann thrombasthenia patients (GT-patients) were studied. This is a platelet dysfunction with a quantitative or qualitative defect of the GPIIb/IIIa complex. Methods: In the present study, platelet shape change was investigated in a total of ﬁve healthy donors and six GT patients. For this purpose, platelets were allowed to spread on ﬁbrinogen under different conditions (without activator, with ADP, or with TRAP), the actin cytoskeleton was stained with phalloidin, and then 40 immunoﬂuorescence images were acquired per condition and time point. The immunoﬂuorescence images were then evaluated using an algorithm (automated quantitative morphometry analysis) by determining various parameters such as area, fractal dimension, number of pseudopodia, etc. In addition, the morphometry of the platelets was examined by electron microscopy. Results: Analysis of the immunoﬂuorescence images shows that the GT platelets have a spreading defect, which is particularly characterized by the absence of lamellipodia formation. Whereas the healthy platelets have mostly a fully spread shape after 45 min, the GT platelets persist in the early phase of spreading, which is characterized by a large number of long pseudopodia. Overall, the results of the algorithm show that the individual parameters (such as number of pseudopodia, FD, circularity, and area) describing the morphometry differ signiﬁcantly between the studied collectives (healthy and GT). Here, the differences are particularly distinct at the late spreading time points. Results: We observed reduced EB extravasation in APChigh (aPC overexpression) mice in a chronic kidney disease mouse model as compared to WT or TMPro/Pro mice. Single nuclei RNA-sequencing of wildtype and TMPro/Pro murine brains revealed striking differences in various cell type clusters, comprising neurons, microglia cells, glia cells, pericytes and endothelial cells. Functional annotation revealed downregulation of genes related to “learning” in the TMPro/Pro mice. Indeed, we saw impairment in behavioral testing of TMPro/Pro mice. Discussion: Our data establish that coagulation proteases differentially regulate the BBB in vivo. Altered coagulation activation is associated with structural defects in the CNS and with altered cognition. Recent data suggest that different anticoagulants differentially regulated coagulation protease dependent signaling, which may affect disease outcome. A prime example is activated protein C (APC), a blood protease with anticoagulant activity and cell-signaling activities. Of note, receptors for coagulation proteases are widely expressed at the blood brain barrier (BBB) and by various cells in the central nervous system (CNS). APC variants have shown beneﬁts in preclinical models of ischemic stroke, brain trauma, multiple sclerosis and amyotrophic lateral sclerosis. Our data may show, for the ﬁrst time, how coagulation may affect cognition. Influence of coagulation proteases and signalling on cognitive function Silke Zimmermann; Gyulten Mangova; Akash Mathew; Shihai Jiang; Berend Isermann Universitätsklinikum Leipzig, Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, Leipzig, Germany Aims: Since effects of thrombin on the blood brain barrier (BBB) have been proposed, we want to deﬁne the relevance of the thrombomodulin (TM) - activated protein C (aPC) system for BBB integrity and neuronal cell function. In Germany, one million patients are treated with lifelong anticoagulant therapy (anticoagulants). We speculate that anticoagulants, via inﬂuencing coagulation factors, may interfere with neuronal function, impair cognition or convey other CNS-related side- effects. Clinical reports suggested that anticoagulation affects cognition. We want to deﬁne (i) the impact of thrombin on neuronal function and cognitive performance in mice and (ii) whether the changed behavior of the animals is a conse- quence of the increased coagulation activation. Material and Methods: We performed snRNA sequencing of wildtype (WT) versus TMPro/Pro (mutation leads to endothelial dysfunction due to reduced function of the TM-protein C pathway murine brains. We use mice with genet- ically altered activity of the TM-PC system to study its impact on cognitive function in mice, investigating locomotor activity, fear-related exploratory behavior, sensorimotor gating and learning patterns. Evans Blue (EB) extravasation test will show us, how (i) genotypes are affected regarding BBB integrity and (ii) whether anticoagulation affects integrity. Using CRISPR/Cas9 mediated cell-speciﬁc deletion of EPCR or PARs (PAR1-4) we want to identify the receptor through which aPC and thrombin modulate the function of brain cells, given the available data on IIa`s and aPC`s effect on endothelial barrier. eA34 German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 Using quantitative morphometry data, the impaired formation of lamellipodia in Glanzmann thrombasthenia patients could be evaluated Thus, the GT platelets are much smaller due to the large number of pseudopodia, and the FD is increased. Electron microscopy also shows the altered morphometry of the GT platelets. eA35 German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 Conclusion: Spreading analysis show that platelet shape change is impaired in GT platelets. Both immunoﬂuorescence microscopy and electron microscopy could show an absence of lamellipodia formation. Quantitative morphometry analysis was used to better describe cytoskeletal reorganization and to show the differences between the two collectives. Overall, quantitative morphometry analysis is a useful tool to better describe the different stages of platelet shape change in patients with thrombocytopathy. Conclusion: Spreading analysis show that platelet shape change is impaired in GT platelets. Both immunoﬂuorescence microscopy and electron microscopy could show an absence of lamellipodia formation. Quantitative morphometry analysis was used to better describe cytoskeletal reorganization and to show the differences between the two collectives. Overall, quantitative morphometry analysis is a useful tool to better describe the different stages of platelet shape change in patients with thrombocytopathy. Predictors of hypercoagulability in prediabetes Sebastian Hörber1; Karina Althaus2; Tamam Bakchoul2; Jürgen Machann³; Norbert Stefan⁴; Andreas Birkenfeld⁴; Andreas Fritsche⁴; Andreas Peter1 1Universitätsklinikum Tübingen, Institute for Clinical Chemistry and Pathobiochemistry, Department for Diagnostic Laboratory Medicine, Tübingen, Germany; 2Universitätsklinikum Tübingen, Institute for Clinical and Experimental Transfusion Medicine, Tübingen, Germany; ³Universitätsklinikum Tübingen, Section on Experimental Radiology, Department of Radiology, Tübingen, Germany; ⁴Universitätsklinikum Tübingen, Department of Internal Medicine IV, Division of Diabetology, Endocrinology and Nephrology, Tübingen, Germany Background: Obesity and insulin resistance predispose for arterial and venous thrombosis that can be explained by a hypercoagulable state. However, data about underlying associations of the hypercoagulability with metabolic alterations is limited. Therefore, the aim of the present study was to identify metabolic predictors of hypercoagulability in prediabetes. Methods: Endogenous thrombin potential (ETP) was determined in 141 subjects with impaired glucose tolerance and/or impaired fasting glucose using a commercially available thrombin generation assay. All subjects were metabolically characterized including an oral glucose tolerance test. Furthermore determination of body fat distribution and liver fat content was performed using magnetic resonance imaging and spectroscopy, respectively. Results: ETP was signiﬁcantly associated with fasting plasma glucose, insulin sensitivity and body fat distribution. In particular, increased amounts of total adipose tissue, visceral adipose tissue and subcutaneous adipose tissue were signiﬁcantly associated with an increase in ETP. Increased liver fat content was also related to higher ETP. Subjects with fatty liver had higher levels of ETP compared to subjects without fatty liver. Adjusting for insulin sensitivity, fasting plasma glucose and body fat compartments ETP remained signiﬁcantly and independently elevated in subjects with fatty liver compared to controls. Conclusion: ETP is closely linked to metabolic alterations in prediabetes. Body fat distribution, particular increased liver fat content is signiﬁcantly and independently associated with hypercoagulability in prediabetes and may therefore contribute to the increased risk for arterial and venous thrombosis. Further analysis will focus on the underlying mechanisms including molecular associations of hypercoagulability and liver fat content in prediabetes. eA36 Background: Thrombin is a liver-derived serine protease involved in hemostasis, acting through catalytic activation of soluble substrates (ﬁbrinogen) and circulating cells (platelets). In addition, thrombin has a host of actions on cells with functions in development, angiogenesis, wound healing, inﬂammation, atherosclerosis, brain disorders, and tumour biology through activation of membrane-bound G-protein coupled protease-activated receptors (PARs). Previously, we uncovered extrahepatic prothrombin expression in emerging ﬁbrosarcoma tumours, which drives tumour proliferation and invasiveness (Nourse et al.,bioxiv 2021). Here we investigated the interaction between endogenous tumour-derived (pro)thrombin and PAR1 on the surface of these cells by using bioluminescence resonance energy transfer (BRET). Methods: To establish the BRET reporter assay system, we produced a prothrombin-luciferase (emission max 535 nm) and a PAR1-turbo ﬂuorescent protein (emission at 635 nm) fusion construct, which were then used in transient trans- fection experiments of HEK cells. A bioluminogenic substrate (coelenterazine) was added to the co-transfected cells to elicit the energy transfer between the prothrombin-luciferase and the PAR1 turbo FP, resulting in a speciﬁc red ﬂuorescent signal upon the interaction of (pro)thrombin with PAR1. Subsequently, we transfected endogenously prothro-mbin-luciferase expressing fibrosarcoma cells (obtained from a newly generated transgenic reporter mouse model after chemical tumor induction with methylcholanthrene (Nourse et al.,bioxiv 2021)) with the PAR1 turbo FP construct to study the interaction between endogenous (pro)thrombin and PAR1 using the established BRET assay system. Results: First, we conﬁrmed the production of a functional prothrombin-luciferase fusion protein and the presence of PAR1-turbo FP on the membrane of the cells. After establishing optimal BRET assay conditions, we were able to observe red-shifted signal in transfected cells, which could be modulated with ecarin (increase in red signal) and hirudin (reduced red signal), respectively. These ﬁndings conﬁrm a successful function of the established BRET assay prin- ciple. Furthermore, we could show an emission stroke shift in endogenously prothrombin expressing cancer cells, suggesting that prothrombin binds to PAR1 receptors located on the membrane of these cells. Conclusion: We successfully established a BRET reporter assay system to monitor (pro)thrombin-PAR1 interaction. We demonstrate that tumor-derived-prothrombin binds to PAR1 receptors expressed on the membrane of the tumor cells. Regarding the wide-spread clinical use of thrombin-targeting by direct oral anticoagulants, determination of the role and underlying mechanisms of thrombin in tumour growth may reveal previously unidentiﬁed beneﬁts of selective thera- peutic targeting of the hemostatic system in cancer. Tumour derived prothrombin interacts with tumour PAR1 receptors Yaman Mouhish1; Jamie Nourse2; Essak S. Khan2; Sven Danckwardt2 1Universitätsmedizin der Johannes Gutenberg-Universität Mainz, Center for Thrombosis and Hemostasis (CTH), Mainz , Germany; 2Universitätsmedizin der Johannes Gutenberg-Universität Mainz, Center for Thrombosis and Hemostasis (CTH), Mainz, Germany eA36 German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 eA37 suitability for DOAC screening on a routine 24/7 basis. We compared the method for three of the available DOACs using the LCMS-8050 system coupled to CLAM-2030 (both Shimadzu) versus commercially available chromogenic tests. Methods: The plasma samples from 56 anesthesiologically and nephrologically supervised patients were collected. DOAC plasma concentrations of Apixaban, Dabigatran and Rivaroxaban were measured on a LCMS system through an automated sample preparation module. Sample protein precipitation and chromatographic separation with a sharp linear gradient on a fused core column at 45°C were performed automatically by CLAM-2030. The target compounds were identiﬁed by parent ions and optimized MRM transitions. Quantiﬁcation was performed by using deuterated internal standards. Quality controls were checked twice a day. In parallel, the quantitative determination of DOACs were assessed using conventional automated chromogenic tests (DTI-Assay (Dabigadran), Anti-Xa-Assay (Apixaban, Rivaroxaban), HemosIL (Werfen Company) on a IL Coagulation System (ACL TOP 750). Patient plasma samples were placed on the devices according to their arrival in the lab. Results: The Screening results have shown a good correspondence with the patients’ data. Passing–Bablok regression analysis revealed good comparability between the methods (Apixaban r=0.984, y=1.019x–1.354; Rivaroxaban r=0.986, y=1.063x–1.663; Dabigadran r=0.988, y=0.856x–0.362). The precision of calibrations (range about 10 to 500ng/ml) and controls was within the manufacturer limit. The automated system was straightforward and proved easy to handle after short training periods. Running time including sample preparation was approx. six minutes. Conclusion: The presented method is convincing in its easy handling and is conceivable for (24/7) routine measure- ments. In contrast to previously used methods, particularly the contemporaneous assignment and quantiﬁcation of different DOACs is innovative. Automated 24/7 screening and quantification of DOACs in plasma in a single run on CLAM2030 – LCMS8050 Anna Abratis1; Moritz Schnelle1; Frank Streit1; Manuel Wallbach2; Nils Kunze-Szikszay³; Sebastian Uwe Schnitzler³; Julie Schanz1; Andreas Fischer1; Ivana Markovic1 1Universitätsmedizin Göttingen, Interdisziplinäres UMG Labor, Göttingen, Germany; 2Universitätsmedizin Göttingen, Department of Nephrology and Rheumatology, Göttingen, Germany; ³Universitätsmedizin Göttingen, Department of Anesthesiology, Göttingen, Germany Aims: In recent years, therapeutic drug monitoring (TDM) as part of DOAC therapy has gained in importance, since the outcome can be improved through individual dose adjustment, especially when treating critically ill emergency patients. At present, the effects of various DOACs are usually assessed indirectly and insufﬁciently (e.g. by determining the thromboplastin time or the activated partial thromboplastin time). Liquid chromatography with tandem mass spec- trometry (LC-MS/MS) is an appropriate system for simultaneous measurement of multiple drugs. Until today, however, the use of LC-MS/MS Systems in emergency labs were not established. Therefore, this study aims to develop and to validate a new LCMS-based method to screen and quantify different DOACs simultaneously in a single run. Similar methods have so far been restricted to research purposes, usually presupposing trained staff and long running times. Connecting the LCMS to an automated sample preparation module, we assessed its German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 Procoagulant platelets as a diagnostic tool for heparin-induced thrombocytopenia using platelet-rich plasma by flow cytometer Lisann Pelzl1; Aleyna Karakuyu1; Hisham Jaffal1; Anurag Singh1; Irene Marini1; Jan Zlamal1; Stefanie Hammer2; Karina Althaus³; Tamam Bakchoul³ 1Universitätsklinikum Tübingen, Institute for Clinical and Experimental Transfusion Medicine, Medical Faculty of Tue- bingen, Tuebingen, Germany; 2Universitätsklinikum Tübingen, Centre for Clinical Transfusion Medicine, University Hospital of Tuebingen, Tuebingen, Germany; ³Universitätsklinikum Tübingen, Universitätsklinikum Tübingen, Institute for Clinical and Experimental Transfusion Medicine, Medical Faculty of Tuebingen; Centre for Clinical Transfusion Medicine, University Hospital of Tuebingen, Tuebingen, Germany Heparin-induced thrombocytopenia (HIT) is caused by anti- PF4/heparin IgG antibodies, which activate the platelets and lead to thrombocytopenia and thrombosis. The diagnosis of HIT is usually confirmed by using functional assays such as the Heparin-Induced Platelet Activation assay (HIPA assay). However, functional assays are time consuming and routinely available only in specialized laboratories. The aim of the current study was to establish a flow cytometer-based assay to determine procoagulant platelets in platelet-rich plasma (PRP) for the diagnosis of HIT. Sera samples from patients with HIT (HIT group) were incubated with PRP from healthy donors for different durations with (30, 60, 90 minutes). Procoagulant platelets were determined by analyzing double expression of P-selectin (CD62p) and phosphatidylserine (PS) externalization by ﬂow cytometry. CD32a- mediated cross-link and platelet stimulation with ionomycin were used as positive controls. Sera from HIT-diagnosed patients but not from the control-group induced a significant increase in the procoagulant platelet subpopulation in the presence of 0.2 U/mL heparin (% double positive CD62/PS: 1.2±1.1 vs 18.5±8.1, p=0.0021). The optimal incubation time was 60 minutes. A donor dependency of the ﬂow cytometric method was not observed (control vs HIPA+: 0.7±0.59 vs 19.0±3.2, p=0.0129). In addition, the use of washed platelets and PRP with HIT-sera led to the same results in this ﬂow cytometric method (34.2±6.3 vs 30.1±2.2, ns). Our data reflects towards suitability of aPRP-based protocol that can be used to detect the ability of HIT antibodies to induce procoagulant platelets by flow cytometry. In our ongoing studies, we are currently investigating the potential clinical implementation of this protocol in the diagnostic work up for HIT. Review: The Peripheral Blood Smear Evadne Parulan Holzhueter Achterwehr, Germany Review: Peripheral Smear Review: Peripheral Smear Case Report: This is a case of a 74 year old female on a yearly medial check.up on the 25th of October 2021. Except for a mild to moderate hypochromic anemia the diferential blood picture showed the following cellular percentage distribution as follows: • Neutrophil — 52% • Eosinophil — 3.5% • Basophil — 1.2% • Monocyte — 12.3% • Lymphocyte – 31% • Neutrophil — 52% • Eosinophil — 3.5% • Basophil — 1.2% • Monocyte — 12.3% • Lymphocyte – 31% Examination of the peripheral blood smear through the microscope revealed presence of plasma cells of varying stages of maturation. Furthermore a request for Immune Fixation test confirmed the diagnosis of Monoclonal Gammopathie, IgM Lambda. The increased percentage of Monocytes done through automation gave me the impression that the plasma cells are recognized as such. Therefore i like to present the following points of view relevant to consider as follows: Examination of the peripheral blood smear through the microscope revealed presence of plasma cells of varying stages of maturation. Furthermore a request for Immune Fixation test confirmed the diagnosis of Monoclonal Gammopathie, IgM Lambda. The increased percentage of Monocytes done through automation gave me the impression that the plasma cells are recognized as such. Therefore i like to present the following points of view relevant to consider as follows: g p g p 1. The examination of the peripheral blood smear through the microscope remains a standard operating procedure to detect early morphological, behavioral cellular changes in the human blood cells 1. The examination of the peripheral blood smear through the microscope remains a standard operating procedure to detect early morphological, behavioral cellular changes in the human blood cells 2. To develop our skills and competence to recognize the presence of normal and abnorm 2. To develop our skills and competence to recognize the presence of normal and abnormal cells as early as possible. 3. To emphasize the importance of laboratory diagnosis in health care and clinical decision makin German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 eA38 Results: The local calcium concentration forms a gradient in the BMM with highest calcium concentrations close to the endosteum. The calcium concentration in the BMM, CaSR expression on leukaemia cells, CaSR sensitivity to extracellular calcium, adhesion and migration in various concentrations of calcium differ between leukaemia types. CaSR acts as tumor suppressor or an oncogene in different leukaemias. In acute myeloid leukaemia (AML), limiting dilution transplantation of CaSR-deﬁcient AML-initiating cells revealed a 7-fold reduction of leukaemic stem cells. Downstream of CaSR, we implicated ﬁlamin A and other proteins as important signaling molecules. Treatment of mice with a CaSR agonist or antagonist differentially impacted myeloid leukaemias. Results: The local calcium concentration forms a gradient in the BMM with highest calcium concentrations close to the endosteum. The calcium concentration in the BMM, CaSR expression on leukaemia cells, CaSR sensitivity to extracellular calcium, adhesion and migration in various concentrations of calcium differ between leukaemia types. CaSR acts as tumor suppressor or an oncogene in different leukaemias. In acute myeloid leukaemia (AML), limiting dilution transplantation of CaSR-deﬁcient AML-initiating cells revealed a 7-fold reduction of leukaemic stem cells. Downstream of CaSR, we implicated ﬁlamin A and other proteins as important signaling molecules. Treatment of mice with a CaSR agonist or antagonist differentially impacted myeloid leukaemias. Conclusion: In summary, our results suggest that the CaSR and possibly calcium ions from the BMM strongly and differentially inﬂuence leukaemia progression, with ﬁlamin A playing an essential role in AML. As an adjunct to existing treatment strategies, targeting of CaSR with speciﬁc pharmacologic agents may be beneﬁcial in different leukaemias. Liquid profiling of circulating tumor DNA in colorectal cancer: steps needed to achieve its full clinical value as standard care Maren Hedtke1; Matthias Frölich2; Angelika Duda1; Laura Mirbach1; Volker Ast1; Victor Costina1; Uwe Martens³; Ralf Hofheinz⁴; Michael Neumaier1; Verena Haselmann1 1Universitätsmedizin Mannheim, Institut für Klinische Chemie, Mannheim, Germany; 2Universitätsmedizin Mannheim, Klinik für Radiologie und Nuklearmedizin, Mannheim, Germany; ³SLK Kliniken Heilbronn, Medizinische Klinik III, Heilbronn, Germany; ⁴Universitätsmedizin Mannheim, III. Medizinische Klinik, Tagestherapiezentrum, Mannheim, Germany Introduction: The analysis of circulating tumor DNA (ctDNA) is at the threshold of implementation into standard care for colorectal cancer (CRC) patients. However, data about the clinical utility of liquid proﬁling (LP), its acceptance by clinicians, and its integration into clinical workﬂows in real-world settings remain limited. Methods and Results: In total, 243 LP tests for 168 CRC patients were performed as part of standard care for RAS using beads, emulsiﬁcation, ampliﬁcation, and magnetics (BEAMing), for BRAF V600 by digital droplet PCR (ddPCR), or for both molecular targets. LP tests requested as part of routine care since 2016 were retrospectively evaluated. Results show restrained request behavior that improved moderately over time, as well as reliable diagnostic performance comparable to translational studies, with an overall agreement of 91.7%. Extremely low ctDNA levels at < 0.1% in over 20% of cases, a high frequency of concomitant driver mutations (in up to 14% of cases), and ctDNA levels reﬂecting the clinical course of disease were revealed. However, certain limitations hampering successful translation of ctDNA into clinical practice were uncovered, including the lack of clinically relevant ctDNA thresholds, appropriate time points of LP requests, and integrative evaluation of ctDNA, imaging, and clinical ﬁndings. Conclusion: These results highlight the potential clinical value of LP for CRC patient management and demonstrate issues that need to be addressed for successful long-term implementation in clinical workﬂows. Calcium, calcium-sensing receptor and its role in leukaemia progression Daniela S. Krause Uniklinik Marburg und Georg-Speyer-Haus Frankfurt, Laboratoriumsmedizin, Marburg, German Introduction: 99% of the body’s calcium is stored in bone, and calcium is released during bone remodeling. The calcium sensing receptor (CaSR) plays a role in the localization of normal haematopoietic stem cells in the BM microenvironment (BMM). However, the role of this receptor and its associated pathways for leukaemia development, therapy success and whether modulation of this receptor may be beneﬁcial therapeutically, is not known. Methods: Hypothesizing that the CaSR contributes to development, progression and response to therapy in leukaemia, we employed various in vitro assays, in vivo microscopy, leukaemia induction and in vivo treatment assays to test this question. German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 eA39 eA39 Aims: Inﬂammatory bowel disease (IBD) affects up to 1.3% of the western population and is characterized by chronic or relapsing intestinal inﬂammation. Biologic tumor necrosis factor (TNF) blockers, pioneered by the therapeutic antibody inﬂiximab (IFX), opened up the possibility of achieving disease remission. Yet, all biologics can provoke formation of anti-drug antibodies (ADA) that may cause therapy failure. 65% of patients on IFX develop ADA and thus, monitoring of both IFX and ADA is recommended. Currently used assays for TNF blocker and ADA assessment lack harmonization and could so far not sufﬁciently elucidate the impact that ADA quantity and quality have on patient-individual disease progress. Addressing the need for more powerful analytics, we present a surface plasmon resonance (SPR)-based biosensor. Materials and Methods: An assay for rapid ADA puriﬁcation from serum via magnetic IFX beads followed by SPR analysis was developed. We present the dissociation ratio (DissR) as an indicator of ADA:IFX binding kinetics, which is assessed within the ADA quantiﬁcation run. 45 sera from IBD patients under IFX therapy were analyzed by SPR and a diagnostics-approved ELISA. The individual ADA kinetics (only accessible by SPR) were evaluated and correlated with therapeutic outcomes. Results: The SPR assay exhibited robust drug-tolerance in presence of up to 10-fold molar excess of IFX. LOD and LQO of ADA quantiﬁcation were determined as 0.15 μgEq/mL and 0.40 μgEq/mL. Semi-quantitative ADA assessment was possible between LOD and LOQ. The ADA detection rate in the patient sera was similar for SPR and ELISA (52% and 55%). The moderate correlation of ADA concentrations between SPR and ELISA (R2=0.69) is owed to the methodological differences. DissR was shown to be independent of ADA concentration and unbiased by serum-individual matrix effects. Interest- ingly, ADA concentrations determined by ELISA showed stronger positive correlation with DissR (R2=0.66) compared with SPR quantiﬁcation (R2=0.57), i.e., high-afﬁnity ADA were observed in higher concentrations. This afﬁnity bias may be associated with determination of falsely low ADA titers in patients with lower-afﬁnity ADA. Monitoring of larger patient cohorts with the less biased SPR assay will show if this ﬁnding is clinically relevant. [Association studies of DissR with therapy success are in progress. At the congress, a larger cohort size and accordingly adjusted results will be presented.] Conclusion: Enabling parallel ADA quantiﬁcation and binding kinetic analysis, SPR biosensors deliver more informa- tion about ADA than currently used assays do. Larger studies could contribute to a better understanding of ADA occurrence in IBD patients treated with biologics. If future studies suggest clinical relevance, ADA kinetics may aid to re- evaluate the diagnostic suitability of currently used methods for ADA quantiﬁcation. Quantity or quality? Deciphering the diagnostic relevance of anti-drug antibody analysis by surface plasmon resonance Melina Grasmeier; Susanne Weber; Markus Thaler; Peter Luppa Melina Grasmeier; Susanne Weber; Markus Thaler; Peter Luppa Klinikum rechts der Isar der TU München, Institut für Klinische Chemie und Pathobiochemie, Münch Klinikum rechts der Isar der TU München, Institut für Klinische Chemie und Pathobiochemie, München, Germany eA40 German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 eA40 Results: Excellent correlation between the two LC-MS/MS methods could be found. The Passing-Bablok regression analysis revealed an intercept of -0.02 mg/L (95% conﬁdential interval (95% CI), -0.07 to 0.01 mg/L) and a slope of 1.00 mg/L (95% CI, 0.97 to 1.02 mg/L). Cusum test for linearity shows that there was no signiﬁcant deviation from linearity (P = 0.89). The correlation coefﬁcient was 0.99. Furthermore, comparing measurements were performed between two Xevo TQD tandem mass spectrometers using the faster MPA LC-MS/MS method. Very good correlation between these comparing measurements could also be found. The Passing-Bablok regression analysis revealed an intercept of -0.06 mg/ L (95% CI, -0.08 to 0.04 mg/L) and a slope of 1.02 mg/L (95% CI, 1.01 to 1.03 mg/L). Cusum test for linearity shows that there was no signiﬁcant deviation from linearity (P = 0.98). The correlation coefﬁcient was 0.99. Results: Excellent correlation between the two LC-MS/MS methods could be found. The Passing-Bablok regression analysis revealed an intercept of -0.02 mg/L (95% conﬁdential interval (95% CI), -0.07 to 0.01 mg/L) and a slope of 1.00 mg/L (95% CI, 0.97 to 1.02 mg/L). Cusum test for linearity shows that there was no signiﬁcant deviation from linearity (P = 0.89). The correlation coefﬁcient was 0.99. Furthermore, comparing measurements were performed between two Xevo TQD tandem mass spectrometers using the faster MPA LC-MS/MS method. Very good correlation between these comparing measurements could also be found. The Passing-Bablok regression analysis revealed an intercept of -0.06 mg/ L (95% CI, -0.08 to 0.04 mg/L) and a slope of 1.02 mg/L (95% CI, 1.01 to 1.03 mg/L). Cusum test for linearity shows that there was no signiﬁcant deviation from linearity (P = 0.98). The correlation coefﬁcient was 0.99. Conclusion: In summary, we could show that within the framework of a change control a signiﬁcantly faster LC-MS/MS measurement of MPA without loss of quality was possible. The new method was more than twice as fast as the fully validated old method and the correlation between the two LC-MS/MS methods was excellent, respectively. Comparison of two LC-MS/MS methods for quantification of mycophenolic acid in the sera of patients Joachim Kuhn; Cornelius Knabbe Institut für Laboratoriums- und Transfusionsmedizin, Herz- und Diabeteszentrum Nordrhein-Westfalen, Universitätsklink der Ruhr-Universität Bochum Aims: Measurement of mycophenolic acid (MPA) concentration in human blood is an important feature within the framework of therapeutic drug monitoring (TDM) of heart transplantation patients. Methods:Atotalof100clinicalsamplesfromhearttransplantedpatientswhichweretreatedwithmycophenolatemofetil(MMF) were used to compare a fully validated MPA LC-MS/MS method with a new faster one. The MPA measurements of the serum samples were performed after sample preparation using liquid/liquid extraction on a Quattro micro tandem mass spectrometer within a run time of 3.5 min on the one hand and, on theother, on aXevo TQD tandem mass spectrometerwith atotal run timeof only 1.6 min. MPA-D3 was used as internal standard. German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 eA41 eA41 Simplified preanalytical handling of plasma samples in patients treated with High-dose Methotrexate therapy and Glucarpidase rescue nörnschild2; Sven Liebig2; Immanuel Merz2; Peggy Kießling³; Monika Prpic1; Ulrich Keller2; Stefan 1 Amir Jahic1; Franz Knörnschild2; Sven Liebig2; Immanuel Merz2; Peggy Kießling³; Monika Prpic1; Ulrich Keller2; Stefa Schwartz2; Kai Kappert1 1Charité-Universitätsmedizin Berlin, Institut für Laboratoriumsmedizin, Klinische Chemie und Pathobiochemie, Berlin, Germany; 2Charité-Universitätsmedizin Berlin, Department of Hematology, Oncology and Cancer Immunology, Berlin, Germany; ³Labor Berlin - Charité Vivantes GmbH, Laboratoriumsmedizin & Toxikologie, Berlin, Germany Aims: High-dose methotrexate (HD-MTX) therapy is toxic and therefore requires an obligatory rescue approach combining leucovorin and, depending on individual patient history, glucarpidase administration. The exogenous folate derivative leucovorin provides a source of intracellular tetrahydrofolates and counters the toxic effect of the folate antagonist methotrexate (MTX). The recombinant bacterial enzyme glucarpidase is a rescue agent that speciﬁcally cleaves MTX and to a minor extent its active metabolite 7-OH-MTX extracellularly, forming the inactive metabolites 2,4-diamino-N10-methylpetroic acid (DAMPA) and OH-DAMPA in vivo. Glucarpidase theoretically might continue to metabolize MTX and 7-OH-MTX after blood withdrawal ex vivo. However, no consensus about preanalytical sampling and processing of blood collecting tubes intended for laboratory MTX and 7-OH-MTX testing after HD-MTX has been established. Material and Methods: One hundred ten plasma specimens from hematooncological patients (count: four, age: 74-86 years) that received HD-MTX, leucovorin rescue and glucarpidase, are selected as samples without (i.e. native) and with supplements (sodium chloride, NaCl; hydrogen chloride, HCl). Patient samples are further stored at various temperature conditions, prior to measuring MTX and MTX metabolites by liquid chromatography (LC) - tandem mass spectrometry (MS), LC-MS/MS. LC-MS/MS (MTX, 7-OH-MTX, DAMPA) measurements are compared to immunoassay (MTX) for selected conditions. Results: We will systematically evaluate different preanalytical conditions, including the effect of acid (HCl) addition, aiming at presumed glucarpidase inactivation ex vivo. MTX measurements determined by an immunoassay and the LC-MS/MS are expected to be different. MTX immunoassay (ARK diagnostics, Inc.) is expected to show a positive bias compared to the gold standard approach (LC-MS/MS). LC-MS/MS based quantiﬁcation of MTX and MTX metabolites in acid pretreated, i.e. inac- tivated specimens is expected to exhibit signiﬁcant differences when compared to measurements obtained from their non-inactivated comparative samples. Moreover, speciﬁc processing conditions are suspected to affect the stability of MTX and MTX metabolites in plasma samples when stored at different temperatures. Discussion and Conclusions: Preanalytical laboratory specimen management is still challenging in routine diagnostics. Simplified preanalytical handling of plasma samples in patients treated with High-dose Methotrexate therapy and Glucarpidase rescue Even for well-established laboratory parameters in hematooncological diagnostic settings, the storage temperature and the pretreatment of blood collecting tubes could be of high relevance for further clinical and therapeutic decisions. Fully automated application of the LCMS-8060NX system coupled to CLAM-2030 in 24/7 routine using HL-7 interface (beta-version) standards Frank Streit; Andreas Fischer; Ivana Markovic Universitätsmedizin Göttingen, Interdisziplinäres UMG Labor, Göttingen, Germany versitätsmedizin Göttingen, Interdisziplinäres UMG Labor, Göttingen, Germany Background and Aims: Liquid chromatography coupled with mass spectrometry (LC-MS/MS) offers exceptional sensi- tivity and speciﬁcity and is widely used for therapeutic drug monitoring and toxicological screening. The development of preanalytical and analytical sample management improved the efﬁciency of sampleprocessing,the precision and quality of manually work, as well as automated validation and interpretation of data. Recent advancements in LC-MS/MS develop- mentofferinnovativeanalytical platforms, however routine applications arestilllimited. Inour laboratory,there are several laboratory developed tests (LDT) using automated LC-MS/MS on Clam2030-LCMS8060 NX established (i.e. the measure- ment of antibiotics, anticonvulsive drugs and direct oral anticoagulants concentrations (DOACs)). Therefore, we aimed to develop and validate a fully automated application of the LCMS-8060NX system coupled to CLAM-2030 in 24/7 routine by using the HL-7 interface (beta-version, for research only) standards. Materials and Methods: The clinical laboratory automated module CLAM2030 combined with an LCMS8060NX (both Shimadzu) was tested for efﬁciency of preanalytical and analytical sample management for three different LDT-procedures (sample preparation, analyzing, calibration identiﬁcation and calculation of quality controls (Alsachim and Recipe)). HL-7 interface (beta-version) standards were used for bidirectional communication between the laboratory information system (OSM, Germany) and Clam2030-LCMS8060 NX. Results: Patient samples and quality control samples for three different LDT-procedures (TDM of antibiotics, anticon- vulsive drugs and DOACs) were measured 5 times in a randomized manner. Variation of results and retention times are presented. Different acceptance criteria for analytes, as well as stable isotopic labelled internal standards were used to verify analytical processes in terms of identiﬁcation, calculation and result transmission. We demonstrate an effective work ﬂow from requesting a laboratory test until transmission of the data using a bidirectional HL-7 interface (beta- version) protocol between the laboratory information system and the Clam2030-LCMS8060 NX instrument. Conclusion: Simultaneous measurement of different LDT-procedures makes CLAM-2030-LCMS8060 NX an attractive device for 24/7 use in clinical and toxicological diagnostics. Automated randomized measurements of patient samples with the LCMS-8060NX system coupled to CLAM-2030 requesting different LDT procedures were successfully transferred to the laboratory information system (LIS) using HL-7 Interface (beta-version) standards, enabling its applications in 24/7 routine. eA42 German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 eA42 The impact of the EMA DPYD variant analysis recommendations from 2020 on requesting behavior and number of analyses The impact of the EMA DPYD variant analysis recommendations from 2020 on requesting behavior and number of analyses Maren Hedtke; Romy Eichner; Laura Mirbach; Michael Neumaier; Verena Haselmann Universitätsmedizin Mannheim, Institut für klinische Chemie, Mannheim, Germany German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 eA43 Introduction: A major cause of toxic or potentially lethal side effects of ﬂuoropyrimidine therapy (like 5 ﬂuorouracil or capecitabine) is a deﬁciency or a complete lack of the dihydropyrimidine dehydrogenase (DPD) activity. DPD is the main enzyme for the degradation of the aforementioned drugs. Mutations that lead to a decreased enzyme activity are found in up to 9% of the population, 0.1 – 0.5% show a complete lack of DPD activity. Therefore, since 2020 the European Medicines Agency (EMA), the Federal Institute for Drugs and Medical Devices (BfArM) and the German society for hematology and medical oncology (DGHO) recommend testing for four DPYD mutations that affect enzyme activity before the ﬁrst administration of ﬂuoropyrimidines. Introduction: A major cause of toxic or potentially lethal side effects of ﬂuoropyrimidine therapy (like 5 ﬂuorouracil or capecitabine) is a deﬁciency or a complete lack of the dihydropyrimidine dehydrogenase (DPD) activity. DPD is the main enzyme for the degradation of the aforementioned drugs. Mutations that lead to a decreased enzyme activity are found in up to 9% of the population, 0.1 – 0.5% show a complete lack of DPD activity. Therefore, since 2020 the European Medicines Agency (EMA), the Federal Institute for Drugs and Medical Devices (BfArM) and the German society for hematology and medical oncology (DGHO) recommend testing for four DPYD mutations that affect enzyme activity before the ﬁrst administration of ﬂuoropyrimidines. Methods: In a retrospective data evaluation 386 patient results (PCR and subsequent pyrosequencing) were analyzed for the frequency and the distribution of the variants 2A, 13, haplotype B3 (or c.1129-5923C>G) and c.2846 A>T (or p. D949V) of DPYD. Furthermore, the number of requests from clinicians between 2015 and 2021 is evaluated with regard to the change of requesting behavior with the introduction of mandatory DPYD testing. Results: The most frequently found variant was a heterozygous mutation for haplotype B3 that was found in 4.6% of the patients. Heterozygosity for 2A and c.2846A>T was found in 0.78% and 1.3% respectively, whereas 13 was not detected at all. Homozygous mutations were not found for any of the four variants. The impact of the EMA DPYD variant analysis recommendations from 2020 on requesting behavior and number of analyses Identiﬁed variant frequencies are in accordance with previously published data. In 2019 we had 8 requests for DPYD analysis while in 2021 there were 233. Conclusion: We want to emphasize the value of DPYD genotyping for patients before therapy with ﬂuoropyrimidines. The drastic increase of test requests shows how many patients were not analyzed before 2020 and how many side effects or even fatal outcomes could have been prevented. A simple LC-MS/MS method for iohexol quantification in serum Jonathan Stefi; Katharina Habler; Michael Vogeser; Michael Paal Klinikum der Universität München, LMU München, Institut für Laboratoriumsmedizin, München, Germany Introduction: The measurement of glomerular ﬁltration rate (GFR) is a very important tool for the assessment of renal function and an indispensable indicator for the progression of chronic kidney disease. Measuring GFR by iohexol clearance is increasingly applied in clinical practice (e.g. critically ill patients) given that estimations of the GFR with creatinine and cystatine do not necessarily reﬂect the actual GFR. The aim of the current study was to develop a highly selective LC-MS/MS method for the quantiﬁcation of iohexol in human serum to allow accurate GFR measurements with this nonionic contrast medium. Methods: After cleanup of 50 μL serum by protein precipitation, iohexol was chromatographically separated within 3.5 minutes on a Acquity BEH-C18 column using water-formic acid and methanol-formic acid as mobile phases for gradient elution. Iohexol was quantiﬁed within the range of 5 – 500 mg/L using iohexol-D5 as internal standard. The method was comprehensively validated according to the European Medicines Agency (EMA) bioanalytical method validation protocol. Results: Inaccuracy and the imprecision coefﬁcient of variation were ≤7.2 % and ≤4.6 % for all quality controls. The internal standard consistently compensated for matrix effects. Analysis of external quality control (EQA) samples gave deviations of ≤1.9 % from expected nominal concentrations. Conclusion: We hereby present a robust and accurate LC-MS/MS method that is suitable for iohexol quantiﬁcation in human serum. Due to the simple sample preparation protocol and chromatography setup, the method is readily implementable in clinical LC-MS/MS laboratories. An European IVD conform LC-MS-based method for monitoring of Azathioprine metabolites in whole blood Verena Horneffer-van der Sluis Universitätsklinikum Hamburg-Eppendorf (UKE), Institut für Klinische Chemie und Laboratoriumsmedizin, Hamburg, Germany Introduction: Azathioprine is a widely used immunosuppressant drug with broad indications, including treatment of inﬂammatory bowel disease and organ rejection in transplanted patients. Its metabolites are cytotoxic and metabolic rates differ highly among individuals. TDM is needed to assess treatment efﬁcacy and dosage regime. However, there is a lack in commercially available CE marked tests in the EU and laboratory developed tests (LDT) remain to be established. Here, we present the implementation of a liquid chromatography mass spectrometry based assay for quantifying Azathi- oprine metabolites 6-methylmercaptopurine (6-MMP) and 6-thioguanine nucleotides (6-TGN) in whole blood samples according to European 2017/746 on In-Vitro-Diagnostics regulation (IVDR). Materials and Methods: EDTA samples were prepared as described previously [1]. A seven point calibration curve and two quality control samples of different concentration ratios are processed in parallel to the samples before injected into a Waters Acquity UPLC-system hyphenised with a Waters TQS triple quadrupole mass spectrometry instrument equipped with electrospray ionisation. Compound speciﬁc multiple reaction monitoring transitions are acquired during a 6 min Reversed Phased chromatographic gradient. Method documentations are obtained and maintained as required by the European IDVR guidelines. Results: Validation of the method with regards to sensitivity, linearity, accuracy, bias, and precision according the CLSI recommendation given in EP05, 06, 09, and 17 based on more than 100 sample injections revealed following: a LLOQ of 50 pmol/0.2mL (4 samples in duplicate at 2 days) and a linearity of > 0.995 up to a ULOQ of 10,000 pmol/0.2mL (5 replicates per point for 2 operators). An accuracy and precision of better than 15% is achieved for within laboratory performance including within-run, between days, and inter-operator comparison (5 samples in 5 replicates at 3 days and 2 operators). The bias is determined to be within 3%. The method is applied to routine sample analysis of more than 40 samples. German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 eA44 Discussion and Conclusion: In conclusion, we describe an IVDR consistent UPLC–MS/MS based method that is sen- sitive, linear, and reproducible for the simultaneous quantiﬁcation of 6-MMP and 6-TGN in whole blood samples. Discussion and Conclusion: In conclusion, we describe an IVDR consistent UPLC–MS/MS based method that is sen- sitive, linear, and reproducible for the simultaneous quantiﬁcation of 6-MMP and 6-TGN in whole blood samples. Work content, platform and methods: The main goal of this working group is to teach its members the basic principles of being able to efﬁciently perfom workﬂow-oriented tasks using programming languages. Colleagues get trained to independently analyze laboratory data. Work content, platform and methods: The main goal of this working group is to teach its members the basic principles of being able to efﬁciently perfom workﬂow-oriented tasks using programming languages. Colleagues get trained to independently analyze laboratory data. To this end, our main medium-term goal is to learn the programming language R, which specializes in statistics and data analytics. In addition, simple "supporting" tools such as writing Markdown text for e.g. static analyses documents or websites as well as version control (Git and GitHub) in order to be able to work together on projects are tought. In addition, well-founded statistical knowledge will be required, which is to be imparted through the participation of external lecturers in the regularly held online sessions. As a working environment, the working group uses the platform "GitHub", a website on which a graphical interface for the version management software "Git" can be used free of charge. For this purpose, the organization "DGKL Junges Labor - AG: Digitale Kompetenz" was founded on GitHub. Additionally, in order to keep track of the jungle of "IT buzzwords" (e.g. deep learning, blockchain, cryptography, etc.), current "trending words" are presented in an easy-to-access format of an "IT buzzword bingo" (5 - 8-minute presentations) and then discussed. Outlook: The working group is initially planned to run for about two years. In addition to the training of working group members, further training offers are to be created in order to facilitate the introduction of the programming language R for the members of the DGKL. After the two-year initial phase, it is planned to create a multi-day "Digital Competence" course that could be offered by the DGKL. Working Group: Digital Competence - Strengthening digital competence in laboratory medicine Jakob Adler1; Alexander Tolios2; Ronald Biemann³ 1Medical Laboratory for Clinical Chemistry, Microbiology, Infectious Disease and Genetics “Prof. Schenk/Dr. Ansorge & Colleagues”, Clinical Chemistry, Endocrinology, Therapeutic Drug Monitoring, Magdeburg, Germany; 2Medical Univer- sity of Vienna, Department of Blood Group Serology and Transfusion Medicine, Wien, Austria; ³University of Leipzig, Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, Leipzig, Germany Aims: More and more digital applications are ﬁnding their way into everyday medical practice. In recent years tech giants such as Google are developing Deep Learning-based algorithms for diagnosing and classifying diseases such as diabetes mellitus through retinal image analysis and are even running ahead of the universities in some ﬁelds of medical research. Since digital competence among physicians and professionals in laborory medicine is neither taught during medical graduate training nor part of the already existing range of competences, the Young Laboratory Section (Sektion “Junges Labor”) of the German Society of Laboratory Medicine (“DGKL”) created a working group to provide support in that ﬁeld. German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 eA45 Ramona Christina Dolscheid-Pommerich1; Sarah Verlage2; Birgit Stoffel-Wagner1; Ingo Gräff³ Ramona Christina Dolscheid-Pommerich1; Sarah Verlage2; Birgit Stoffel-Wagner1; Ingo Gräff³ 1University Hospital Bonn, Institute of Clinical Chemistry and Clinical Pharmacology, Bonn, Germany; 2University of Cologne, Department of Rehabilitation and Special Education, Köln, Germany; ³University Hospital Bonn, Emergency Department, Bonn, Germany Introduction: Determination of hemoglobin (Hb) concentration with either POCT co-oximetry or central laboratory SLS detection is critical regarding diagnosis of anemia or critical transfusion limits. At the emergency department of the University Hospital Bonn (UKB), blood gas analysis, including co-oximetry, is performed during a patient’s ﬁrst medical examination. At the same time, venous blood is taken and immediately sent via pneumatic tube for further analysis, including blood cell count, to the central laboratory (CL). Given the importance of properly determining hemoglobin concentrations for diagnosing anemia, it is essential to know whether the two methods yield comparable results and whether both are equally appropriate for different age groups. In the present study, we examined this question using a large-scale sample. Methods: N = 19182 patients were included (10439 male, 8743 female, mean age: 57.4 years, min 18, max 102 years). The cohort of patients was divided into the following three subgroups: 1 (age > 18-64 years, n = 11201), 2 (65-84 years, n = 6500) and 3 (age > 85 years, n = 1481). For each patient, POCT Hb concentration (RapidLab 1265, Siemens Healthineers) and CL concentration (XN1000, Sysmex) were compared. The whole collective as well as the subgroups were analyzed regarding diagnosis of anemia, according to the WHO deﬁnition, and critical anemia (Hb < 8 g/dl). Results: Overall, a signiﬁcant correlation was found for Hb concentrations between both methods, r = .96, p < 0.001. There were signiﬁcant differences between the two methods, with POCT yielding higher Hb concentrations (average value: 13.43 g/dl, SD ± 2.41) than CL (average value, 13.1 g/dl SD ± 2.35), p < 0.001. For the whole sample, both methods differed signiﬁcantly in their classiﬁcation of anemia according to WHO (male p < 0.001, female p < 0.001) and critical anemia (p < 0.001). In the three subgroups, classiﬁcation of anemia differed for all age groups (1: male p < 0.001, female p < 0.001, 2: male p < 0.001, female p < 0.001, 3: male p < 0.001, female p < 0.001). Regarding classiﬁcation of critical anemia, the two methods differed depending on age group. Ramona Christina Dolscheid-Pommerich1; Sarah Verlage2; Birgit Stoffel-Wagner1; Ingo Gräff³ For subgroups 1 and 2, POCT and CL Hb concentrations differed signiﬁcantly in their classiﬁcation of critical anemia (1: p = 0.025, 2: p = 0.025), while for subgroup 3, there was no signiﬁcant difference between both methods used (3: p = 0.054). Conclusion: Overall, POCT and CL values were highly correlated, however, both methods also yielded relevant differ- ences concerning diagnosis of anemia. Hemoglobin concentrations determined with SLS CL are more likely to yield a diagnosis of anemia compared to Hb determined with POCT co-oximetry at UKB. eA46 German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 Development of a photonic microring resonator sensor system for the fast detection of biomolecules in point-of-care testing devices Christina Hoffmann1; Peter Hoffmann1; Jakob Reck2; Moritz Kleinert2; Klara Mihov2; Norbert Keil2; Vera Froese³; Ulrich Kertzscher³; Kristina Mykhailiuk⁴; Julia Michaelis⁴; Hans-Jürgen Heupke⁵; Sören Scholand⁵ 1Charité - Universitätsmedizin Berlin, Institut für Laboratoriumsmedizin, Klinische Chemie und Pathobiochemie, Berlin, Germany; 2Fraunhofer Heinrich-Hertz-Institut, Photonic Component Department, Berlin, Germany; ³Charité - Uni- versitätsmedizin Berlin, Institut für Kardiovaskuläre Computer-assistierte Medizin, Berlin, Germany; ⁴SCIENION GmbH, SCIENION GmbH, Berlin, Germany; ⁵Eschweiler GmbH & Co. KG, Eschweiler GmbH & Co. KG, Kiel, Germany Aims: Microring resonators are round optical waveguides which resonate with a characteristic wavelength. If an analyte attaches to the surface, the optical properties change, and a wavelength shift can be observed. This shift is proportional to the mass (amount) of the attached analyte. The aim of the optION sensor project is to research and develop photonic microring resonators as analytic tools for in vitro diagnostics. Materials and Methods: The photonic sensor chips were fabricated from silicon nitride wafers using photolithography. The ring resonators were functionalized with sodium ionophore as capture molecule. As a reference, one ring resonator remained without functionalization. Aqueous solutions with NaCl concentrations between 500 mM and 125 mM were applied to the optION sensor. Between measurements, the chip was rinsed with Triton X-405 and ddH2O. Results: The maximal wavelength shift occurred after about 10 seconds. It is possible to observe the binding curve of the analyte. The repeated usage of the same sensor after washing was successful. Sodium ions were detected in the inves- tigated concentration range between 500 mM and 125 mM, which reaches physiologically relevant concentrations. Conclusion: The photonic microring resonators are a promising technology for fast and label-free point-of-care measurements. Funding: Federal Ministry of Education and Research, grant 13GW0243F Conclusion: The photonic microring resonators are a promising technology for fast and label-free point-of-care measurements. Funding: Federal Ministry of Education and Research, grant 13GW0243F 1Institute of Clinical Chemistry and Laboratory Medicine, University Hospital Regensburg, Franz-Josef-Strauss-Allee 11, 93053 Regensburg, Germany; 2Singapore Lipidomics Incubator (SLING), Life Sciences Institute, National University of Singapore, Singapore Introduction: The analysis of human faecal metabolites can provide an insight into metabolic interactions between gut microbiota and host organism. The creation of metabolic proﬁles in faeces has received little attention until now and reference values, especially in the context of dietary and therapeutic interventions, are missing. The aim of the present study is to give concomitant concentration ranges of faecal sterol species, bile acids and short chain fatty acids based on a large cohort. Methods: Sterol species, bile acids and short chain fatty acids in human faeces from 165 study participants were quantiﬁed by LC-MS/MS. For standardization, we refered all values to dry weight of faeces. Results and Discussion: Based on the individual intestinal sterol conversion we classiﬁed participants into low and high converters according to their coprostanol/cholesterol ratio. Based on a large number of study participants we give a general quantitative overview of several metabolites in human faeces that can be used as reference values. The intestinal cholesterol conversion is a distinctive feature to evaluate SCFA and bile acid concentrations. Patient stratiﬁcation into high or low sterol converter groups is associated with signiﬁcant differences in faecal metabolites with biological activities. Such stratiﬁcation should then allow assessing faecal metabolites better before therapeutic interventions. Low converters excrete signiﬁcantly more straight chain fatty acids and bile acids than high converters. 5th, 95th percentile and median of bile acids and short chain fatty acids were calculated for both groups. The strength of our calculation is that our data base on i) a large cohort, ii) an uncontrolled diet which should reflect the behaviour of the normal population and iii) a comprehensive data set from various countries in Europe. Short chain fatty acids and bile acids in human faeces are associated with the intestinal cholesterol conversion status Silke Matysik Regensburg, Germany Silke Matysik1, Sabrina Krautbauer1, Gerhard Liebisch1, Hans-Frieder Schött2 German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 eA47 eA47 High-Throughput Sample Preparation in NMR-Spectroscopy Marc Fenzlaff1; Ann-Kristin Henning1; Kathrin Budde1; Friederike Gauß1; Daniel Rosenkranz2; Astrid Petersmann2; Matthias Nauck1 1University Medicine Greifswald, Institute of Clinical Chemistry und Laboratory Medicine, Greifswald, Germany; 2University Medicine Oldenburg, Institute of Clinical Chemistry und Laboratory Medicine, Oldenburg, Germany Background and objectives: Sample preparation in nuclear magnetic resonance (NMR)-spectroscopy can be time- consuming, especially if a huge amount of samples should be measured (e.g. in epidemiological studies). Furthermore, the inter-observervariability has animpact onthequalityoftheprepared samplesandconsequently onthe spectra quality.This study aimed to compare the manual and the automated sample preparation for NMR measurements in terms of time, reference concentration and congruence of the spectra. Study design and methods: To achieve comparable results, we used a plasma pool for both manual and automated sample preparation. Manual sample preparation consists of mixing 300 μL of phosphate buffer [prepared with D2O and contained sodium 3-trimethylsilyl-(2,2,3,3-D4)-1-propionate (TSP) as reference, (pH 7.4)] with 300 μL plasma directly in a 5 mm NMR-tube. Automated sample preparation was realised by a pipetting robot (Tecan Freedom Evo) which is equipped with four needles. Spectra were recorded on Bruker Avance Neo 600 MHz NMR-spectrometer at 310 K as a standard one- dimensional 1H-NMR pulse sequence including water suppression. Whenever four samples were completely prepared with both preparation methods, the time of sample preparation was monitored and compared. The TSP signals of both prepa- ration methods were overlapped to have a visual impression on spectra quality. In addition to the visual evaluation, the means of the TSP concentrations were calculated and compared to the reference concentration. Results: Manual pipetting of 60 plasma samples took about 49 minutes compared to the automated pipetting which only took about 37 minutes in average. Thus, automated pipetting resulted in an average time saving of 25 %. In addition, the hands-on time dropped from 49 to 37 minutes. With regard to the quality of the spectra, we observed that the overlapped TSP-signals in both methods were almost congruent. The measured TSP-concentrations of both sample preparations were nearly identical after measurement of 16 plasma samples (mean manual preparation = 35.10 mmol/L, relative standard deviation RSD = 0.54 %; mean automatic preparation = 35.05 mmol/L, RSD = 0.79 %). The reference value of TSP is 35.90 mmol/L. Conclusion: The present study has shown that high-throughput sample preparation provides a convenient tool in NMR-spectroscopy. eA48 Conclusion: In clinical lipidomics a time span of 4h at 4°C between blood drawing and separation of plasma for transportation and handling is acceptable. We detected a high variability between and within distinct lipids and classes and recommend therefore careful assessment of the sample history before performing high resolution clinical lipidomics investigations. Conclusion: In clinical lipidomics a time span of 4h at 4°C between blood drawing and separation of plasma for transportation and handling is acceptable. We detected a high variability between and within distinct lipids and classes and recommend therefore careful assessment of the sample history before performing high resolution clinical lipidomics investigations. High-Throughput Sample Preparation in NMR-Spectroscopy It minimizes the time-consuming work for the staff and affords a comparable spectra quality compared to the manual preparation. Therefore, an automated sample preparation in NMR-spectroscopy should be considered, especially for the analysis of huge amounts of samples. Preanalytical considerations for clinical lipidomics Qingqing Wang1; Miriam Hoene2; Chunxiu Hu1; Andreas Peter2; Guowang Xu1; Rainer Lehmann2 1Dalian Institute of Chemical Physics, Chinese Academy of Sciences (CAS), Dalian 116023, China, CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian, China; 2University Hospital Tübingen, Institute for Clinical Chem- istry and Pathobiochemistry, Tübingen, Germany Introduction: The interest in clinical lipidomics increased continuously during recent years. Most commonly big cohort studies are performed with plasma or serum biobank samples collected years ago. Those blood samples were transported, handled and processed at that time according to various preanalytical protocols or standard operating procedures. In view of the fact that clinical lipidomics cover lipid species of various stabilities we investigated alterations of the lipidome in whole blood after drawing based on pre-analytical variabilities. This is the ﬁrst report performed in a considerable number of samples from different individuals. Methods: More than 400 lipid species of 14 classes were proﬁled in > 800 EDTA plasma samples by UHPLC-MS. Before plasma separation whole blood samples were exposed to various conditions (three different temperatures (4°C, 21°C, 30°C) and six time-intervals from 30 min up to 24h). These lipidomes were compared to lipidomes of at once after blood collection prepared plasma samples. Signiﬁcant alteration of a lipid was deﬁned as >10% changes in the lipid level (p < 0.05, FDR < 0.05). Results: Summer time conditions, meaning sample handling or transportation at 30°C, led within 60 min to signiﬁcant changes in > 6% of lipid species. Most instable lipid classes are (O-acyl)-hydroxy fatty acids (FA), free FA, and LPCs. In at once cooled blood samples only 0.7% of all lipid levels were altered after 4h and transportation for 24h is possible (< 6% changes in lipid levels at 4°C). Even at 30°C for 24h several lipid classes were stable. Detailed information for distinct lipid classes and species will be provided. eA48 German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 Cognitive impairment coincides with increased circulating levels of endocannabinoids. Alexander Gaudl; Uta Ceglarek Universitätsklinikum Leipzig, Institut für Laboratoriumsmedizin, Klinische Chemie und Molekulare Diagnostik, Leipzig, Germany German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 eA49 Introduction: Impairment of cognitive functioning is a concomitant in most neurodegenerative disorders (NDD). Still, their cause and genesis remain mostly undeﬁned. Several mechanisms depending on cell type, stage of development, and brain region have been proposed. The endocannabinoid system (ECS), including at least the cannabinoid receptors type 1 and type 2 as well as endogenous cannabinoids (eCB) and enzymes facilitating their synthesis and degradation, has been shown to contribute to the development of NDDs in animal experiments. The aim of this study was to associate the eCBs 2-arachidonoyl glycerol (2-AG), arachidonoyl ethanolamide (AEA), oleoyl ethanolamide (OEA), and palmitoyl ethano- lamide (PEA) with cognitive functioning in a case/control cohort within the LIFE Adult study. Introduction: Impairment of cognitive functioning is a concomitant in most neurodegenerative disorders (NDD). Still, their cause and genesis remain mostly undeﬁned. Several mechanisms depending on cell type, stage of development, and brain region have been proposed. The endocannabinoid system (ECS), including at least the cannabinoid receptors type 1 and type 2 as well as endogenous cannabinoids (eCB) and enzymes facilitating their synthesis and degradation, has been shown to contribute to the development of NDDs in animal experiments. The aim of this study was to associate the eCBs 2-arachidonoyl glycerol (2-AG), arachidonoyl ethanolamide (AEA), oleoyl ethanolamide (OEA), and palmitoyl ethano- lamide (PEA) with cognitive functioning in a case/control cohort within the LIFE Adult study. Methods: An LC-MS/MS method was established to simultaneously quantify 2-AG, AEA, OEA, and PEA in human plasma within a total run time of 10 minutes. From the LIFE Adult study 500 probands were selected by the inclusion criteria of an age ≥60 years and the participation in a neuro-psychological test battery to address cognitive performance in a sur- rounding of Alzheimer’s disease or dementia. Hereby, the SISCO score is determined, which derives from summation of distinct items from the unit „Strukturiertes Interview für die Diagnose einer Demenz vom Alzheimer-Typ, der Multiinfarkt- (oder vaskulären) Demenz und Demenzen anderer Ätiologie nach DSM-III-R, DSM-IV und ICD-10“ (SIDAM), and caps at 55 points. Subcohorts depicting high cognitive performance (SISCO ≥53) as well as slight cognitive impairment (SISCO ≤45) were used for association analysis. Results: Statistically signiﬁcant concentration differences between the two subcohorts were found for 2 AG, AEA, and OEA. Cognitive impairment coincides with increased circulating levels of endocannabinoids. Inﬂuencing factors were found to be sex (for AEA, OEA) and BMI (for 2 AG, AEA). After adjusting for these confounders AEA and OEA remained signiﬁcantly different and showed both a negative association with cognitive performance (odds ratio per standard deviation [OR] 1.33, 95 % CI 1,17-1,46), OEA (OR 1.23, 95 % CI 1.06-1.37). Conclusion: The negative association of AEA with cognitive performance opposes to former results from nimal experiments, which showed for example increased memory performance during increased half-life of AEA in the synaptic cleft. This might indicate an inverse relation of total concentration and residence time and, therefore, a much more complex role of eCBs in cognitive functioning. eA50 Materials and Methods: Serum samples from eleven healthy men were obtained from the 4-day laboratory BOTI (BOdy TIme) study, which was conducted at Charité Universitätsmedizin Berlin. The study consisted of an adaption night, a baseline night, a 40-hour episode of sleep deprivation and an 8-hour recovery sleep episode. Before the baseline night current gold standard for internal circadian time, the dim-light melatonin onset (DMLO), was determined in saliva samples. The sleep deprivation period was carried out under constant routine conditions. The concentration of up to 630 metabolites from 26 biochemical classes (e.g., acylcarnitines, amino acids, bile acids, ceramides, cholesteryl esters, lipids) were measured using LC MS/MS. To create and evaluate predictive models of time-based metabolomics data four different methods of machine learning (Random Forest, Cubist, ZeitZeiger, least absolute shrinkage and selection operator (LASSO)) were applied. Results: After data processing concentrations of 494 out of 630 metabolites were used for building a model for determination of the internal time. The predictor determination was performed with two different predicted variables (external and internal time) and four metabolite data formats (1-sample: single measurement in the time series or 2-sample: ratio of two measure- ments 6 hours apart in the time series and scaled or scaled and denoised data). All types of predictors performed comparably within one method of machine learning: Random Forest, Cubist, ZeitZeiger and LASSO. Comparing the different machine learning methods LASSO and LASSO with maximum 10 features showed the best performance with 77.27-93.51% and 67.53- 90.26% of predictions showing an error ≤2 hours, respectively. Conclusion: The study showed a highly accurate model to estimate the internal circadian time from a single blood sample. Only a few metabolite concentrations are needed, and the model is as comparable with the current gold standard. Thus, the described metabolite model provides an accurate tool for personalization of health care according to the patient’s circadian clock. Determination of internal circadian time with metabolites Sophie Neugebauer1; Wolfgang Schmidt-Heck2; Mirko Peitzsch³; Gianni Panagiotou2; Achim Kramer⁴; Michael Kiehntopf1 1Jena University Hospital, Department of Clinical Chemistry and Laboratory Diagnostics, Jena, Germany; 2Hans-Knoell- Institute, Leibniz Institute for Natural Product Research and Infection Biology, Jena, Germany; ³University Hospital and Medical Faculty Carl Gustav Carus, Technical University Dresden, Institute of Clinical Chemistry and Laboratory Medi- cine, Dresden, Germany; ⁴Charité Universitätsmedizin Berlin, Laboratory of Chronobiology, Institute for Medical immunology, Berlin, Germany Aims: The circadian clock is a fundamental biological program that coordinates 24-hour rhythms in physiology, metabolism, and behavior, and has a major impact on health. The beneﬁts of therapy adapted to the time of day (chronotherapy) have been demonstrated in many studies. Since internal circadian time is different for each individual, the basis of any personalized chronotherapy is to determine the individual chronotype. In this regard, internal time is not a stable characteristic, but is inﬂuenced by several factors (e.g., age, gender, ambient light, season). Currently, a simple diagnostic tool for determining individual internal circadian time is lacking. In this project, mass spectrometric methods will beusedtodetermineselectedsmallmoleculesandmetabolitestoestablishachronotoolthatcanbeusedtodetermineinternal circadian time. eA50 German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 Sterol characterization of lipid rafts by targeted mass spectrometry Ilijana Begcevic Brkovic1; Madlen Reinicke1; Soroth Chey1; Ingo Bechmann2; Uta Ceglarek1 lijana Begcevic Brkovic1; Madlen Reinicke1; Soroth Chey1; Ingo Bechmann2; Uta Ceglarek1 1Leipzig University, Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, Leipzig, Germany; 2Leipzig University, Institute of Anatomy, Leipzig, Germany Introduction: Cholesterol is an essential component of the mammalian plasma membrane and is highly concentrated in specialized microdomains of the plasma membrane termed as lipid rafts. Non-cholesterol sterols such as lanosterol (LA), desmosterol (DE) and 7-dehydrocholesterol (7-DHC), and nutritional derived plant sterols such as campesterol (CA), sitosterol (SI) and stigmasterol (ST) are structurally and functionally similar to cholesterol (CH) and are therefore incor- porated in cell membranes, too. There is some evidence that membrane ﬂuidity and signaling may be affected by the non- cholesterol content. The aim of this study was to develop targeted liquid chromatography mass spectrometry (LC-MS/MS) assay for analysis of sterol compounds in lipid rafts of microglia cell membranes. Methods: Human EDTA-plasma (pooled and individual) was used for method development and validation purpose. Samples were prepared according to the established protocol, involving precipitation with methanol/isopropanol (50/50, v/v) solution, containing relevant internal standards. Following, samples were separated and analyzed over the 14-min LC gradient, utilizing a Shimadzu Nexera XR HPLC system (Duisburg, Germany), online coupled to a QTRAP 6500 (Sciex, Framingham, MA, USA) mass spectrometer. Lipid rafts were isolated from mouse microglia SIM A9 cells applying the OptiPrep™discontinuousdensitygradientwith 20-hourultracentrifugation.Extractedlipidswereanalyzed with developed MS-assay, while lipid rafts marker ﬂotillin-1 was determined by Western blot. Results: All sterol compounds had good linear response with R2 ≥0.9982. The estimated LLOQs were: 0.05 mg/L (CA, SI and LA), 0.1 mg/L (BR and ST) and 2.5 mg/L (CH). Recovery was in the range of ± 15% and within-day/between-day precision of native and spiked plasma was below 10% and 7%, respectively. Plasma sterols were stable at 10 °C for at least 48 hours and were resistant to repeated freeze and thaw cycles. Method comparison between LC-MS/MS and gas chromatography-MS method showed good agreement (Spearman correlation r=0.8912, p < 0.001). Similar abundance proﬁle was observed for sterols and ﬂotili-1 in the isolated lipid rafts fractions. DE and LA had the highest concentration in the lipid rafts, followed by the SI, ST and CA. German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 eA51 Conclusion: Overall, this study demonstrates successful development, validation and implementation of the quanti- tative, multiplex LC-MS/MS assay for sterol analysis. Sterol characterization of lipid rafts by targeted mass spectrometry The method has wide application e.g. in human plasma, lipid rafts, but also other biological samples, using simple preparation protocol and small sample volumes (10 μL). Future work will be focused to reveal functional importance of sterol compounds in the lipid rafts fractions. Conclusion: Overall, this study demonstrates successful development, validation and implementation of the quanti- tative, multiplex LC-MS/MS assay for sterol analysis. The method has wide application e.g. in human plasma, lipid rafts, but also other biological samples, using simple preparation protocol and small sample volumes (10 μL). Future work will be focused to reveal functional importance of sterol compounds in the lipid rafts fractions. Targeting ABCC6 in Mesenchymal Stem Cells: Impairment of Differentiated Adipocyte Lipid Homeostasis Ricarda Plümers; Michel Robin Osterhage; Christopher Lindenkamp; Isabel Faust-Hinse; Cornelius Knabbe; Doris Hendig Herz- und Diabeteszentrum NRW, Institut für Laboratorium- und Transfusionsmedizin, Bad Oeynh Introduction: Ectopic calciﬁcation of connective tissue, especially elastic ﬁbers, is the main characteristic of pseu- doxanthoma elasticum (PXE, OMIM 264800), a rare genetic multisystem disorder. Mutations in ABCC6, an ATP-binding cassette (ABC) transporter with so far unknown function were found to cause PXE. The lipid transporting properties of a variety of ABC transporters as well as metabolic studies suggest an involvement of ABCC6 in lipid homeostasis. To bring light onto the participation of ABCC6 in adipocyte metabolism, our aim was to generate an ABCC6 knockout by using genome editing in human mesenchymal stem cells (hMSCs) and performe adipogenic differentiation experiments with this cell line. Methods: Successful adipogenic differentiation was conﬁrmed via ﬂuorescent staining for lipids with Bodipy and gene expression analysis via quantitative real-time PCR (qRT-PCR) of adipocyte master regulator PPARg. ABCC6 gene expression was evaluated during differentiation. An ABCC6 deﬁcient cell culture model was generated using clustered regulatory interspaced short palindromic repeat (CRISPR-Cas9) system. Adipocyte lipid homeostasis was analyzed via triglyceride measurement in cell culture supernants and mRNA expression of low density lipoprotein receptor (LDLR), lipoprotein lipase (LPL), adipose triglyceride lipase (ATGL; also known as palatin-like phospholipase domain containing protein 2, PNPLA2), fatty acid elongase 3 (ELOVL3) and ABC transporter C 1 (ABCA1). Results: Induction of adipogenic differentiation goes along with signiﬁcantly elevated ABCC6 gene expression reaching highest level in mature adipocytes. To further clarify the role of ABCC6 in lipid homeostasis, CRISPR-Cas9 mediated ABCC6 knockout was performed and resulted in signiﬁcantly reduced ABCC6 mRNA expression. ABCC6 deﬁciency in MSCs did not disturb lipid droplet formation and did not inﬂuence gene expression of differentiation markers of adi- pogenesis but resulted in decreased triglyceride content in cell culture supernants. Results of gene expression analysis in mature adipocytes revealed diminished gene expression levels of LDLR, LPL, ATGL, ELOVL3 and ABCA1 indicating impaired lipid uptake, lipolysis, and cholesterol efﬂux. Conclusion: Although ABCC6-deﬁciency does not disturb adipogenic differentiation, lipid homeostasis in mature adi- pocytes in dysregulated. Quantiﬁcations of triglycerides in cell culture supernants and gene expression analysis indicate an impaired lipolysis and lipid trafﬁcking in adipocytes. eA52 Background: The autosomal-recessive disorder Pseudoxanthoma elasticum (PXE, OMIM #264800), characterized by calciﬁcation and fragmentation of elastic ﬁbres in the skin, retina and vessel walls, can be caused by mutations in the ABCC6 gene. This gene encodes for ATP-Binding Cassette subfamily C member 6 (ABCC6), a transporter mainly localized in die basolateral membrane of hepatocytes and kidney cells. It has been shown that fibroblasts from PXE patients exhibit a senescence-like phenotype with elevated β-galactosidase activity and increased mRNA expression of the cell cycle inhibitor p21 and interleukin (IL) 6. Furthermore, signs of oxidative stress have been reported in the serum of PXE patients and in PXE fibroblasts. Due to the possible role of p21 in the aging process of stem cells, followed by a loss of regenerative potential, human mesenchymal stem cells (hMSCs) were chosen for these investigations. Methods: To induce cellular aging, hMSCs were incubated with 1 mM H2O2 for 1 h following 72 h of incubation. Removal of apoptotic cells was performed by reseeding the hMSCs in a density of 8.000 cells/cm2. Senescence was validated by evaluating the mRNA expression of senescence markers using qRT-PCR, measuring the β-galactosidase activity and immunoﬂuorescence staining of p21. In addition, the mRNA expression of ABCC6 was determined by qRT-PCR. Methods: To induce cellular aging, hMSCs were incubated with 1 mM H2O2 for 1 h following 72 h of incubation. Removal of apoptotic cells was performed by reseeding the hMSCs in a density of 8.000 cells/cm2. Senescence was validated by evaluating the mRNA expression of senescence markers using qRT-PCR, measuring the β-galactosidase activity and immunoﬂuorescence staining of p21. In addition, the mRNA expression of ABCC6 was determined by qRT-PCR. Results: The β-galactosidase activity of the hMSCs increased by 50% following treatment with H2O2 with elevated mRNA expression of p21 and senescence-associated cytokines IL1β and IL8 further conﬁrming the senescent phenotype. The immunostaining of p21 revealed an increase in the portion of p21-positive cells to 100%. The mRNA expression of ABCC6 increased up to 10-fold. Results: The β-galactosidase activity of the hMSCs increased by 50% following treatment with H2O2 with elevated mRNA expression of p21 and senescence-associated cytokines IL1β and IL8 further conﬁrming the senescent phenotype. The immunostaining of p21 revealed an increase in the portion of p21-positive cells to 100%. The mRNA expression of ABCC6 increased up to 10-fold. Conclusion: The elevated mRNA expression of ABCC6 in hMSCs following treatment with H2O2 indicates an association of ABCC6 with the process of cellular senescence and oxidative stress. This pathomechanistic link should be further investigated by knockout of ABCC6 in hMSCs and evaluation of markers of senescence and oxidative stress. Evaluating the mRNA-Expression of ABCC6 in human mesenchymal stem cells during induced oxidative stress hel Robin Osterhage; Ricarda Plümers; Christopher Lindenkamp; Isabel Faust-Hinse; Cornelius Knab Michel Robin Osterhage; Ricarda Plümers; Christopher Lindenkamp; Isabel Faust-Hinse; Cornelius Knabbe; Doris Hendig Herz- und Diabeteszentrum NRW, Institut für Laboratoriums- und Transfusionsmedizin, Bad Oeynhausen, Germany Herz- und Diabeteszentrum NRW, Institut für Laboratoriums- und Transfusionsmedizin, Bad Oeynhausen, Germany eA52 German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 Results: Treatment of NHDF with H2O2 signiﬁcantly reduced XYLT1 and XYLT2 mRNA expression in comparison to control. Based on the results of the mass spectrometry activity assay, the signiﬁcantly decreased XT protein expression determined by western blot, was assigned to the XT-I isoform. Characterization of the SA secretory phenotype revealed signiﬁcantly increased gene expressions of interleukin 1β and matrix metalloproteinase 1. In contrast, collagen I, ﬁbronectin and decorin expressions were signiﬁcantly reduced. However, on TGF-β1 gene expression senescence in- duction had no physiologically relevant effect. Results: Treatment of NHDF with H2O2 signiﬁcantly reduced XYLT1 and XYLT2 mRNA expression in comparison to control. Based on the results of the mass spectrometry activity assay, the signiﬁcantly decreased XT protein expression determined by western blot, was assigned to the XT-I isoform. Characterization of the SA secretory phenotype revealed signiﬁcantly increased gene expressions of interleukin 1β and matrix metalloproteinase 1. In contrast, collagen I, ﬁbronectin and decorin expressions were signiﬁcantly reduced. However, on TGF-β1 gene expression senescence in- duction had no physiologically relevant effect. Conclusion: In summary, our study demonstrated an anti-ﬁbrotic effect in acute senescent myoﬁbroblasts leading to a suppressed XYLT1 and XT-I expression. It can be concluded that XT-I plays an important role in the switch between physiological and pathological wound healing process. Nevertheless, further studies are needed to elucidate the un- derlying signaling pathways. Suppression of Human Xylosyltransferase I in H2O2-Induced Acute Senescent Myofibroblasts Vanessa Schmidt; Justus Maximilian Ohmes; Thanh-Diep Ly; Bastian Fischer; Anika Kleine; Cornelius Knabbe; Isabel Faust-Hinse Herz- und Diabeteszentrum Nordrhein-Westfalen, Universitätsklink der Ruhr-Universität Bochum, Institut für Labo- ratoriums- und Transfusionsmedizin, Bad Oeynhausen, Germany Introduction: Fibrosis represents a dysregulated wound healing process in which extracellular matrix (ECM)-producing myoﬁbroblasts, after completion of the repair process, do not induce apoptosis or cellular senescence but persist in the tissue. Cellular senescence of myoﬁbroblasts during the physiologic wound healing process is referred to as acute senescence and serves to protect against ﬁbrosis. Cellular senescence, by deﬁnition, is an irreversible form of cell cycle arrest preventing damaged cells from undergoing proliferation and precluding potential neoplastic transformation. The xylosyltransferase isoform XT-I represents a biomarker of myoﬁbroblast differentiation. The isoforms XT-I and XT-II catalyze the initial rate-limiting step of proteoglycan (PG) biosynthesis by transferring xylose from UDP-xylose to speciﬁc serine residues of the PG core protein. Known mediators of XT-I regulation in context to ﬁbrotic processes are transforming growth factor β1 (TGF-β1), activin A or interleukin 1β. Although the regulation of XT- I in proﬁbrotic processes is mainly understood, little is known about the XT-I regulation during the transition of myoﬁbroblasts into an acute senescent cell state. The aim of this study was to determine whether XT-I is regulated during this transition and, consequently, has an impact on ECM remodeling at the end of the physiological wound healing process. Methods: To investigate XT-I regulation in acute senescent myoﬁbroblasts, primary normal human dermal ﬁbroblasts (NHDF) were treated with H2O2 and cultured in a myoﬁbroblast cell culture model. XT-I regulation was examined at gene, protein and enzyme activity levels by quantitative real-time PCR, western blot analysis and a XT-I selective mass spectrometric activity assay. The induction of an acute senescence state was veriﬁed by a senescence-associated (SA) β-galactosidase activity assay and the determination of p21 and p16 protein expressions by immunoﬂuorescence microscopy. German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 eA53 Maintaining automatic 24/7 post-requests and proper workflows on the laboratory automation system in the event of recurring delivery shortages for separation tubes Bozena Zechmeister; Andreas Fischer; Gry Helene Dihazi Universitätsmedizin Göttingen, Interdisziplinäres UMG Labor, Göttingen, Germany Universitätsmedizin Göttingen, Interdisziplinäres UMG Labor, Göttingen, Germany Aims: Due to recently recurring delivery bottlenecks for laboratory mateials (lithium heparin plasma separation tubes) and the impact on laboratory results of additional requests in an automated 24-hour operation, the effects of sample storage of tubes with and without separation material were examined. Materials and Methods: Blood was collected from 20 healthy volunteers using gel (routine) and non-gel (spare) plasma separation tubes. A total of 23 clinical-chemical parameters were determined at 0 h, 4 h, 8 h, 24 h, 48 h and 72 h. The samples were processed on the laboratory automation system (LAS), centrifuged but not separated by a ﬁlter. Between the measurements, the samples were automatically stored at 4°C in the connected archive. Statistical differences were evaluated using a generalized estimating equation regression model. Results: For most parameters, there was no signiﬁcant change in the measurement results within the tested time, regardless of the collection tube. Signiﬁcant differences in the comparison of the measurements over time could be detected for some parameters, but there were no signiﬁcant differences in the results in the ﬁrst 8 h and there were hardly any differences between the different tubes. Parameters known to be sensitive to interference from erythrocyte or platelet contamination were stable for at least 8 hours after the ﬁrst measurement. Discussion (Conclusion): These data indicate that sample storage in the primary tube, both with and without the plasma separation tube, might cause relevant changes for certain parameters. However, there was little difference between using tubes with or without plasma separation. To maintain an effective workﬂow in the routine laboratory and to ensure the possibility of additional requests within 24 hours in the event of supply shortages, the use of tubes without separation material is sufﬁcient for most parameters. eA54 German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 eA54 Impact of manufacturer-dependent differences on in vitro speciﬁc IgE results in low concentrated serum samples Nathalie Wojtalewicz1; Laura Vierbaum1; Kathrin Kabrodt2; Ingo Schellenberg³ 1INSTAND e.V., wiss. Mitarbeiter, Düsseldorf, Germany; 2Hochschule Anhalt, Institute of Bioanalytical Science, Bern- burg, Germany; ³INSTAND e.V., Vorstand, Düsseldorf, Germany Aims: The in vitro detection of speciﬁc IgE (sIgE) against different allergen sources in serum is a widely used method to identify possible sensitizations to this source which could manifest in allergic disease. In the past, INSTAND repeatedly reported huge manufacturer-dependent differences for important allergen sources like hymenopteran venoms, food allergens and inhalant allergens. The aim of this study was the evaluation of the current status quo of manufacturer- dependent differences in the in vitro detection of sIgE for allergy diagnostics observed in external quality assessment (EQA) schemes. Materials and Methods: We evaluated, manufacturer-dependent, the semiquantitative EQA results for sIgE for the allergen sources European house dust mite, cow’s milk protein and wasp venom obtained between 2018 and 2022. For this analyzes, samples with low sIgE concentrations were chosen, especially when at least one manufacturer collective showed sIgE levels around the cut-off value of 0.35 kU/L. We analyzed the differences in mean for the three biggest manufacturer collectives, including 95 % conﬁdence intervals. Results: For all reviewed allergen sources, we observed samples where at least one manufacturer collective reported a negative result (< 0.35 kU/L), while others reported a clear positive result (> 0.35 kU/L). The differences were higher in case of house dust mite and cow’s milk protein, while the results for wasp venom show a trend to harmonization. In case of house dust mite allergens, the three biggest manufacturer collectives showed mean-differences up to 3.8-fold. Discussion (Conclusion): While the manufacturer-dependent differences in low concentrated samples have declined for the allergen source wasp venom, they diverge in case of the allergen sources house dust mites and cow’s milk protein. Unfortunately, there is no positive trend of harmonization for both allergens, for these differences have been observed since 1995 and were reconﬁrmed in 2017. As currently lyophylized sample material is used for this EQA, the introduction of liquid samples from individual donors in future EQA surveys is planned with the intention to eliminate the possibility of matrix effects inﬂuencing the EQA results. Without a proper harmonization, clinicians are dependent on a thorough clinical anamnesis to accompany the in vitro diagnostics, especially when a positive sIgE value is close to the cut-off value of 0.35 kU/L. Anika Kleine; Phil Lange; Vanessa Schmidt; Thanh-Diep Ly; Isabel Faust-Hinse; Cornelius Knabbe; Bastian Fischer Anika Kleine; Phil Lange; Vanessa Schmidt; Thanh-Diep Ly; Isabel Faust-Hinse; Cornelius Knabbe Herz- und Diabeteszentrum Nordrhein-Westfalen, Institut für Laboratoriums- und Transfusionsmedizin, Bad Oeynhausen, Germany Background: The xylosyltransferase isoforms XT-I and XT-II catalyse the initial step of the proteoglycan (PG) tetra- saccharide linker region to bind glycosaminoglycans (GAG) to the PG-core protein. Through this essential step, the XT isoforms crucially contribute to the homeostasis of ECM remodelling. Spondylo-ocular syndrome is a rare autosomal recessive inherited disease with features such as skeletal fragility, short stature, developmental delay as well as cardiac and ocular manifestations. Various XYLT2 mutations have been identified to be causative concerning disease manifestation. Due to the low availability of patient cells, the aim of this work was to generate an isolated CRISPR/Cas9-mediated XYLT2 knockout in neonatal human dermal fibroblasts to analyse the effects of XT-II deficiency on the cells. In addition, this approach could help to better understand the hitherto unexplained relevance of two different XT-isoforms in all higher organisms. Methods: CRISPR/Cas9 based XYLT2 knockout: normal human dermal ﬁbroblasts (NHDF) were revers transfected with 10 nM of a ribonucleoprotein (RNP)-complex targeting the XYLT2 gene. After 24 h the cells were sorted using FACS technology, genomic DNA was isolated and afterwards analysed using sanger sequencing. Analysis of relative mRNA expression and XT activity: relative mRNA expression levels were determined using quanti- tative real-time PCR. Intra- (cell culture lysates) and extracellular (cell culture supernatants) XT-activity was measured using an in-house UPLC-MS-assay by specifically detecting the xylosylated peptide. Migration assay: An artificial scratch within a confluent monolayer of control and XYLT2-deficient fibroblasts was created. The migration of cells into the area was observed for 96 h. Senescence assay: NHDF and XYLT2-deficient cells were cultivated for 72 h and subsequently harvested. The senescence- associated-β-galactosidase-activity was observed photometrically. Results: CRISPR/Cas9-based genome editing was successful due to a single thymine base deletion, resulting in a shortened amino acid sequence (p.Val95Glyfs3). Compared to controls, XT-II deﬁcient cells showed reduced mRNA expression levels of different glycosyltransferases, as well as a signiﬁcantly reduced XT activity. In addition, the XYLT2-deﬁcient cells closed the artiﬁcial gap more slowly and showed a signiﬁcantly increased senescence-associated- β-galactosidase activity in comparison to the control ﬁbroblasts. Conclusion: Using the CRISPR/Cas9 system, a XYLT2-deﬁcient ﬁbroblast culture was successfully obtained. The lack of XT activity also lowers the mRNA expression of the remaining transferases involved in the tetrasaccharide linker syn- thesis. Establishing reference intervals for NMR spectra in human plasma Daniel Rosenkranz1; Kathrin Budde2; Ann-Kristin Henning2; Gunnar Brandhorst1; Sarah Schäfer1; Friederike Gauß2; Nele Friedrich2; Astrid Petersmann1; Matthias Nauck2 1Carl-von-Ossietzky University, Institute for clinical chemistry and laboratory medicine, Oldenburg, Germany; 2University Medicine Greifswald, Institute for clinical chemistry and laboratory medicine, Greifswald, Germany Background: Reference intervals are an important tool for the clinical interpretation of laboratory results. The usual approach for establishing reference intervals implies the calculation of designated percentiles after the measurement of a single measurand in a deﬁned “healthy” reference population. For multi-measurand determinations within one analytical run, e.g. from NMR spectra, the calculation of reference intervals becomes more complex and is not well established. Material and Methods: In this work, 600 MHz NMR spectra from approximately 400 human plasma samples out of the study of health in Pomerania (SHIP) serve as the data base for the calculation of a reference spectrum. The NMR-measurement provides 4000 measuring points per spectrum, which refers to a spectral resolution of 0.0025 ppm (1.5 Hz). Percentiles of intensities are calculated at each measuring point and the reference spectrum is depicted as color- coded intensities. At this time point, no disease-speciﬁc exclusions were applied, so that the results are preliminary. German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 eA55 Results and Discussion: The established reference spectrum represents the distribution of the intensity of a given ppm across a large population. The intensity percentiles at a given ppm are color-coded, so that we are able to describe regions at the spectra which are uniform up to regions, which are very variable between participants. Results and Discussion: The established reference spectrum represents the distribution of the intensity of a given ppm across a large population. The intensity percentiles at a given ppm are color-coded, so that we are able to describe regions at the spectra which are uniform up to regions, which are very variable between participants. Conclusion: According to the high standardization and comparability of the NMR spectra in human plasma samples, reference spectra are needed, to improve the medical value of this technology in the health care setting. Here we show a ﬁrst approach to calculate a reference spectrum out of a large epidemiological study for plasma, measured by NMR spectroscopy. RESULTS OF THE EXTERNAL QUALITY ASSESSMENT SCHEME (EQA) FOR ISOLATION AND ANALYSIS OF CIRCULATING TUMOR DNA (ctDNA) Romy Eichner1; Parviz Ahmad-Nejad2; Sascha Neumann³; Wolf J. Geilenkeuser³; Laura Mirbach1; Angelika Duda1; Vere Haselmann1 Romy Eichner1; Parviz Ahmad-Nejad2; Sascha Neumann³; Wolf J. Geilenkeuser³; Laura Mirbach1; Ang Haselmann1 1Universitätsmedizin Mannheim, Institut für Klinische Chemie, Mannheim, Germany; 2HELIOS Klinikum Wuppertal, Witten/Herdecke University, Institut für Medizinische Labordiagnostik, Wuppertal, Germany; ³Stiftung für Patho- biochemie und Molekulare Diagnostik (SPMD), Referenzinstitut für Bioanalytik (RfB), Bonn, Germany Background: Circulating tumor DNA (ctDNA) analysis, commonly referred to as liquid biopsy, has the potential to revolutionize oncologic diagnostics by helping to stratify targeted therapies, serving as a personalized tumor marker for treatment monitoring, and ultimately facilitating the detection of emerging resistance mechanisms. However, several obstacles hamper successful translation into standard care, with the lack of harmonized preanalytical and analytical issues being the most signiﬁcant concern. This external quality assessment (EQA) scheme aimed to address issues of analytical quality as prerequisite for an urgently needed standardization of laboratory workﬂows. Methods: In each EQA schemes offered, three samples containing 2-3 mL EDTA-plasma spiked with fragmented genomic DNA isolated from tumor cell lines were provided for analysis of sequence variations in KRAS p.G12/p.G13, BRAF p.V600E and EGFR p.T790M. The variant allele frequency (VAF) ranged from 0% to 10%. Laboratories were asked to use their routine procedures and report following: 1) time elapsed for processing of samples, 2) storage temperatures, 3) method used for extraction and quantiﬁcation, 4) genotyping methods and results. Results: A total of 184 laboratories from 13 European countries participated in these EQAs. Neither the median shipment time to participants of 2.8 days nor the total time elapsed before analysis of 7 days affected the overall diagnostic performance. 73.5% reported to isolate cfDNA manually, and the most commonly used isolation kit was the QIAamp circulating nucleic acid kit (52.9%). On average, 2.6mL plasma were used for cfDNA isolation, with 83.3% of laboratories using buffer for cfDNA elution. Isolated cfDNA was quantiﬁed by Qubit in 57.8% of cases, followed by 9.8% using ddPCR and 9.3% using NanoDrop. Importantly, cfDNA equivalent to 640μL plasma was used on average for ctDNA analysis, representing only 21.3% of the maximum possible input. For analysis, ddPCR (45.2%) was used most frequently, followed by qPCR (16.5%), and MassArray (12.1%). Overall, 95/1152 genotypes were determined inaccurately, resulting in an overall error rate of 8.3%. The false-positive rate was 4.2% and increased with time. Anika Kleine; Phil Lange; Vanessa Schmidt; Thanh-Diep Ly; Isabel Faust-Hinse; Cornelius Knabbe; Bastian Fischer The migration capacity of the cells is impaired and there is a marked increase in cellular senescence. Our data reveal an impact of XYLT2 expression on the cellular metabolism. Further effects need to be analysed in the future. eA56 eA56 German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 RESULTS OF THE EXTERNAL QUALITY ASSESSMENT SCHEME (EQA) FOR ISOLATION AND ANALYSIS OF CIRCULATING TUMOR DNA (ctDNA) The false-negative rate was 8.9%, with the error rate increasing with decreasing VAF, e.g., exceeding 30% at a VAF of 0.1%. Noteworthy, the error rate varied signiﬁcantly depending on the method used, with the lowest error rate observed for ddPCR and BEAMing. Conclusion: This EQA schemes illustrate the current variability in multiple stages of cfDNA processing and analysis of ctDNA,resultingin an overallerror rate of 8.3%. Importantly, the error rateclearlydepends onthe method usedand the VAF of the target gene. Of note, the false-negative rate has improved over time, suggesting that laboratories are using more sensitive techniques, while the false-positive rate is increasing, indicating problems in accurately determining the detection limit. eA57 Klinik für Kinder- und Jugendmedizin, Göttingen, Germany; ⁴Universitätsmedizin Oldenburg, Institut für Klinische Chemie und Laboratoriumsmedizin, Oldenburg, Germany Klinik für Kinder- und Jugendmedizin, Göttingen, Germany; ⁴Universitätsmedizin Oldenburg, Institut für Klinische Chemie und Laboratoriumsmedizin, Oldenburg, Germany Aims: Multiple studies show the potential of amino acids (AA) to serve as biomarkers in prognosis and diagnosis of various diseases, e.g. diabetes. 1H-NMR-platforms are increasingly used in metabolic proﬁling studies due to their inherent advantages like robust and reproducible measurements. Still, NMR methods are not used in clinical settings. In order to evaluate the use of NMR methods for AA measurement in research and routine patient care, we compared levels of AA measured on a 1H-NMR platform with those measured on a well established HPLC platform. Materials and Methods: Eleven AA were measured in 90 EDTA-plasma samples from patients of the University Medicine Greifswald. Measurements were conducted on a HPLC platform (Biochrom 30+ system, Biochrom, Cambridge, UK) and on a 600 MHz 1H-NMR platform (Bruker AVANCE-III, Bruker Biospin, Ettlingen, Germany). All measurements were per- formed in duplicates. For evaluation of precision of the two methods, coefﬁcients of variation (CV%) were calculated from duplicates and Passing-Bablok regression analysis was conducted. For the comparison of NMR and HPLC measurements, we determined Pearson correlation coefﬁcients and performed Passing-Bablok regression. Results: CV based on duplicates were lower on average for HPLC than for NMR measurements: median CV were 2.79% (interquartile range IR: 2.75% – 3.25%) and 10.22% (IR: 6.73% – 13.77%) for HPLC and NMR, respectively. Further, Passing-Bablok analysis revealed no or only small bias between the ﬁrst and second measurement with very few exceptions for NMR-measured AA. The comparison of NMR and HPLC measured levels showed a median correlation coefﬁcient of r=0.82 (IR 0.76 – 0.91). Highest correlation was found for phenylalanine (r=0.99), tyrosine (r= 0.98), glycine (r= 0.92) and alanine (r = 0.89). In Passing-Bablok regression, no or only very small bias was found for phenylalanine and alanine. Discussion: In the comparison of AA levels measured on a NMR and a HPLC platform, two AA, namely phenylalanine and alanine, were highly comparable in terms of high correlation and no or only small bias. Thus, NMR methods have the potential to be applied in clinical context. In research context, NMR measurements of AA, which correlate well with HPLC levels, may be used in proﬁling studies, e.g. in the epidemiological ﬁeld, to detect signiﬁcant associations to clinical outcomes despite some bias between the exact AA levels. Generally, precision of NMR measurements and correlation between HPLC and NMR measured levels were higher for those AA with the highest measured concentration levels. Still, also AA with low concentrations, such as tyrosine, showed a good performance, which indicates, that improvement of quantification algorithms or measurement time may lead to a higher analytical quality of NMR methods. NMR-based quantification of amino acids – a method comparison Friederike Gauß1; Kathrin Budde1; Ann-Kristin Henning1; Angelika Hafke2; Ralph Krätzner³; Matthias Nauck1; Astrid Petersmann⁴ 1Universitätsmedizin Greifswald, Institut für Klinische Chemie und Laboratoriumsmedizin, Greifswald, Germany; 2Universitätsmedizin Göttingen, Institut für Klinische Chemie, Göttingen, Germany; ³Universitätsmedizin Göttingen, German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 AI in smart laboratory processes - medicalvalues and smartLIS Change management in the laboratory and at the sender will also be addressed. Results: Using the example of different areas such as endocrinology, coagulation and infectiology, it will be shown how on the one hand Results: Using the example of different areas such as endocrinology, coagulation and infectiology, it will be shown how on the one hand ‒ Medical guidelines ‒ Requirement in accordance with guidelines ‒ Formal principles (EBM rules) ‒ Formal principles (EBM rules) can be taken into account in the request, thus preventing or avoiding inquiries and incorrect requests, and on the other hand how the laboratory physician can be supported in the validation of the results. Discussion and Conclusion: For a sustainable implementation, international standards and interfaces such as FHIR and LOINC were used. This is supported by a modern software architecture, but also by a cooperative approach / mindset among all project participants. The presentation will also critically examine and discuss the medical device requirements. AI in smart laboratory processes - medicalvalues and smartLIS Detlef Pechermeyer1; Jan Kirchhoff2 1labdock GmbH, Geschäftsführung, Hamburg, Germany; 2medicalvalues GmbH, Geschäftsführung, Karlsruhe, Germany 1labdock GmbH, Geschäftsführung, Hamburg, Germany; 2medicalvalues GmbH, Geschäftsführung, Introduction and Objectives: The power of modern laboratory medicine allows a wide range of analytes and mea- surements. In the short time available, it is not always possible for the sender to exhaust the complete diagnostic potential and also to consider new, innovative analytes. The focus is on targeted and personalized diagnostics for patients. We want to show how networking of controlled requisition/CDS in the laboratory order system with processing in the laboratory system and guided validation in the practice can function, where added value is created and content can be managed. Introduction and Objectives: The power of modern laboratory medicine allows a wide range of analytes and mea- surements. In the short time available, it is not always possible for the sender to exhaust the complete diagnostic potential and also to consider new, innovative analytes. The focus is on targeted and personalized diagnostics for patients. and also to consider new, innovative analytes. The focus is on targeted and personalized diagnostics for patients. We want to show how networking of controlled requisition/CDS in the laboratory order system with processing in the laboratory system and guided validation in the practice can function, where added value is created and content can be managed. The special feature here is the connection of an AI system with the process in the LIS. The following 3 areas should be covered: The following 3 areas should be covered: g ‒ Initial request: supporting the physician in selecting correct parameters in an intuitive interface. ‒ Initial request: supporting the physician in selecting correct parameters in an intuitive interface. eA58 German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 ‒ Reporting in the laboratory: support of the laboratory physician in the reporting of ﬁndings ‒ Reporting in the laboratory: support of the laboratory physician in the reporting of ﬁndings ‒ Reporting in the laboratory: support of the laboratory physician in the reporting of ﬁndings ‒ Reporting at the physician’s site: information and possibilities for targeted reﬂex testing p g y pp y p y p g g ‒ Reporting at the physician’s site: information and possibilities for targeted reﬂex testing ‒ Reporting at the physician’s site: information and possibilities for targeted reﬂex testing Change management in the laboratory and at the sender will also be addressed. Eight months of experiences in newborn screening for sickle cell disease (SCD) and spinal muscular atrophy (SMA) Friederike Hörster; Ulrike Mütze; Joachim Janda; Rafael Tesorero; Patrik Feyh; Georg F. Hoffmann; Jürgen G. Okun Friederike Hörster; Ulrike Mütze; Joachim Janda; Rafael Tesorero; Patrik Feyh; Georg F. Hoffmann; Universitätsklinikum Heidelberg, Dietmar-Hopp-Stoffwechselzentrum Neugeborenen Screening, H Introduction: Since October 2021, SCD and SMA are included in the German national newborn screening (NBS) panel. Our center started a 3-months NBS pilot study in July 2021. For SCD screening, blood transfusion and prematurity (< 34 weeks of gestation) have been announced as major diagnostic pitfalls. Aims: To report ﬁrst experiences on prevalence, disease conﬁrmation and diagnostic pitfalls. Materials and Methods: Real-time quantitative PCR (RT-qPCR) investigation of SMA, SCD and TRECs is performed using a common platform to facilitate high-throughput analysis. For SMA screening, the SMN1 gene is used as a target, while beta-actin is used as an internal control for successful ampliﬁcation. For SCD screening, the common mutation is targeted in RT-PCR. In a second-tier approach for SCD, all samples revealing a suspicious ﬁrst tier result undergo ESI-MS/MS analysis of characteristic hemoglobin fragments using a commercial kit (SpOtOn Clinical Diagnostics Limited). Results: 84 769 newborns (26 764 within the pilot phase and 58 005 within the regular screening program) have already been investigated. Due to low beta-actin levels in 24 cases a control card was requested. beta actin is used as an internal control for successful ampliﬁcation. For SCD screening, the common mutation is targeted in RT-PCR. In a second-tier approach for SCD, all samples revealing a suspicious ﬁrst tier result undergo ESI-MS/MS analysis of characteristic hemoglobin fragments using a commercial kit (SpOtOn Clinical Diagnostics Limited). Results: 84 769 newborns (26 764 within the pilot phase and 58 005 within the regular screening program) have already been investigated. Due to low beta-actin levels in 24 cases a control card was requested. y Results: 84 769 newborns (26 764 within the pilot phase and 58 005 within the regular screening program) have already been investigated. Due to low beta-actin levels in 24 cases a control card was requested. SMA: We identified 13 newborns affected by SMA (birth prevalence about 1: 6 500), 12 of them already confirmed by MLPA (multiplex ligation dependent probe amplification) revealing 0 SMN1 copies and 1-4 SMN2 copy numbers, NBS revealed no false positive results. Evaluation of hemolysis index thresholds in seven routine biochemistry assays Nicole Hoffmeier1; Martin Mühlbauer2; Angelika Carl1; Gunnar Brandhorst1; Astrid Petersmann1 1Klinikum Oldenburg AöR, Universitätsinstitut für Klinische Chemie und Laboratoriumsmedizin, Oldenburg, Germany; 2MVZ Medizinisches Labor Oldenburg GmbH, MVZ Medizinisches Labor Oldenburg GmbH, Oldenburg, Germany Background: In vitro hemolysis in laboratory samples is one of the most frequent preanalytical errors and bears the risk of clinical misinterpretation of test results and consequently false patient treatment. Modern laboratory analysers are able to assess the degree of hemolysis to prevent reporting of impaired results. Manufacturers provide hemolysis index (HI) thresholds for each measurand. High reliability of these thresholds is important for patient safety by two means: 1. too forgiving thresholds may cause erroneous results to be released; 2. too strict thresholds may cause unnecessary sup- pression of test results which in turn may put the patient at risk as well. Therefore, the aim of this study was to evaluate the reliability of the manufacturer’s thresholds in view of patient safety. Methods: Samples were collected from anonymized residual patient blood (n=15). A dilution series was then prepared with deﬁned degrees of haemolysis from 4 mg/dl up to 167 mg/dl and seven measurands susceptible to haemolysis namely potassium, aspartate aminotransferase (AST), lactate dehydrogenase (LDH), haptoglobin, total and direct bili- rubin and high-sensitive troponin T were measured in each dilution on the cobas pro system (Roche Diagnostics, Mannheim, Germany). For each measurand distribution of results was depicted as boxplots. Relative changes in mea- surement results across the dilution series were compared to various relative limits, for both, increase and decrease of measurement results with increasing hemolysis. Results: Relative result increase and decrease of the investigated measurands was evaluated for 5, 10, 15, and 20% and compared to different HI levels in the dilutions. A 5% increase or decrease – depending on the measurand - was found at HI levels of 92 for potassium (20), 34 for AST (20), 19 for LDH (15), 577 for haptoglobin (10), 179 for total bilirubin (800), 8 for direct bilirubin (10) and 3305 for troponin T (100), respectively (manufacturer’s thresholds are given in parentheses). For some measurands like potassium, troponin T and haptoglobin the effect of hemolysis was found to be considerably smaller compared to the manufacturer’s recommendations. For direct and total bilirubin effects of hemolysis were more severe than recommendations of the manufacturer reﬂected. Evaluation of hemolysis index thresholds in seven routine biochemistry assays German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 eA59 Conclusion: Hemolysis levels impacting measuring results deviated considerably from the manufacturer’s recom- mendations. For ﬁve of the investigated measurands the given HI appeared too strict, for two measurands too forgiving. Further studies can help to conﬁrm these ﬁndings and assess their clinical signiﬁcance. Conclusion: Hemolysis levels impacting measuring results deviated considerably from the manufacturer’s recom- mendations. For ﬁve of the investigated measurands the given HI appeared too strict, for two measurands too forgiving. Further studies can help to conﬁrm these ﬁndings and assess their clinical signiﬁcance. Eight months of experiences in newborn screening for sickle cell disease (SCD) and spinal muscular atrophy (SMA) SCD: We identified 21 newborns with SCD (birth prevalence about 1:4 000): n=15 homozygous Hb S/S, n= 4 compound heterozygotes Hb S/Hb C, n=2 compound heterozygotes HbS/ beta thalassemia major- conﬁrmation was mostly done by molecular genetics. NBS revealed no false positive results. One recall card needed to be requested due to blood transfusion and three recall cards due to gestational age below 34 weeks (total number of newborns < 34 weeks of gestation in the observed period was 3 502, number of transfusions estimated 298). Conclusion: NBS for SMA and SCD has been successfully established as a Multiplex-qPCR in parallel with the detection of TRECs. In SCD screening, a second-tier approach based on RT-qPCR for the common SCD mutation and subsequent ESI-MS/MS analysis of hemoglobin fragments is feasible and facilitates dealing with the two major pit-falls blood- transfusion and prematurity. eA60 Introduction: A new carrier for pneumatic tube systems (PTS) was developed by Aerocom®, which supports the automated unloading of blood samples. This carrier is a further development in a line of various prototypes. It is equipped with eight transparent tubes containing a ﬁxing system for blood sample tubes. Up to 24 blood sample tubes can be transported at the same time in one carrier. There is no need for special packing or unpacking of the transported samples – neither on the ward nor in the laboratory. The carrier has been developed to work together with an automated unloading station. After unloading a direct transfer of the sample tubes into a bulk loader is feasible. In this study we examine the inﬂuence of the transport and unloading process using the new carrier on blood sam-ple quality and compare it to courier transport. Methods: Duplicate blood sample sets (1 EDTA, 1 Lithium Heparin, 1 Citrate) of 20 volunteers were split among the different transportation methods: 1. courier and 2. new PTS carrier. After transport, 51 measurands from clinical chem- istry, hematology and coagulation were measured and compared. We determined the median concentration (1st - 3rd quartile) for each measurand for courier and PTS transportation, respectively. In addition, Spearman correlation co- efﬁcients and conducted Wil-coxon rank sum tests were determined. Statistical signiﬁcance was assumed at FDR-adjusted p-value < 0.05. Results: All investigated measurands showed a good concordance between the two transport methods: median corre- lation coefﬁcient was 0.95 (interquartile range 0.88 – 0.97). In Wilcoxon rank sum tests no signiﬁcant differences between courier and PTS transport were found. Especially the hemolysis sensitive measurands potassium, lactate dehydrogenase (LDH) and free hemoglobin (fHb) showed no statistically signiﬁcant difference, however a trend toward higher levels after PTS transport was observed for fHb and LDH. Conclusion: Our results show, that the new carrier by Aerocom® is suitable for transporting diagnostic blood samples. Sample transport and sample processing can be carried out faster and measurement re-sults are more rapidly available for patient treatment. However, we see further improvement po-tential for the prototypes. The mechanical components of the carriers are exposed to physical stress and tend to wear out quickly. Also the weight of the new carriers is clearly higher compared to conventional PTS carriers. This may be one reason for the mentioned trend of higher LDH and fHb levels in the new carriers. Evaluation of a new PTS prototype for automatic unloading Juliane Schöpfel; Friederike Gauß; Matthias Nauck University Medicine Greifswald, Germany, Institute of Clinical Chemistry and Laboratory Medicine, Greifswald, Germany versity Medicine Greifswald, Germany, Institute of Clinical Chemistry and Laboratory Medicine, Grei eA60 German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 Results: The values of the investigated patient self-monitoring systems show good agreement with the laboratory method. Correlation coefﬁcients were ≥0.959. The systematic measurement deviation was below 5 % for eight of the systems examined but above 5 % and up to 14% for the remaining ﬁve systems. Nine systems for patient self-monitoring were also comparable with the laboratory method in terms of imprecision < 5%, as recommended by the “Kommission für Labordiagnostik in der Diabetologie (KLD)”. However, four of the systems had a considerably higher imprecision with a coefﬁcient of variation of up to ± 10 %. Results: The values of the investigated patient self-monitoring systems show good agreement with the laboratory method. Correlation coefﬁcients were ≥0.959. The systematic measurement deviation was below 5 % for eight of the systems examined but above 5 % and up to 14% for the remaining ﬁve systems. Nine systems for patient self-monitoring were also comparable with the laboratory method in terms of imprecision < 5%, as recommended by the “Kommission für Labordiagnostik in der Diabetologie (KLD)”. However, four of the systems had a considerably higher imprecision with a coefﬁcient of variation of up to ± 10 %. Conclusion: The investigated systems for patient self-monitoring showed variable analytical results, which has to be conﬁrmed in further studies. About one third of the investigated devices for patient self-monitoring showed an unac- ceptable high imprecision and a clinically relevant systematic measurement deviation. This should be taken into account when such systems are used for patient-self monitoring. Commutability as a qualitative pre-requirement for a valid evaluation of proficiency testing schemes using the example of steroid hormones Conclusion: The results of this study emphasize that the deviations of the collective showing repeatedly unsatisfactory passing rates in past PTs are not induced by the sample additives but rather due to an insufﬁcient calibration or insufﬁcient speciﬁcity of the respective test method. After error analysis in cooperation with a manufacturer, deviations of TEST detection from the RMV have notably decreased. This positive effect could be due to recalibration effort in terms of metrological traceability. Based on this development, we recommend the initiation of error analysis for PROG detection as well. Results: For the fresh serum samples, a total of 390 TEST results were reported in two surveys and 342 PROG results in three surveys. A comparison of the relative differences between the two sample types showed variations in means around 16 % to 27 % for TEST and around 16 % to 58 % for PROG in dependence of the manufacturer. Constant relative differences between the sample types indicate no problems with commutability for individual manufacturer collectives. Results: For the fresh serum samples, a total of 390 TEST results were reported in two surveys and 342 PROG results in three surveys. A comparison of the relative differences between the two sample types showed variations in means around 16 % to 27 % for TEST and around 16 % to 58 % for PROG in dependence of the manufacturer. Constant relative differences between the sample types indicate no problems with commutability for individual manufacturer collectives. Conclusion: The results of this study emphasize that the deviations of the collective showing repeatedly unsatisfactory passing rates in past PTs are not induced by the sample additives but rather due to an insufﬁcient calibration or insufﬁcient speciﬁcity of the respective test method. After error analysis in cooperation with a manufacturer, deviations of TEST detection from the RMV have notably decreased. This positive effect could be due to recalibration effort in terms of metrological traceability. Based on this development, we recommend the initiation of error analysis for PROG detection as well. Conclusion: The results of this study emphasize that the deviations of the collective showing repeatedly unsatisfactory passing rates in past PTs are not induced by the sample additives but rather due to an insufﬁcient calibration or insufﬁcient speciﬁcity of the respective test method. After error analysis in cooperation with a manufacturer, deviations of TEST detection from the RMV have notably decreased. Large Performance Differences in Glucose Measurement Devices used in Patient Self- Monitoring Marla Hiepler1; Friederike Gauß2; Daniel Rosenkranz1; Angelika Carl1; Astrid Petersmann1 1Universitätsmedizin Oldenburg, Institut für klinische Chemie und Laboratoriumsmedizin, Oldenburg, Germany; 2Universitätsmedizin Greifswald, Institut für klinische Chemie und Laboratoriumsmedizin, Greifswald, Germany Background: Blood glucose systems for patient self-monitoring are not covered by the guideline of the German Medical Association for the quality assurance of laboratory tests. Analytical quality is assessed as part of the approval process but is not mandatory thereafter. Consequently, patients often do not apply and monitor internal quality controls on a regular basis e.g. like in medical laboratories. Materials and Methods: In our study we investigate the analytical performance of 13 glucose measurement devices from 7 different manufacturers for patient self-monitoring and compare them with glucose concentrations obtained from a system used in hospital care (Cobas pro, Roche Diagnostics, Mannheim). Two approaches were chosen: a method comparison and an imprecision study. For method comparison, 15 EDTA-whole blood from left over samples were measured on the patient self-monitoring systems. Within 15 min these whole blood samples were centrifuged and the EDTA plasma was used for the determination of the comparison glucose concentration at the Cobas pro platform. Thereafter, the plasma-referenced values of the patient self-monitoring systems were compared to the glucose concen- trations from the hospital care platform. For comparison, we calculated correlation coefﬁcients and conducted linear regression analysis. For determination of imprecision, EDTA plasma pools were prepared from left-over material. The pools encompassed six different glucose concentrations which were measured in duplicates for five consecutive days. Coefficients of variation were calculated for each method. German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 eA61 Commutability as a qualitative pre-requirement for a valid evaluation of proficiency testing schemes using the example of steroid hormones Laura Vierbaum1; Nathalie Wojtalewicz1; Thomas Keller2; Stephan Weber³; Ingo Schellenberg⁴; Peter B. Luppa⁵ 1INSTAND e.V., wissenschaftliche MA, Düsseldorf, Germany; 2ACOMED statistik, Geschäftsführer, Leipzig, Germany; ³ACOMED statistik, MA, Leipzig, Germany; ⁴INSTAND e.V., Leitung der Geschäftsstelle, Vorstand, Düsseldorf, Germany; ⁵Klinikum rechts der Isar der TU München, Institut für Klinische Chemie und Pathobiochemie, München, Germany Introduction: High quality of proﬁciency testing (PT) samples is a basic necessity to allow conclusive statements on the quality and homogeneity of medical laboratory analysis. Sample materials for interlaboratory comparison often require artiﬁcial processing, e.g. spiking of sufﬁcient analyte concentration or the addition of stabilizing additives. These supplements might hamper the laboratory detection of the analytes thus the commutability of such PT materials must be veriﬁed. In past PT surveys, INSTAND notices manufacturer-dependent differences in the detection of the steroid hormones testosterone (TEST) and progesterone (PROG). Methods: To determine whether these differences were due to artiﬁcial processing of the sample or whether they have test-speciﬁc causes, native fresh pooled sera were tested for TEST and PROG in several PT surveys, in addition to the regularly processed INSTAND PT samples. In parallel to the PT sample shipment and data collection, stability testing was performed for the fresh sample material. Methods: To determine whether these differences were due to artiﬁcial processing of the sample or whether they have test-speciﬁc causes, native fresh pooled sera were tested for TEST and PROG in several PT surveys, in addition to the regularly processed INSTAND PT samples. In parallel to the PT sample shipment and data collection, stability testing was performed for the fresh sample material. As reference method procedures are available for TEST and PROG, the manufacturer-collective results were normalized to the reference method value (RMV). A comparative analysis of the relative differences was performed for any combination of fresh and stabilized samples. Results: For the fresh serum samples, a total of 390 TEST results were reported in two surveys and 342 PROG results in three surveys. A comparison of the relative differences between the two sample types showed variations in means around 16 % to 27 % for TEST and around 16 % to 58 % for PROG in dependence of the manufacturer. Constant relative differences between the sample types indicate no problems with commutability for individual manufacturer collectives. Product problems of In-vitro diagnostics for COVID-19 disease – Analysis of FSN published by the BfArM 2020-2021 Rüdiger Siekmeier; Jürgen Hannig Pharmazeutisches Institut der Universität Bonn, Drug Regulatory Affairs, Bonn, Germany Pharmazeutisches Institut der Universität Bonn, Drug Regulatory Affairs, Bonn, Germany Introduction: Directive 98/79/EC and Regulation 2017/746 regulate marketing and market surveillance of In-vitro diagnostics (IVD) in the European Economic Area. In cases of incidents and ﬁeld safety corrective actions (FSCA) manufacturers have to inform responsible Competent Authority (D: BfArM) and public by ﬁeld safety notices (FSN). FSN are published by BfArM. In December 2019 a new disease termed 2019-nCoV (now COVID-19) by the World Health Organization (WHO) on Feb. 11th 2020 was ﬁrstly described. Until April 26th 2022 at least 511 million infections/6.2 million deaths were caused worldwide. Many tests were developed for disease detection (PCR, RT-PCR, other molecular and antigen (AG) tests (laboratory tests, rapid tests (lateral ﬂow assays, LFA))) for professional/lay users and immunological tests (antibody (AB) tests (laboratory tests, LFA)). Study aim was to analyze FSCA/FSN for COVID-10 tests/analyzers for product problems, resulting risks and type of FSCA. Materials and Methods: All FSCA/FSN for IVD for COVID-19 diagnostics published by BfArM (http://www.bfarm.de/DE/ Medizinprodukte/riskinfo/kundeninfo/functions/kundeninfo-node.html) until 31.12.2021 were analyzed. Results: 39 FSCA for COVID-19 IVD were found, i. e. 9.8 % of all FCSA for IVD (2020: 11 (5.9 %), 2021: 28 (13.3 %)). FSCA 2020/2021 affected molecular IVD for virus detection in nose/throat swabs (PCR, RT-PCR, Multiplex-PCR: 4/7, PCR controls: 0/2, PCR software: 2/1, racks for PCR analyzer: 0/1, saliva sampling system: 0/1), IVD for AB detection in blood/ serum (LFA: 2/0, ELISA: 1/0, tests/test speciﬁc software/reagent/calibrator for immune analyzer: 1/4, blot 1/0) and IVD for AG detection in swabs/saliva (LFA: 0/9, test for immune analyzer: 0/1, POCT with analyzer: 0/2). FSCA affected (both years, mult. entries) reagents/calibrators/controls (30), software (7), sample materials (1), analyzer racks (1) and ana- lyzers (1). Product failures were (both years, mult. entries) errors in labeling/instructions for use (IFU; 11), production errors (12; e. g. ﬁlling, packaging, product release), software errors (7), material errors (5), ﬂuid leaks (3), microbial contamination (1) and temperature error (1) and no information (3). Potential consequences of product failures (both years, mult. entries) were false-positive/false-negative results (19/12), invalid/inconclusive results (8), normal function (6), delayed function (1) and no information (5). Corrective measures (both years, mult. Product problems of In-vitro diagnostics for COVID-19 disease – Analysis of FSN published by the BfArM 2020-2021 entries were) recalls (32; FSN mandatory), customer recommendations (29; of these with information for control of results: 14), software-upgrade (9), modiﬁcation of labeling/IFU (5) and modiﬁcation of production/quality control (4). Conclusion: FSCA of IVD for COVID-19 diagnostics represent a relevant number of all FSCA. Product failures and corrective measures differ in respect to analytical principles and type of IVD. FSN play an important role for risk reduction in cases of product failure. Commutability as a qualitative pre-requirement for a valid evaluation of proficiency testing schemes using the example of steroid hormones This positive effect could be due to recalibration effort in terms of metrological traceability. Based on this development, we recommend the initiation of error analysis for PROG detection as well. The knowledge of specimen characteristics is critical to evaluate the causes of observed non-permissible deviations and thus provide the scientific background for initiating appropriate corrective action. eA62 German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 eA62 Determination of Age- and Sex-specific Reference Intervals for Cholinesterase and Gamma- Glutamyl Transferase Using Real-World Data of the Marienhospital in Stuttgart, Germany: Comparison of Three Different Indirect Methods Anne Meyer1; Robert Müller1; Markus Hoffmann1; Jürgen Bartz1; Matthias Orth2 1Abbott GmbH, Core Diagnostics, Wiesbaden, Germany; 2Vinzenz von Paul Kliniken gGmbH, Marienhospital Stuttgart, Instituts für Laboratoriumsmedizin, Stuttgart, Germany German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 eA63 Introduction: Accurate reference intervals are crucial for the correct interpretation of laboratory results. A fast and cost- effective alternative to direct reference interval estimation is provided by indirect methods using routine laboratory data. The objective of this study was to compare reference intervals provided by the manufacturer to estimates calculated with three indirect methods. For this comparison, we used the reference intervals stated in Abbott’s instruction for use and real-world data of cholinesterase (CHE) and gamma-glutamyl transferase (GGT) provided by the Marienhospital in Stuttgart. Introduction: Accurate reference intervals are crucial for the correct interpretation of laboratory results. A fast and cost- effective alternative to direct reference interval estimation is provided by indirect methods using routine laboratory data. The objective of this study was to compare reference intervals provided by the manufacturer to estimates calculated with three indirect methods. For this comparison, we used the reference intervals stated in Abbott’s instruction for use and real-world data of cholinesterase (CHE) and gamma-glutamyl transferase (GGT) provided by the Marienhospital in Stuttgart. Methods: Routine laboratory data of CHE and GGT that had been analyzed on the Alinity c platform were used to determine refence intervals. The datasets were cleaned, and data pre-processing steps were applied to decrease the number of presumably diseased subjects in the data. This helps the indirect methods to identify and mathematically model the result distribution of the healthy population. Reference intervals were estimated with a modiﬁed Hoffmann method (RefLim), based on a quantile-quantile plot, the Truncated Maximum Likelihood method (TML), and an inverse modeling approach (reﬁneR) for both sexes and for different age groups. Results: For CHE, all three indirect methods showed a good agreement with comparable results. However, for women, the lower reference limits were consistently higher than the expected values provided by the manufacturer, and for men the same was observed for the upper reference limits. For GGT, the situation was more complex due to a poor agreement between the three methods and an age-dependent increase of the upper reference limits for both sexes. In general, RefLim tended to estimate wider and refineR narrower reference intervals, while TML was in between. Determination of Age- and Sex-specific Reference Intervals for Cholinesterase and Gamma- Glutamyl Transferase Using Real-World Data of the Marienhospital in Stuttgart, Germany: Comparison of Three Different Indirect Methods All three methods confirmed the lower reference limits reported by the manufacturer and obtained different age-dependent upper reference limits. Conclusion: Our study reinforces the call of numerous guidelines and recommendations that reference intervals reported by a manufacturer should be compared with results from the laboratory’s own subject population. Using CHE as an example, we showed that three different indirect methods based on completely different statistical procedures yield comparable results. The derived statistical models of the healthy population were similar, which indicates that they reﬂect the true reference population. The example of GGT, on the other hand, showed that these methods can result in different and possibly false reference limits if the presumably pathological results in a mixed population cannot be correctly identiﬁed and separated with the statistical techniques of the indirect methods. gnosis Paths for Medical Diagnosis based on an Augmented Knowledge Graph Jan Kirchhoff1; Florian Stumpe1; Niclas Heilig1; Lucie Flek2; Joan Plepi2; Heiko Paulheim³ 1medicalvalues GmbH, Research, Karlsruhe, Germany; 2Philipps Universität Marburg, Language Technologies, Marburg, Germany; ³Universität Mannheim, Data and Web Science Group, Mannheim, Germany Medical diagnosis is the process of making a prediction of the disease a patient is likely to have, given a set of symptoms and observations. This requires extensive expert knowledge, in particular when covering a large variety of diseases. Such knowledge can be coded in a knowledge graph – encompassing diseases, symptoms, and diagnosis paths. Since both the knowledge itself and its encoding can be incomplete, reﬁning the knowledge graph with additional information helps physicians making better predictions. At the same time, for deployment in a hospital, the diagnosis must be explainable and transparent. In this paper, we present an approach using diagnosis paths in a medical knowledge graph. We show that those graphs can be reﬁned using latent representations with RDF2vec, while the ﬁnal diagnosis is still made in an explainable way. Using both an intrinsic as well as an expert-based evaluation, we show that the embedding-based prediction approach is beneﬁcial for reﬁning the graph with additional valid conditions. In this paper, we have introduced the medicalvalues knowledge graph, which is used for medical diagnosis using so- called diagnosis paths. Those paths allow for a transparent prediction of a patient’s disease. Since the paths are developed manually, they are notoriously incomplete.To tackle this incompleteness, we have introduced an approach which first enriches the medicalvalues knowledge graph into a augmented graph, connecting it to a large dataset of patient records. On that augmented graph, we have trained vector embeddings with RDF2vec, which are used to predict completions. Both in an internal validation as well as in an expert evaluation, we have shown that the prediction of such extensions is possible with high precision. This methodology of enriching the graph and producing predictions therewith is inde- pendent of the task and domain at hand. One key limitation of the approach is the external data used, which is data gathered from intensive care units. Therefore, diseases which do rarely lead to treatments in intensive care are not well covered. In order to augment diagnosis paths for as versatile diseases as possible, other external datasets should be considered as well. A tool for plausibility checks of reference intervals Sandra Klawitter1; Georg Hoffmann2; Stefan Holdenrieder³; Tim Kacprowski⁴; Frank Klawonn⁵ 1Ostfalia University of Applied Sciences, Department of Computer Science, Braunschweig, Germany; 2Trillium GmbH, Medizinischer Fachverlag, Grafrath, Germany; ³German Heart Centre, Institute of Clinical Chemistry, München, Germany; ⁴Peter L. Reichertz Institute for Medical Informatics of Technical University of Braunschweig and Hanover Medical School, Data Science in Biomedicine, Braunschweig, Germany; ⁵Ostfalia University of Applied Sciences / Helmholtz Centre for Infection Research, Biostatistics, Braunschweig, Germany Background: Laboratory information systems typically contain hundreds or even thousands of reference limits stratiﬁed by sex and age. Since under these conditions a manual plausibility check is hardly feasible, we have developed a simple algorithm that facilitates this check. A user-friendly open-source R tool is available as a Shiny application at github.com/ SandraKla/Zlog_AdRI. Methods: Based on the zlog standardization, we can possibly detect critical jumps at the transitions between age groups, regardless of the analytical method or the measuring unit. Its advantage compared to the standard z-value is that means and standard deviations are calculated from the reference limits rather than from the underlying data itself. The purpose of the tool is illustrated by the example of reference intervals of children and adolescents from the Canadian Laboratory Initiative on Pediatric Reference Intervals (CALIPER). eA64 German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 eA64 Results: The Shiny application identiﬁes the zlog values, lists them in a colored table format and plots them additionally with the speciﬁed reference intervals. The algorithm detected several strong and rapid changes in reference intervals from the neonatal period to puberty. Remarkable jumps with absolute zlog values of more than 5 were seen for 29 out of 192 reference limits (15.1%). This might be attenuated by introducing shorter time periods or mathematical functions of reference limits over age. Results: The Shiny application identiﬁes the zlog values, lists them in a colored table format and plots them additionally with the speciﬁed reference intervals. The algorithm detected several strong and rapid changes in reference intervals from the neonatal period to puberty. Remarkable jumps with absolute zlog values of more than 5 were seen for 29 out of 192 reference limits (15.1%). This might be attenuated by introducing shorter time periods or mathematical functions of reference limits over age. A tool for plausibility checks of reference intervals Discussion: Age-partitioned reference intervals will remain the standard in laboratory routine for the foreseeable future, and as such, algorithmic approaches like our zlog approach in the presented Shiny application will remain valuable tools for testing their plausibility on a wide scale. RefLim: A Graphical and Numerical Approach to Reference Interval Verificatio Georg Hoffmann1; Frank Klawonn2 Georg Hoffmann1; Frank Klawonn2 1Trillium GmbH Medizinischer Fachverlag, Trillium Akademie, Grafrath, Germany; 2Helmholtz Centre for Infection Research, Biostatistics, Braunschweig, Germany Aim: There is a wide range of direct and indirect methods, by which reference limits can be determined [1]. Comparably little attention has been paid to the veriﬁcation of speciﬁed reference intervals from test package inserts and other external sources. The respective guideline procedure [2] is simple, but due to the low number of just 20 values, it suffers from unacceptably wide conﬁdence limits [3], and the criterion that ≥90% of the measured values must fall within the speciﬁed limits cannot detect excessively wide reference intervals [4]. We aimed to develop an alternative method that is as easy to perform, but works with a moderate number of routine laboratory data and detects all kinds of deviations between expectation and observation. Methods: We suggest a modiﬁcation of a previously published iterative boxplot method [5] to create substantially stronger truncation. We derive the quantiles q of the expected central 95% of reference values from the quartiles Q1, Q2, and Q3 according to the following algorithm: var = min(Q2 −Q1, Q3 −Q2) q(0.025, 0.0975) = Q2 ± f · var where f is 2.91 for the first truncation step and 3.08 for all subsequent iterations. where f is 2.91 for the first truncation step and 3.08 for all subsequent iteration This algorithm is applied to log-transformed values assuming that unknown distributions of reference values can be modelled as lognormal [6]. Outliers beyond the estimated quantiles are removed and the algorithm is iterated, until no more outliers are detected. From the truncated data, zlog values are calculated [7] with reference to the specified limits, and a normal quantile-quantile plot with 39 equidistant quantiles is generated [5]. A linear regression line y = a + b ⋅x is calculated from the central 33 data points, and the deviations of a and b from the expected values 0 and 1, resp., are used as measures of discordance between expectation and observation. Results: Based on public data (archive.ics.uci.edu/ml/datasets/HCV+data) for routine liver biomarkers (e. g. gnosis Paths for Medical Diagnosis based on an Augmented Knowledge Graph Here, the connectors to clinic information systems (CIS) and laboratory information systems (LIS) may also add large-scale instance data in the future, which can also be exploited with the same methodology. So far, drugs are not represented in the medicalvalues knowledge graph. In the future, we would like to include them, both as a part of a patient’s medical history (i.e., existing medication), as well as possible treatments once a diagnosis is made. To that end, we plan to augment the graph with existing datasets on drugs and drug interactions. Full paper: https://arxiv.org/pdf/2204.13329.pdf Full paper: https://arxiv.org/pdf/2204.13329.pdf German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 eA6 eA65 RefLim: A Graphical and Numerical Approach to Reference Interval Verificatio trans- aminases, bilirubin) from 540 blood donors and 75 hepatitis C patients, we demonstrate four types of deviations, which can be quantiﬁed by our method: the intercept a signiﬁes positive or negative shifts of the given reference interval, whereas the slope b indicates whether that interval is too narrow or too wide. We use empirical thresholds for both constants to visually represent the magnitude of the deviations by trafﬁc light colors. Comparable results are obtained for blood donors and mixed data. Out of 16 manufacturer-derived reference intervals, nine should be rejected (red), and four should at least be given more careful consideration (yellow). Conclusion: Our method provides an easy and robust way to verify given reference intervals using routine data and intuitive graphics. Our little study makes it likely that a substantial proportion of manufacturer-derived limits needs to be reevaluated using more sophisticated methods for reference interval determination [1]. eA66 Aims: Artiﬁcial intelligence and machine learning have made substantial advances during the last decade in a broad ﬁeld of applications ranging from fraud detection over speech recognition to self-driving cars. In healthcare machine learning approaches are state of the art in medical imaging. In laboratory science however applications are uncommon despite the fact that laboratory medicine with its numerical and structured data would be a very suitable ﬁeld for machine learning. Meaningful but yet unknown correlations are expected to be discovered. First models e.g. for estimation of iron deﬁciency anemia, liver function parameters or low-yield repetitive laboratory tests are already described in basic research. However, their validation in prospective studies or their application in clinical routine seems to be inhibited by rigid laboratory and clinical information systems. Therefor we evaluated technical possibilities for establishing data science approaches in routine diagnostics. Methods: The authors evaluated possible work ﬂows for the application of data science and machine learning methods in laboratory routine or prospective study settings. Results: Interfaces to a plethora of devices and applications are a key feature of every LIS. However most LIS providers tend to distribute these as so-called highly customized software solutions at a high price, even if the connected device is used in virtually every laboratory. One possible and cost-effective workaround could be intercepting interface commu- nication. Another more desirable and efﬁcient approach would be the usage of standardized resources and application programming interfaces like HL7 FIHR. Conclusions: While granular and inconsistent interfaces resulted from the ﬁrst steps of digitalization in laboratory medicine, nowadays well-structured and consistent standards for exchanging health care data like HL7 FIHR are available as open source standards. Therefor we demand that laboratory and hospital information systems provider anticipate in the evaluation and validation of innovative data science techniques by catalyzing their development through deployment of open-source interfaces or free application programming interfaces for research use or develop- ment and validation of novel data science techniques in laboratory diagnostic routine. Artificial Intelligence in laboratory medicine and the responsibility of laboratory information system providers Julian Gebauer; Anja Prantz; Daniel Lasch; Boris Rolinski Elblab GmbH, Elblandklinikum Meißen, Meißen, Germany Julian Gebauer; Anja Prantz; Daniel Lasch; Boris Rolinski Elblab GmbH, Elblandklinikum Meißen, Meißen, Germany eA66 German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 Identification of Putative Non-Substrate-Based XT-I Inhibitors by Small Molecule Library Screening Thanh-Diep Ly; Anika Kleine; Bastian Fischer; Vanessa Schmidt; Doris Hendig; Joachim Kuhn; Cornelius Knabbe; Isabel Faust-Hinse Herz- und Diabeteszentrum Nordrhein-Westfalen, Universitätsklinik der Ruhr-Universität Bochum, Institut für Labo- ratoriums- und Transfusionsmedizin, Bad Oeynhausen, Germany Instruction: Fibroproliferative disorders are characterized by excessive accumulation of extracellular matrix (ECM) components, including collagens and proteoglycans (PGs), which can cause organ dysfunction. The transition of ﬁ- broblasts to ECM-synthesizing myoﬁbroblasts in the presence of ﬁbrotic mediators such as transforming growth factor-β1 (TGF-β1) is a key event of this process. The rise in myoﬁbroblast content is marked by an increase in intracellular and extracellular activity of xylosyltransferase-I (XT-I), which is the initial enzyme of PG biosynthesis. Therefore, human XT-I resembles not only a myoﬁbroblast marker but also a serum biomarker for accessing the proteoglycan biosynthesis rate under ﬁbrotic conditions. Accordingly, the inhibition of XT-I would be a promising treatment option for ﬁbrosis reducing ECM accumulation. Methods: We used a natural product-derived molecular library to identify non-substrate-based inhibitors of the human XT-I by ultra-performance liquid chromatography/electrospray ionization tandem mass spectrometry. We combined this cell-free approach with virtual and molecular biological analyses to conﬁrm and prioritize the inhibitory potential of the identiﬁed compounds. The characterization of the compound’s efﬁcacy in TGF-β1-mediated XYLT1 transcriptional regulation in primary human dermal ﬁbroblasts, the key cells of ECM remodeling, was investigated by gene expression analyses. German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 eA67 Results: Through this approach, we identiﬁed amphotericin B and celastrol as novel non-substrate-based XT-I protein inhibitors. The XT-I inhibitory effect of amphotericin B was mediated by a non-competitive inhibition mode, while that of celastrol was based on a competitive mode of inhibition. Both compounds reduced the XYLT1 mRNA-expression levels and cellular XT-I activity of the primary cells. Furthermore, we demonstrated that the cellular effects mediated by amphotericin B and celastrol were due to inhibitor-induced changes in the TGF-β and microRNA-21 signaling pathway. Conclusion: The results of this study provide a promising basis for the optimization and future use of the XT-I inhibitors amphotericin B and celastrol as therapeutic agents for the treatment of ﬁbroproliferative diseases. Results: Through this approach, we identiﬁed amphotericin B and celastrol as novel non-substrate-based XT-I protein inhibitors. The XT-I inhibitory effect of amphotericin B was mediated by a non-competitive inhibition mode, while that of celastrol was based on a competitive mode of inhibition. Erythropoietin determinations in proficiency tests - variation in laboratory results and method precision Luisa Toll1; Folker Wenzel1; Nathalie Wojtalewicz2; Laura Vierbaum2; Ingo Schellenberg2; Mario Th 1Furtwangen University, Faculty of Medical and Life Sciences, Villingen-Schwenningen, Germany; 2INSTAND e.V., So- ciety for Promoting Quality Assurance in Medical Laboratories, Düsseldorf, Germany; ³German Sport University Cologne, Institute of Biochemistry/ Center for Preventive Doping Research, Cologne, Germany Luisa Toll1,3,, Folker Wenzel1,2, Nathalie Wojtalewicz2, Laura Vierbaum2, Ingo Schellenberg2, Mario Thevis3 Luisa Toll1,3,, Folker Wenzel1,2, Nathalie Wojtalewicz2, Laura Vierbaum2, Ingo Schellenberg2, Mario 1Faculty of Medical and Life Sciences, Furtwangen University, Villingen Schwenningen, Germany; 2INSTAND e.V., Society for Promoting Quality Assurance in Medical Laboratories, Düsseldorf, Germany; 3Institute of Biochemistry/ Center for Preventive Doping Research, German Sport University Cologne, Cologne, Germany; hor. E-mail address: tolu@hs-furtwangen.de Corresponding author. E-mail address: tolu@hs-furtwangen.de *Corresponding author. E-mail address: tolu@hs-furtwangen.de Aims: The aim of the study was to summarize the results of the proﬁciency tests for Erythropoietin (EPO) determination managed by INSTAND e.V. and to evaluate the measurement quality of the speciﬁc methods used by the participating laboratories. Materials and Methods: In each of a total of nine test series, two samples (sample a and b) with different EPO concentrations were send to the participating laboratories. The median of each round was calculated for sample a and b, respectively. Deviation from the median and the relative error-values was calculated. The criteria of acceptance were set to 20 % around the median. In consideration of the used methodology, the relative deviation of one method around the total mean as well as the mean without the respective method was calculated and the outcomes were contrasted. Results: The ﬁrst proﬁciency test was performed with a number of 10 participants and in the following test series it has increased up to a maximum of 85 participating laboratories in series 8. The average fraction of measurements not meeting the deﬁned criteria of acceptance was 14.3 ± 17.4 % (sample a) and 8.2 ± 6.1 % (sample b). The relative deviation tended to be higher in a lower concentration range. Enzyme linked immunosorbent assay (ELISA) showed signiﬁcantly higher values compared to chemiluminescent immunoassay (CLIA) and luminescence-enhanced enzyme immunoassay (LEIA) in all cases. Conclusion: The results appeared to be well harmonized in most cases. However, individual proﬁciency tests showed higher scatterings of the measured values. This should be observed in future test series. Identification of Putative Non-Substrate-Based XT-I Inhibitors by Small Molecule Library Screening Both compounds reduced the XYLT1 mRNA-expression levels and cellular XT-I activity of the primary cells. Furthermore, we demonstrated that the cellular effects mediated by amphotericin B and celastrol were due to inhibitor-induced changes in the TGF-β and microRNA-21 signaling pathway. Conclusion: The results of this study provide a promising basis for the optimization and future use of the XT-I inhibitors amphotericin B and celastrol as therapeutic agents for the treatment of ﬁbroproliferative diseases. eA68 Städtisches Klinikum Dresden, IKL - Institut für Klinische Chemie und Labormedizin, Dresden, Germany Städtisches Klinikum Dresden, IKL - Institut für Klinische Chemie und Labormedizin, Dresden, Germany Aims: The COVID-19 pandemic brought enormous challenges for clinical laboratories: demand for timely and precise SARS-CoV-2-PCR results increased rapidly while consumables for sampling and testing were in ever shorter supply. Laboratories had to build up adequate testing capacities in terms of machines and personnel under great time pressure. We implemented a multiple-swab pool testing method without any volume dilution [1] in a hospital lab setting, which increased efﬁciency dramatically, providing economic SARS-CoV-2-PCR screening without loss of sensitivity. Materials and Methods: Up to 10 nasopharyngeal swabs were pooled directly into one 3,0 ml UTM (Universal Transport Medium) tube. Additionally, dry nasopharyngeal swabs were collected from each participant of the pool, serving as secondary samples for eventual re-testing. After RNA extraction and RT-qPCR, negative pool test results were reported. For positive pool tests, the corresponding secondary samples were tested to identify the infected individual(s) causing the positive pool test result. Results: From April 2020 to March 2022 a total of 25.243 pool tests were analyzed, resulting in 770 positive pool samples with 840 participants found SARS-CoV-2-PCR positive with a mean difference between pool and secondary sample of 0,1 Ct values (standard deviation: 3,6 Ct values). In approx. 4 % of positive pool tests no positive participant could be identiﬁed. Overall, 7 results per one PCR pool test were produced using this protocol, which reduces the price per result accordingly. Time to result is very short for SARS-CoV-2-PCR negative pools (approx. 3 h) and still acceptable for individual SARS-CoV-2-PCR results for the participants of positive pools (approx. 7 h). Discussion: Our pool testing method shows good sensitivity for time efﬁcient SARS-CoV-2-PCR screening. The disad- vantage of our pool testing method is the necessity to collect secondary samples, which in consequence may be of different sampling quality, as seen in cases where in spite of positive pool tests no positive participant could be identiﬁed, probably due to low viral load in early or late infection – SARS-CoV-2-PCR can be (weakly) positive for several weeks to months after infection. The widely used alternative method by Dorfman et al. [2] is based on pooling individual samples in the lab. No secondary samples are required, but sensitivity is reduced markedly due to the dilution effect [3]. Also, sample preparation takes longer because of the additional pooling step, and instrumentation complexity and lab consumable use are higher, e.g. for some PCR clean ﬁlter pipette tips in short supply up to today. SARS-CoV-2-PCR pool testing: high efficiency without sensitivity loss Konstantin Gröber; Katja Graul; Angela Bessert; Thomas Demant eA68 German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 eA69 German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 Results: From 2nd of August 2021 to 1st of May 2022, we observed a total of 218’815 SARS-CoV-2 positive samples, with 168’101 samples matching the deﬁned criteria. Out of these, 194 specimen with NGTF were identiﬁed (0.12 %). The samples originated from all regions of Switzerland and were mostly clonally unrelated. In large part, samples with NGTF were collected from October to December 2021 correlating with the infection wave attributable to the Delta variant (B.1.617.2) and its sub-lineages. During this time, a proportional increase of NGTF among all positive samples with a peak frequency of 1 % was observed. Frequency of NGTF swiftly fell once the Omicron lineages BA.1 and BA.2 became prevalent. Sequencing revealed the nucleotide substitution G28922T (A217S) in 148 cases (88.6 %). 10 samples (6 %) carried the deletion 28913 – 28918 (del214/215), 8 samples (4.8 %) the deletion 28913 – 28915 (del214) and 1 sample (0.6 %) the deletion 28892 – 28930 (del207 – 219). Samples with intact N gene ampliﬁcation lacked the speciﬁed mutations. Lineages included the Delta variant parental lineage B.1.617.2 (n=4), sub-lineages thereof (AY.4 (n=99), AY.4.3 (n=4), AY.33 (n=2), AY.36 (n=9), AY.39 (n=1), AY.43 (n=7), AY.43.3 (n=2), AY.46.6 (n=1), AY.98.1 (n=4), AY.122 (n=1) and AY.125 (n=11)) as well as the Omicron lineage BA.2 (n=1). The lineage of 24 specimen could not be determined. Conclusion: The substitution A217S, as well as the deletions G214-, G214/215- and del207-219 in SARS-CoV-2 appear to be associated with the NGTF in the TaqPath Kit. These mutations were identiﬁed in several sub-lineages of the Delta variant as well as the Omicron BA.2 lineage and were apparently not linked to a particular variant. Importantly, a selection advantage associated with the NGTF could not be identiﬁed. The N gene is a common target in RT-PCRs e.g., Cepheid’s Xpert Xpress SARS CoV 2 or the VIASURE SARS-CoV-2 (N1 + N2) Real Time PCR Detection Kit for BD MAX. Continuous monitoring and timely re-assessment using whole genome sequencing can thus improve the development of diagnostic tests. SARS-CoV-2 nucleocapsid protein mutations disrupt N gene amplification in frequently employed multiplex RT-PCR assays Dominique Hilti1; Faina Wehrli1; Anna Roditscheff2; Martin Risch³; Lorenz Risch³; Adrian Egli⁴; Thomas Bodmer2; Nadia Wohlwend1 1Dr. Risch, Medizinische Mikrobiologie, Buchs SG, Switzerland; 2Dr. Risch, Medizinische Mikrobiologie, Bern, Switzerland; ³Dr. Risch, Allgemeine Innere Medizin, Buchs SG, Switzerland; ⁴Universitätsspital Basel, Klinische Bakteriologie und Mykologie, Basel, Switzerland Aims: Absence of SARS-CoV-2 PCR target signals may indicate the presence of new variants. We analysed the increase of N gene dropouts in the TaqPath COVID-19 CE-IVD RT-PCR Kit from Thermo Fisher Scientiﬁc (TaqPath) covering the viral ORF1ab, N and S genes. Whole genome sequencing was used to identify potential issues with the N gene PCR efﬁcacy. Materials and Methods: We analysed nasopharyngeal swabs and saliva samples from symptomatic patients as well as asymptomatic carriers from mass testing programs by RT-PCR with the TaqPath Kit. Complete N gene target failures (NGTF) were deﬁned as missing N gene ampliﬁcation in the presence of intact ampliﬁcation of ORF1ab and S genes. Ultimately, whole genome sequencing was performed on matching samples with CT-values < 30 using a GridIon Nanopore sequencer (Oxford Nanopore Technologies - ONT, Oxford, UK). MULTIMODAL SPATIALLY RESOLVED INVESTIGATION OF LIPIDS SIGNATURES IN NEEDLE BIOPSIES OF LIVER NEOPLASMS WITHIN THE MANNHEIM MOLECULAR INTERVENTION ENVIRONMENT (M2OLIE) Miriam Rittel1,2, Stefan Schmidt1; Emrullah Birgin³; Carina Ramallo Guevara1; Alexander Marx4; Axel Wellmann5; Cleo Weis4; Nuh Rahbari3; Carsten Hopf1,2,6 Miriam Rittel1,2, Stefan Schmidt1; Emrullah Birgin³; Carina Ramallo Guevara1; Alexander Marx4; Weis4; Nuh Rahbari3; Carsten Hopf1,2,6 1Center for Mass Spectrometry and Optical Spectroscopy (CeMOS), Mannheim University of Applied Sciences; 2Institute of Medical Technology, Heidelberg University; 3Department of Surgery, Medical Faculty Mannheim, Mannheim School of Medicine, Heidelberg University, Mannheim; 4Institute of Pathology, University Medical Centre Mannheim; 5Institute of Pathology Celle; 6Medical Faculties Heidelberg and Mannheim, Heidelberg University Introduction and Aims: Mass spectrometry imaging (MSI) is an indispensable tool for label-free spatially-resolved investigations of biological processes and of the molecular composition of various samples in fundamental research. However, the translation of MSI-based techniques into clinical routine for in-depth medical diagnosis, e.g. molecular pathology, remains a challenging task. Requirements in sample preparation for molecular analysis and time restrictions, as well as feasibility in a clinical context are often on opposing sides of the spectrum. Especially, targeting cancer and cancer sub-types in a fast and reliable way by MSI with diagnostic value for further treatment decisions is of particular interest. Here, we present a multimodal approach aligned with clinical routine practice for the assessment of molecular composition of different tumor tissue samples by fusing modern analytical tools and sophisticated clinical routine. Methods: Within the framework of the M2OLIE research campus, we combined various spatially resolved techniques like infrared spectroscopy, immunohistochemistry and hematoxylin and eosin stainings with MSI-based molecular analysis to investigate the lipid and metabolite composition of primary hepatocellular carcinoma and metastases residing in the liver. Fresh-frozen liver tissue section were measured by means of high-precision untargeted matrix-assisted laser desorption/ionization (MALDI)-MSI in order to reveal the molecular ﬁngerprint of cancer types for biomarker discovery. This includes resected tissue samples as well as biopsies. To this end, we have developed a device for biopsy embedding and MSI sampling, as well as a workﬂow for data fusion of modern molecular analysis with sophisticated clinical routine. By mapping this sample-speciﬁc information onto the MSI data a higher precision in analysis of neoplastic lesions can be accomplished. Results: We analyzed liver cancer tissue sections with a multimodal approach. To this end, we combined the spatial information from infrared spectroscopy and histopathological evaluation to map tissue morphology-speciﬁc features onto the MSI data for in-depth molecular analysis, especially for tumorous regions. eA70 Results: Chromatographic separation was achieved within a total run time of 6.6 min to simultaneously quantify 4 11-OAs as well as 7 other steroid hormones from the original method. Lower limits of quantiﬁcation were well below endogenous ranges (63-320 pmol/l), recoveries ranged from 85% to 117% with CVs ≤15%. The concentration of the analytes were comparable to published values of healthy individuals and showed high stability across ﬁve freeze/thaw cycles (CV ≤3.1%). Clinical veriﬁcation revealed, as expected, an increased concentration of 11-OAs and decreased ratios of androstendione or testos- terone to 11-OAs in CAH patients. Conclusion: We present a robust high-throughput method with high sensitivity for simultaneous quantiﬁcation of four 11-OAs and seven routine steroid hormones using minimal sample preparation. By multiplex-design, ratios of clinically relevant steroids to 11 OAs are instantly accessible, allowing the use of 11-OAs for diagnosis and therapy monitoring in androgen excess-related disorders. In current studies, the method is used for determination of reference intervals (from birth to 80 years) as well as to investigate the relationship between 11-OAs with obesity, metabolic syndrome, and puberty. Liquid chromatography tandem mass spectrometry for quantitative analysis of 11 oxygenated androgens in human serum. Liquid chromatography tandem mass spectrometry for quantitative analysis of 11 oxygenated androgens in human serum. Robert Zeidler; Ronald Biemann; Uta Ceglarek; Juergen Kratzsch; Berend Isermann; Alexander Gau versität Leipzig, Institut für Laboratoriumsmedizin, Klinische Chemie und Molekulare Diagnostik, Le Aims: 11-Oxygenated androgens (11-OAs) are discussed as potential biomarkers for adrenal androgen excess such as congenital adrenal hyperplasia (CAH) and polycystic ovary syndrome (PCOS). However, quantiﬁcation of 11 OAs by LC-MS/ MS still relies on extensive sample preparation including liquid-liquid extraction, derivatization and partial long runtimes, which is unsuitable for high-throughput analysis under routine laboratory settings. Aim of this study was to validate a method to quantify 11-OAs using a clinical routine LC-MS/MS method for steroid hormone proﬁling with minimal sample preparation. Methods: A DIN ISO 15189 accredited LC-MS/MS method for quantitation of 7 serum steroids in daily routine use was extended by 11-ketoandrostendione (11-KA), 11-ketotestosterone (11-KT), 11β-hydroxytestosterone (11-OHT), 11β-hydrox- ytestosterone (11-OHT) and validated. In brief, online solid phase extraction was combined with reverse phase liquid chromatography using methanol/H2O/0.2 mM NH4F as mobile phase. Detection was conducted using a Sciex QTrap 6500plus in positive ionization and multiple reaction mode. Calibrators and controls were produced inhouse by spiking certiﬁed Chromsystems® calibration and control material for steroid hormone analysis covering expected endogenous concentration ranges. Method validation included thorough examination of reproducibility, recovery, linearity, sensitivity and speciﬁcity according to FDA guidelines. Possible effects of freeze/thaw cycles were addressed. For clinical veriﬁcation, 13 CAH patients and healthy controls were compared. eA70 German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 Estimation of Continuous Reference Intervals using Real-World Data and refineR ana Ammer1; André Schützenmeister2; Hans-Ulrich Prokosch³; Manfred Rauh⁴; Christopher Rank2; ndré Schützenmeister2; Hans-Ulrich Prokosch³; Manfred Rauh⁴; Christopher Rank2; Jakob Zierk⁵ 1Friedrich-Alexander-Universität Erlangen-Nürnberg, Chair of Medical Informatics, Erlangen; Roche Diagnostics GmbH, R&D Biostatistics and Data Science, Penzberg, Germany; 2Roche Diagnostics GmbH, R&D Biostatistics and Data Science, Penzberg, Germany; ³Friedrich-Alexander-Universität Erlangen-Nürnberg, Chair of Medical Informatics, Erlangen, Ger- many; ⁴Universitätsklinikum Erlangen, Department of Pediatrics and Adolescent Medicine, Erlangen, Germany; ⁵Uni- versitätsklinikum Erlangen, Department of Pediatrics and Adolescent Medicine; Universitätsklinikum Erlangen, Center of Medical Information and Communication Technology, Erlangen, Germany Introduction: Most biomarkers and their corresponding reference intervals depend on covariates, such as sex, age, or ethnicity. While the derivation of reference intervals for categorical covariates like sex is straightforward, it is substan- tially more challenging to correctly represent the inﬂuence of continuous covariates. Most pronounced during physio- logical development in children, but extending beyond adulthood, many analytes show clinically relevant age- dependent dynamics. One current approach for the estimation of age-speciﬁc reference intervals is a discretization of the continuous covariate. However, this approach leads to unnatural, discontinuous transitions between reference intervals estimated for the artiﬁcial age groups. Therefore, continuous reference intervals are needed to adequately assess test results of these analytes. Here, we propose a novel method leveraging routine measurements and a recently published algorithm, reﬁneR, to establish smooth, continuous reference intervals and percentile charts for biomarkers of interest. Methods: We developed a fully automated pipeline for the generation of continuous reference intervals utilizing solely an indirect method (reﬁneR) and real-world data. First, the input data is divided into ﬁne-grained subgroups, ensuring sufﬁcient amount of data points per group. Second, we apply the reﬁneR algorithm to each group and use the estimated model to determine reference intervals and percentiles for each age bin. These percentiles are then automatically smoothed using a median and bilateral ﬁlter. Subsequently, the reﬁneR algorithm is applied in an iterative way alternating between model estimation and smoothing of the percentile curves. The smoothed percentiles from iteration i then serve as regu- larization for model estimation in iteration i+1 to ﬁnally generate smooth, continuous reference intervals. The presented pipeline was applied to three important biomarkers with extensive pediatric dynamics (hemoglobin, alkaline phosphatase, and creatinine) using data obtained during patient care. MULTIMODAL SPATIALLY RESOLVED INVESTIGATION OF LIPIDS SIGNATURES IN NEEDLE BIOPSIES OF LIVER NEOPLASMS WITHIN THE MANNHEIM MOLECULAR INTERVENTION ENVIRONMENT (M2OLIE) By precise co-registration this guided approach enabled transfer of information between adjacent tissue sections and of data from different modalities. Pre- liminary data revealed a differentiation of the tissue morphology and thus differences in the lipid and metabolite composition of speciﬁc histological features. German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 e eA71 Discussion: These results promote a MSI-based routine that is feasible in clinical practice. Fusion of molecular infor- mation and clinical routine assessment for cancer classiﬁcation could potentially result in beneﬁts for treatment de- cisions based on higher level sample analysis. Discussion: These results promote a MSI-based routine that is feasible in clinical practice. Fusion of molecular infor- mation and clinical routine assessment for cancer classiﬁcation could potentially result in beneﬁts for treatment de- cisions based on higher level sample analysis. Estimation of Continuous Reference Intervals using Real-World Data and refineR Results: The calculated percentile charts for hemoglobin, alkaline phosphatase and creatinine from birth to 18 years of age are in accordance with previously established reference intervals, demonstrating that the presented approach correctly models age-dependency and generates valid continuous reference intervals. Conclusions: The provided pipeline enables the fully automated generation of high-precision percentile charts using real-world data, while requiring no additional tool, except the reﬁneR algorithm. Providing precise percentile charts allows for accurately capturing the pronounced age-dependent dynamics that occur in many biomarkers, facilitating the interpretation of test results and ultimately improving patient care. Das Modul Gesundheitsdaten als Basis für den Bachelor Biomedizinische Labordiagnostik an der ZHAW Lisa Rigassi eA72 German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 eA72 Züricher Hochschule für Angewandte Wissenschaften, Departement Gesundheit, Studiengang Biomedizinische Labor- diagnostik, Winterthur , Switzerland Züricher Hochschule für Angewandte Wissenschaften, Departement Gesundheit, Studiengang Biomedizinische Labor- diagnostik, Winterthur , Switzerland Zielsetzung: Im Herbst 2022 startet ein schweizweit einzigartiger Bachelor Biomedizinische Labordiagnostik [1]. Die interdepartmentelle Ausrichtung zwischen “akademischen Gesundheitsberuf mit naturwissenschaftlichem Hintergrund” soll sich in den Modulen widerspiegeln. Das Modul Gesundheitsdaten im 1. Semester soll als Basismodul gestaltet werden, welches die zentralen Aspekte labordiagnostisch relevanter Daten in Gesundheitsversorgung und Forschung abbildet und eine Verbindung zu wesentlichen Bezugswissenschaften schafft. Fragestellung: Welche Wissensstruktur unterstützt die Zielerreichung des Moduls? Was sind exemplarische Vorschläge zu Inhalten und didaktischen Methoden? Material und Methoden: Die Inhalte des Moduls wurden anhand eines explorativen Brainstormings erarbeitet. Basierend auf den Ergebnissen wurde die Wissensstruktur entwickelt. Als Bezugsgrössen wurden das Modell der CanMEDS-Rollen [2] der labordiagnostische Prozess und ein Modell des Forschungsprozesses genutzt. Diese Wissens- struktur soll mit den involvierten Akteuren evaluiert werden. Als didaktische Methoden wurden u.a. Fallstudien und Workshops vorgesehen. Die Erreichung der Ziele soll durch kritische Validierung der Studierenden und Lehrenden geprüft werden. Ergebnisse: Zur Strukturierung des Moduls Gesundheitsdaten wurden Leitfragen bezüglich Datenerhebung, Bereit- stellung und Auswertung von Daten in der Labormedizin ermittelt und den Phasen des labordiagnostischen Analy- seprozesses zugeteilt. Präanalytisch sind Fragen bezüglich Planung nötig: wer soll welche Daten aus welchem Grund bei wem erheben? In der Analytik ist die Erhebung zentral: z.B. mit welcher Methode und zu welchem Zweck? Post- analytische Fragestellungen befassen sich mit der Datenauswertung: was bedeuten die Daten? Wem sind sie zu melden? In einem zweiten Schritt wurden die Leitfragen den Rollen des CanMEDS-Modells zugeordnet und exemplarische Vorschläge zu konkreten Modulinhalte abgeleitet. Dazu gehören z.B. zum Manager das Managen von Daten in Labori- nformationssystemen und wissenschaftliche Datenbanken, zum Communicator die interprofessionelle Kommunikation mit anderen Professionsangehörigen, Wissenschaftler:innen und Laien sowie zum Collaborator die Zusammenarbeit im Team, zwischen den verschiedenen Akteuren und zwischen Institutionen. Als relevante Bezugswissenschaften wurden Ethik, Recht, Informatik und Kommunikationswissenschaften abgeleitet. Diskussion und Schlussfolgerung: Die Wissensstruktur kann wie folgt beschrieben werden: auf Basis der 7 CanMEDS-Rollen sind Daten in Präanalytik, Analytik und Postanalytik in der Gesundheitsversorgung sowie bei Planung, Erhebung und Auswertung in der Forschung zu managen. Den Rahmen bilden Recht und Ethik sowie Kommunikation und Informatik. Die vorliegende Modulstruktur ist ein erster Entwurf und sollte sowohl intra- als auch interprofessionell mit den Gesundheitsberufen, Naturwissenschaftler und Praxis diskutiert werden. Introduction: Novel ultra-sensitive single molecule array (SiMoA) based detection of neuroﬁlament light (NfL) and other neuroglial proteins is a promising tool for evaluation of neurodestruction. However, clinical applicability is hampered by considerable costs as well as absence of disease speciﬁc cut-off values. eA73 German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 Objective: We aimed to test the clinical validity of reduced cost protocol for SiMoA technology-based assay in two cohorts: 1) patients with relapsing-remitting multiple sclerosis (RRMS) and 2) patients with anti-IgLON5 disease. These inﬂammatory/neurodegenerative disorders were chosen because of their heterogeneous natures and urgent need for standardized biomarkers of disease activity/prognosis. Objective: We aimed to test the clinical validity of reduced cost protocol for SiMoA technology-based assay in two cohorts: 1) patients with relapsing-remitting multiple sclerosis (RRMS) and 2) patients with anti-IgLON5 disease. These inﬂammatory/neurodegenerative disorders were chosen because of their heterogeneous natures and urgent need for standardized biomarkers of disease activity/prognosis. Methods: Serumsamplesweremeasured withNeurology4-plexkit:NfL,Tau,ubiquitinC-terminal hydrolase L1(UCHL-1) and glial ﬁbrillary acidic protein (GFAP) - on fully automated HD-X-Analyzer (Quanterix) using standard and reduced cost protocols (measurements in duplicates vs. singlicates). In the RRMS cohort (N=114), serum samples were obtained pro- spectively at an outpatient clinic (UKSH, Campus Kiel); active disease was deﬁned by new relapses and/or changes on MRI scans observed over the prior six months. In the cohort of treatment naive patients with anti-IgLON5 disease (N=27), the serum samples were recruited retrospectively from German network for Research on autoimmune encephalitis (GENERATE). Healthy controls (N=70) were analyzed for comparison. Biomarker concentrations were compared between patients and controls, correlated with disease activity/prognosis and cut-off values estimated when possible. Results: The intra- and inter-assay variance for NfL and GFAP remained under 10%. In the RRMS cohort, higher NfL values were detected in active patients (N=42) compared to patients in remission (N=72) (12.1 (8.8-26.6) vs. 7.1 (4.8-10.7) pg/ml, p< 0.001) and the controls (9.2 (6.9-12.0)pg/ml, p< 0.001). An age-adapted NfL cut-off value of 8.5pg/ml predicted disease activity with 68% speciﬁcity and 76% sensitivity in patients under the age of 60 years (AUCROC=76.9 ± 0.05%, p< 0.0001). In the anti-IgLON5 cohort, NfL and GFAP concentrations were signiﬁcantly increased (71.1±103.9pg/ml and 126.7±73.3pg/ml, respectively) compared to the controls (N=70) (9.2 (6.9-12.0)pg/ml and GFAP 67.0 (53.1-95.2)pg/ml respectively, p< 0.001). Low pretreatment NfL value was also an independent predictor for better treatment outcome. Tau and UCHL-1 allowed no differentiation between patients and controls. Conclusions: Reduced cost protocols are feasible on fully-automated platform and could facilitate clinical application of serum neuronal biomarkers. While NfL discriminates fairly well between RRMS and controls, sensitivity and speciﬁcity for active disease as standalone marker is insufﬁcient. In patients with anti-IgLON5 disease, low NfL level at treatment initiation may serve as a potential biomarker for response to immunotherapy and prognosis. Clinical feasibility of a novel ultrasensitive multiplex NfL, Tau, UCHL-1 and GFAP assay for the evaluation of neuroaxonal and glial damage in serum samples of patients with multiple sclerosis and anti-IgLON5 disease Justina Dargvainiene1; Thomas Grüter2; Ilya Ayzenberg2; Franziska E. Möllers1; Klaus-Peter Wandinger1; Klarissa Hanja Stürner³; Frank Leypoldt1; Ralf Junker1 1UKSH, Campus Kiel, Institut für klinische Chemie, Kiel, Germany; 2St. Josef Hospital, Ruhr Universität Bochum, Klinik für Neurologie, Bochum, Germany; ³UKSH, Campus Kiel, Klinik für Neurologie, Kiel, Germany Introduction: Novel ultra-sensitive single molecule array (SiMoA) based detection of neuroﬁlament light (NfL) and other neuroglial proteins is a promising tool for evaluation of neurodestruction. However, clinical applicability is hampered by considerable costs as well as absence of disease speciﬁc cut-off values. Introduction: Novel ultra-sensitive single molecule array (SiMoA) based detection of neuroﬁlament light (NfL) and other neuroglial proteins is a promising tool for evaluation of neurodestruction. However, clinical applicability is hampered by considerable costs as well as absence of disease speciﬁc cut-off values. Introduction: Novel ultra-sensitive single molecule array (SiMoA) based detection of neuroﬁlament light (NfL) and other neuroglial proteins is a promising tool for evaluation of neurodestruction. However, clinical applicability is hampered by considerable costs as well as absence of disease speciﬁc cut-off values. Zielsetzung: g (1) Wie ist die TERT Mutationsfrequenz und – verteilung in multiphasisch differenzierten Schilddrüsenkarzinomen? (2) Gibt es hinsichtlich des Auftretens und der Verteilung der TERT Promotermutation Unterschiede zwischen den häuﬁg auftretenden homogenen und den seltenen multiphasisch differenzierten Schilddrüsenkarzinomen? (1) Wie ist die TERT Mutationsfrequenz und – verteilung in multiphasisch differenzierten Schilddrü (2) Gibt es hinsichtlich des Auftretens und der Verteilung der TERT Promotermutation Unterschiede zwischen den häuﬁg auftretenden homogenen und den seltenen multiphasisch differenzierten Schilddrüsenkarzinomen? (2) Gibt es hinsichtlich des Auftretens und der Verteilung der TERT Promotermutation Unterschiede zwischen den häuﬁg auftretenden homogenen und den seltenen multiphasisch differenzierten Schilddrüsenkarzinomen? Nachweis und Verteilung des prognostisch relevanten TERT Promotermutationsstatus bei multiphasisch differenzierten Schilddrüsenkarzinomen Sandra Ehrhardt; Udo Siebolts, Liesa Siemss Universitätsklinikum Halle (Saale), Institut für Pathologie, Halle, Germany Universitätsklinikum Halle (Saale), Institut für Pathologie, Halle, Germany eA74 Results: Upon incubation with the apoptosis inhibitor a signiﬁcant reduction of PS externalization was detected after 10 days of storage (p=0.002). Accordingly, signiﬁcant higher levels of MMP were observed after G04 incubation (p=0.034). Interestingly, the inhibitor did not affect the platelet functionality. In fact, CD62 expression was comparable to untreated cells (p=0.086). While, the responsiveness of CD63 and PAC1 was better conserved in platelets stored with the inhibitor (day 10, CD63 p=0.035 and PAC1, p=0.005). Furthermore, on day 10 no differences in the platelet aggregation ability were detected with or without inhibitor (TRAP, p=0.591 and Ristocetin p=0.998) as well as comparable thrombin formation (p=0.602). Interestingly, a signiﬁcant increase of the number of adherent cells was detected after platelets were treated with G04 (day 7, p= 0.0465). More importantly, a higher percentage of circulating human cold-stored platelets was detected in themouse bloodstream uponincubation with G04after7 daysofcoldstorage comparedtountreatedcells (5h postinjection, p=0.046). Conclusion: Our results show that the cold-induced platelet apoptosis, responsible for the faster destruction of cold- stored platelets in vivo, can be efﬁciently prevented using an apoptosis inhibitor. Furthermore, the inhibitor did not impair platelet functions like activation, aggregation, thrombin formation and adhesion. More importantly, the inhibitor better maintains cell survival in vivo. Therefore, incubation of APCs with apoptosis inhibitor(s) during cold storage, might be a promising strategy to prolong the storage time without impairing platelet’s functionality and improving cell half-life. Einleitung: g Das klinisch relevante Schilddrüsenkarzinom ist das häuﬁgste Malignom des endokrinen Systems. Multiphasische Schilddrüsenkarzinome sind, im Vergleich zu homogenen Tumoren, eine seltene, im wissenschaftlichen Kontext recht unerforschte Tumorentität. Bei diesen Tumoren ﬁndet man neben differenzierten Karzinomanteilen gleichzeitig schlecht- bzw. entdifferenzierte Anteile, wodurch die Tumorheterogenität zu Stande kommt. Ein Gen, welches bei Schild- drüsenkarzinomen eine prognostische Relevanz hat, ist das TERT Gen, welches für das Enzym Telomerase Reverse Transkriptase codiert [1]. In verschiedenen Studien hat man herausgefunden, dass vor allem die zwei somatischen Mutationen im Promoterbereich des Gens c.-124C>T (C228T) und c.-146C>T (C250T) für die Tumorgenese und -progression von besonderer Bedeutung sind [2,3]. Man hat ebenfalls herausgefunden, dass TERT Mutationen häuﬁger in schlecht differenzierten Schilddrüsenkarzinomen zu ﬁnden sind, während ein Auftreten in differenzierten Karzinomen eher selten der Fall ist [4]. Über die TERT Mutationsfrequenz und Verteilung in homogenen Schilddrüsentumoren wurden in der Vergangenheit viele Publikationen verfasst, sehr viel weniger erforscht sind die multiphasisch differenzierten Schild- drüsenkarzinome. Die Bestimmung des TERT Promotermutationsstatus als prognostischer Biomarker soll helfen diese Tumoren in ihrem Progressionsverhalten besser einzuschätzen. Apoptosis inhibition during cold storage of platelets better maintains both platelet functionality and survival Irene Marini1; Yoko Tamamushi1; Lisann Pelzl1; Stefanie Nowak-Harnau2; Tamam Bakchoul³ 1Institut für klinische und experimentelle Transfusionsmedizin, University of Tübingen, Tübingen, Germany, For- schung, Tübingen, Germany; 2Institut für klinische und experimentelle Transfusionsmedizin, University of Tübingen, Tübingen, Germany, Blutspende, Tübingen, Germany; ³Institut für klinische und experimentelle Transfusionsmedizin, University of Tübingen, Tübingen, Germany, Forschung und Blutspende, Tübingen, Germany Introduction: Apheresis-derived platelet concentrates (APCs) is an essential medical therapy use to treat bleeding. Nevertheless, the storage at room temperature increases the risk of bacterial-infection post transfusion. Recently, we reported that cold-stored APCs show better platelet functionality but decreased platelet half-life caused by apoptosis activation (Marini et al., Haematologica 2019). In this study, we investigated the impact of an apoptosis inhibitor on platelet functionality and half-life during cold storage. Methods: APCs were collected from healthy donors and stored for 7 and 10 days at 4°C in the presence or in the absence of an apoptosis inhibitor (G04). Platelet apoptosis was assessed measuring the phosphatidylserine (PS) exposure and the mito- chondrial membrane potential (MMP) by ﬂow cytometry. Platelet functionality was investigated analyzing the expression of CD62, CD63 and PAC1 upon TRAP stimulation, by ﬂow cytometry. Platelet aggregation ability, after TRAP and ristocetin incubation, was tested using an aggregometer. Next, thrombin generation was measured using a thrombogram. While, the adhesion ability was determined performing an in vitro assay. Platelet survival was analyzed using a NSG mouse model. eA74 German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 Ergebnisse: g Die Pyrosequenzierung des multiphasisch differenzierten Schilddrüsenkarzinomkollektivs ergab, dass 71,4% der untersuchten Tumorproben die prognostisch relevante TERT Promotermutation tragen, 28% zeigten einen negativen TERT Status. Im untersuchten Kollektiv wurden die meisten TERT Promotermutationen unerwartet in den PTC Anteilen (75%) detektiert, die wenigsten in den PDTC Arealen (35,7%). Schlussfolgerung: Heterogene Schilddrüsenkarzinome sind Tumoren mit einer ungünstigen Prognose, dies bestätigt der Nachweis der prognostisch relevanten TERT Promotermutation in der Mehrheit der untersuchten Karzinome. Da die meisten TERT Mutationen in den PTC Anteilen detektiert wurden, sollte bei Tumorheterogenität stets der PTC Anteil oder besser jeder der unterschiedlich diff.Karzinomanteile molekularpathologisch untersucht werden. Methodik: Ein Tumorkollektiv aus 14 multiphasisch differenzierten Schilddrüsenkarzinomen wurde mittels eines eigens entwick- elten Pyrosequenzierungsassays auf das Vorhandensein der beiden prognostisch relevanten Hotspot Mutationen C228T und C250T im Promoterbereich des TERT Gens untersucht, die einzelnen Karzinomanteile wurden dabei jeweils getrennt voneinander analysiert. eA75 German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 eA75 EA-02 Uta Nennstiel eA76 German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 eA76 Design und Validierung patientenspezifischer droplet digital PCR (ddPCR) Assays zur Detektion und Quantifizierung zirkulierender Tumor-DNA Laura Mirbach1; Tobias Boch2; Angelika Duda1; Romy Eichner1; Sonja Loges2; Verena Haselmann1 1Universitätsmedizin Mannheim, Institut für Klinische Chemie, Mannheim, Germany; 2DKFZ-Hector Krebsinstitut an der UMM, Abteilung Personalisierte Onkologie, Medizinische Fakultät Mannheim der Universität Heidelberg, Personalisierte Medizinische Onkologie (A420) DKFZ, Mannheim, Germany Zielsetzung: Das Liquid Proﬁling gewinnt als Alternative zur gewebsbasierten Diagnostik zunehmend an Bedeutung. Der klinische Nutzen im Rahmen der Companion Diagnostik zur therapeutischen Stratiﬁzierung konnte bereits für verschiedene Tumorentitäten, wie z.B. das Nicht-kleinzellige Bronchialkarzinom (NSCLC), nachgewiesen werden. Ein weiterer Vorteil des Liquid Proﬁlings liegt in seiner Eignung zum Monitoring von Patienten im Verlauf im Sinne eines personalisierten Tumormarkers. Hierfür müssen patientenspeziﬁsche Sequenzvariationen im Primärtumor identi- ﬁziert und individuelle Assays entwickelt werden. Für dieses Ziel eignet sich insbesondere die droplet digital PCR (ddPCR), da sie neben einer hohen analytischen Sensitivität als offenes System die Etablierung laborentwickelter individueller Assays erlaubt. Methoden: Für die Etablierung individueller ddPCRs müssen zunächst relevante Sequenzvariationen im Primärtumorgewebe identiﬁziert und das Vorliegen in der Keimbahn ausgeschlossen werden. Hieran schließt sich das Primer-/Probe-Design basierend auf einer TaqMan-qPCR an. Hierbei müssen speziﬁsche Punkte im Hinblick auf die ddPCR berücksichtigt werden. Nach experimenteller Ermittlung der optimalen Annealingtemperatur erfolgt die Val- idierung durch Bestimmung des limit of blank, der false-positive-rate, des limit of detection sowie des limit of quanti- ﬁcation mit anschließender Überprüfung von Präzision und Richtigkeit. Ergebnisse: Exemplarisch wird der Fall einer Patientin mit NSCLC vorgestellt. Für diese Patienten wurden zwei relevante Sequenzvariationen im Primärtumorgewebe identiﬁziert, ein Basenaustausch sowie eine Fusion. Für beide Veränder- ungen wurden entsprechende personalisierte ddPCR-Assays etabliert und zur Verlaufskontrolle genutzt. Die Ergebnisse zeigen, dass sich dieser Ansatz eignet, um das Therapieansprechen im Verlauf zu überwachen. Diskussion Und Schlussfolgerung: Das Liquid Proﬁling kann neben der Companion Diagnostik auch als person- alisierter Tumormarker verwendet werden. Hierfür können mittels ddPCR patientenspeziﬁsche Assays entwickelt und zur Verlaufskontrolle eingesetzt werden. Dies stellt einen wichtigen Schritt dar, um neben einer personalisierten Therapie auch eine Personalisierung der Diagnostik als Voraussetzung für das Erreichen einer „Precision Medicine“ in der Onkologie zu erzielen. Uwe Oemüller Uwe Oemüller Qiagen GmbH, MDx Development, Hilden, Germany Qiagen GmbH, MDx Development, Hilden, Germany Molecular in vitro diagnostics and research have allowed great progress in medicine including diagnostics. Further progress is increasingly established by new biomarker tests analyzing specimens’ biomolecule proﬁles such as nucleic acids, proteins, and metabolites. However, proﬁles of these molecules can change signiﬁcantly during specimen collection, transport, storage, and processing including analyte isolation, caused by post collection cellular changes such as gene inductions, gene down regulations, biomolecules modiﬁcations or degradation. This can make the outcome from diagnostics or research unreliable or even impossible because the analytical test will not determine the situation in the patient body but an artiﬁcial specimen analyte proﬁle generated during the pre-analytical workﬂow. Preanalytical variables are a major error source for wrong diagnostic test results. High quality specimens with preserved analyte proﬁles as they were in the patients bodies are therefore crucial for reliable diagnostics, biomedical research and biobanking. Specifying, developing and verifying pre-analytical workﬂow parameters for diagnostics tests has conse- quently become a requirement by new European legislation. The EU SPIDIA Consortium (2008-2013) developed new pre-analytical technologies for preserving molecular profiles in human specimenand generatedbroadevidencethatguidancetolaboratoriesonpre-analyticalworkflowsimprovesanalytical test results. Based on these results, the CEN/TC 140 for “in-vitro diagnostic medical devices” had released first 9 European Technical Specifications for pre-analytical workflows addressing different blood, other body fluids and tissuebased molecular applications. In 2018 and 2019 they progressed to International Standards at the ISO/TC 212 for “clinical laboratory testing and in vitro diagnostic test systems”. The successor EU SPIDIA4P consortium project (2017-2021), supported by a large interna- tional network, has broadened to a ﬁnal portfolio of 22 pre-analytical CEN and ISO Standards intending to improve in vitro diagnostics and biomedical research, has developed corresponding External Quality Assurance (EQA) and is driving inter- national implementation. The new standards can serve to fulﬁll requirements in the new EU In Vitro Diagnostic Regulation 2017/746 (IVDR). The SPIDIA4P project has received several awards including the “CEN-CENELEC Standards+Innovation Project Award 2021” for its important contribution of standardization and innovation to molecular diagnostics. The SPIDIA project received funding from the EU’s FP7 under grant agreement no. 222916. The SPIDIA4P project received funding from the EU’s Horizon 2020 research and innovation program under grant agreement no. 733112. EA-01 Uwe Oemüller Uwe Oemüller eA77 Neugeborenen-Screening“ (ENS) auf zehn metabolische und zwei endokrine Zielkrankheiten in die Kinder-Richtlinie auf und ergänzte es 2018 um Tyrosinämie Typ I, 2019 um schwere kombinierte Immundefekte (SCID) sowie 2021 um Sichelzellkrankheit und Spinale Muskelatrophie (SMA). Mit gesonderten Regelungen wurde das Mukoviszidose- Screening 2016 eingeführt [1]. Daneben unterliegt das ENS dem Gendiagnostikgesetz [2]. Einige Zielkrankheiten können bereits früh zu lebensbedrohlichen Stoffwechselkrisen oder zu bleibenden Schäden führen. Dies setzt eine Diag- nosestellung und den Therapiebeginn bereits in den ersten Lebenstagen bis Wochen voraus. Die Zielkrankheiten des NBS sind selten und treffen in ihrer Gesamtheit eines von etwa 1.000 Neugeborenen [3]. Dies bedeutet wiederum, dass 99,9% der nicht betroffenen „gesunden“ Neugeborenen bei dieser populationsbezogenen Reihenuntersuchung mit untersucht wer- den müssen. Dabei muss neben einer hohen Sensitivität insbesondere eine hohe Speziﬁtät erreicht werden, um nur wenige Familien gesunder Kinder durch einen falsch positiven Befund zu beunruhigen [4]. Das ENS ist somit in erster Linie eine Public-Health-Maßnahme, die nach deﬁnierten Kriterien in einem von der Elternaufklärung bis zur Nachsorge gut struk- turierten und organisierten sowie qualitätsgesichertem Programm durchzuführen wäre [5–9]. Die Screeningdaten aller elf deutschen Screeninglabore werden an die Deutsche Gesellschaft für Neugeborenscreening (DGNS) übermittelt, dort analysiert und in einem jährlichen Screeningreport publiziert [10]. Für den Zeitraum von 2006 bis 2019 liegen für Deutschland Screeningdaten für 10.022.333 Neugeborene vor. Bei 7.995 Kindern wurde die Ver- dachtsdiagnose in der Konﬁrmationsdiagnostik bestätigt, bei 807 Kindern ist unklar, ob die Konﬁrmationsdiagnostik durchgeführt wurde (lost to follow-up). Eine oder mehrere Folgeuntersuchungen waren bei 307.588 Neugeborenen erforderlich und wurden bei 246.253 Kindern (80,059 %) durchgeführt. Die Daten zeigen, dass das ENS insgesamt erfolgreich umgesetzt wird, allerdings fehlen wichtige Komponenten eines qualitätsgesicherten Programms. Optimierungsbedarf ergibt sich beim Loss to follow-up, da von ca. 20 % der Neugebor- enen mit kontrollbedürftigem Screeningbefund nicht bekannt ist, ob dieser jemals abgeklärt wurde. Eine verbesserte Rückmeldung der Diagnostik der behandelnden Ärzte an die Screeninglabore, sowie eine Nachverfolgung kon- trollbedürftiger Screeningbefunde durch ﬂächendeckende Trackingstrukturen ist hier nötig. Die Einführung eines Registers und eine geregelte Evaluation könnten die Qualität des Screening-Programms weiter verbessern. Uta Nennstiel Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit (LGL), Sachgebiet GE4 Gesundheitsberichter- stattung, Epidemiologie, Sozialmedizin, Kindergesundheit, Screeningzentrum, München-Oberschleißheim, Germany Das Neugeborenen-Screening auf angeborene Stoffwechsel- und Hormonstörungen ist seit der Einführung des Screen- ings auf Phenylketonurie (PKU) Ende der 1960er Jahre eine der erfolgreichsten Maßnahmen der Sekundärprävention im Kindesalter und wird für bereits seit. Im Jahr 2004 nahm der Gemeinsamen Bundesausschuss (G-BA) das sog. „Erweiterte German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 e EA-03 Christoph Hiemke Update on Reference Intervals and Decision Limits - Symposium der Sektion Entscheidungsgrenzen/ Richtwerte RIbench: A Benchmark for Evaluating Indirect Methods for Reference Interval Estimation Frank Streit Frank Streit Institut für Klinische Chemie und Interdisziplinäres UMG-Labor, Universitätsmedizin Göttingen Das therapeutische Drug Monitoring (TDM) spielt eine wichtige Rolle in der Patientenversorgung eines Klinikums der Maximalversorgung. Die Bestimmung der aktuellen Medikamenten-Konzentration im Blut ermöglicht eine rasche Anpassung der Dosierung, um optimale Wirkspiegel zu erreichen bzw. um Toxizität zu verhindern. Dazu ist eine zeitnahe Analytik und Rückführung der Ergebnisse notwendig. Viele Medikamentenspiegel werden massenspektroskopisch ermittelt, was, wegen der Komplexität der Methodik, zumeist keinen Einsatz in der 24/7 Routine-Analytik erlaubt. Wir haben TDM in den letzten Jahren mit dem CLAM2030-LCMS8060NX (Fa. Shimadzu) weiterentwickelt, das nun fast den Anforderungen für ein Klinikum genügt, wie wir Sie von den klinisch-chemischen und immunchemischen Analyzern her kennen. Mittels CLAM2030-LCMS8060NX erfolgt eine automatisierte Probenvorbereitung und eine kontinuierliche Analytik im Random-Access-Mode durch aufeinander abgestimmte Methoden. Dies wird durch eine Kalibrations-Stabilität über Wochen, eine hohe Stabilität der Reagenzien auf dem Gerät sowie einer intuitiven Bedienungsoberﬂäche und automa- tisierten Datenauswertung ermöglicht. Die bidirektionale Kommunikation mit unserem LIS-System (OPUS-L) erfolgt mittels HL7-Schnittstelle.Somitistder24/7BetriebauchdurchPersonalohnespeziellemassenspekrometrischeKenntnissemöglich. Die derzeit verfügbaren, IVDR-konformen, automatisierten LCMS-Lösungen für TDM stellen noch keine Option für den Einsatz in der 24/7-Analytik dar, da Sie für Einzelproben ungeeignet sind und eine Abarbeitung und Datenauswertung durch speziell geschultes Personal bedürfen. Um unseren Anforderungen nachzukommen, sind wir daher gewisser- maßen gezwungen, eigene Probenprozessierungen zu etablieren und zu validieren. eA78 von den für orale Applikationsformen empfohlenen Referenzbereichen abzuweichen. TDM kann vor allem bei der Umstellung eines Antipsychotikums von der oralen auf die Depotformulierung nützlich sein, weil es hierdurch besser gelingt, patientenindividuelle pharmakokinetische Eigenschaften und Einﬂussfaktoren bei der Einstellung und bei der Dauertherapie mit einem Depot-Antipsychotikum zu kontrollieren. Wichtig ist es dabei auch, vor der Umstellung auf Depot die individuell optimalen Wirkstoffkonzentrationen zu bestimme, indem mehrfach in Phasen guten Anspre- chens eine Blutspiegelmessung vorgenommen wird. Weitere Indikationen für TDM bei Depotantipsychotika sind Symptomverschlechterung, unerwünschte Arzneimittelwirkungen oder Einnahme von Medikamenten mit Wechselwirkungspotential. versitätsmedizin Mainz, Klinik für Psychiatrie und Psychotherapie, Mainz, Germany Universitätsmedizin Mainz, Klinik für Psychiatrie und Psychotherapie, Mainz, Germany Die Anwendung von Depot-Antipsychotika (Long-Acting Injectable antipsychotics, LAI) ist in den letzten Jahren konstant gestiegen. Anders als bei den meisten oralen Antipsychotika, deren Anwendung oftmals hinsichtlich erwünschter Therapieeffektivität und Arzneimitteltherapiesicherheit durch den Einsatz vom therapeutischen Drug Monitoring (TDM) optimiert wird, ist die Bedeutung von TDM bei Depot-Antipsychotika nach wie vor unklar (Schretsanitis et al. 2021). Weit verbreitetist bei den Behandlerndie Meinung,dassTDM nicht sinnvoll sei, dabei der Depotgabe,andersals beioraler Gabe, stabile Wirkspiegel eingestellt werden. Darüber hinaus wird angezweifelt, ob die Referenzbereiche von Antipsychotika, die fürdieAkutbehandlung etabliertsind, auch fürdieBehandlungmitDepot-Antipsychotikagültigsind. IndiesemBeitragsoll dargestellt werden, dass die Anwendung von TDM zur Therapiesteuerung auch Depot-Antipsychotika nützlich sein kann, weil auch bei Depotgabe die Wirkstoffkonzentrationen bei gleicher Dosis interindividuell hoch variabel sind und wie bei oraler Einnahme Alter, Geschlecht, Begleitmedikation oder metabolischer Phänotyp bzw. Genotyp bedeutsam sind. Mit Blick auf die gut etablierten therapeutischen Referenzbereichen oral verabreichter Antipsychotika scheinen die erwünschten klinischen Wirkungen von Depot-Antipsychotika bereits in niedrigeren Wirkstoffkonzentrationsbereichen erzielt zu werden. Es ist zu diskutieren, ob für Depot-Antipsychotika andere therapeutische Referenzbereiche zu empfehlen sein könnten. Die bisherigen Erkenntnisse hierzu sind aber nicht ausreichend valide, um die Empfehlung auszusprechen, eA78 German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 Tatjana Ammer Tatjana Ammer Universitätsklinikum Erlangen, Kind- und Jugendklinik, Erlangen, Germany German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 eA eA79 Background: Precise reference intervals are essential for the interpretation of laboratory test results in medicine. Indirect methods leverage routine measurements containing a mixed distribution of non-pathological and pathological values to estimate reference intervals, rather than conducting a study with apparently healthy subjects (i.e. ‘direct’ method). In recent years, several such indirect methods have been developed. However, no standardized tool for the evaluation and comparison of indirect methods exists so far that can reveal the strengths and weaknesses of the different methods and guide algorithm selection and application. Background: Precise reference intervals are essential for the interpretation of laboratory test results in medicine. Indirect methods leverage routine measurements containing a mixed distribution of non-pathological and pathological values to estimate reference intervals, rather than conducting a study with apparently healthy subjects (i.e. ‘direct’ method). In recent years, several such indirect methods have been developed. However, no standardized tool for the evaluation and comparison of indirect methods exists so far that can reveal the strengths and weaknesses of the different methods and guide algorithm selection and application. Methods: We provide RIbench, a benchmarking suite that enables quantitative evaluation and comparison of existing and novel indirect methods. The benchmark contains simulated test sets for ten biomarkers mimicking real-world data (routine measurements). The non-pathological distribution of the biomarkers represent four common distribution types observed in laboratory practice: normal, skewed, heavily skewed, skewed-and-shifted. To identify limitations of the indirect methods, we added pathological distributions with varying location, extent of overlap, and fraction to the non- pathological distribution. Further, the sample size was varied to quantify the performance impact of the data set size. Overall, the benchmark suite contains 576 simulated tests sets per biomarker, 5,760 test sets in total. To evaluate the performance, we compute benchmark scores derived from the absolute z-score deviations between the estimated and true reference limits. We showcase the application of RIbench by evaluating ﬁve indirect methods, the Hoffmann method, and four modern approaches: TML, kosmic, TMC, and reﬁneR. The results are compared against each another and a nonparametric direct method (N=120). Results: For all methods, the pathological fraction had a strong inﬂuence on the results. Further, for TML, kosmic, TMC, and reﬁneR, the sample size also strongly affected the performance. Tatjana Ammer With a minimum sample size of 5,000 and a pathological fraction of up to 20%, these indirect methods still achieved results comparable or superior to the direct method. Conclusions: We present RIbench, an open-source R-package that enables a quantitative and systematic evaluation and comparison of existing and novel indirect methods. Covering a variety of tests sets with varying difﬁculty, RIbench can serve as a valuable tool to reveal strengths and weaknesses, and enhance indirect methods, ultimately improving the estimation of reference intervals. EA-06 Martina Sender; Uta Ripperger Martina Sender; Uta Ripperger mio42 GmbH , Berlin, Germany mio42 GmbH , Berlin, Germany Die Kassenärztliche Bundesvereinigung (KBV) trifft die notwendigen Festlegungen für die Inhalte der elektronischen Patientenakte, um deren semantische und syntaktische Interoperabilität zu gewährleisten (§ 355 SGB V). Als Tochter- gesellschaft und im Auftrag der KBV entwickelt und speziﬁziert die mio42 die sogenannten Medizinischen Informa- tionsobjekte (kurz MIOs) für die elektronischen Patientenakte (ePA). MIOs dienen dazu, medizinische Daten nach einem festgelegten Format auf Basis internationaler Standards und Ter- minologien zu dokumentieren. Sie können als kleine digitale Informationsbausteine verstanden werden, die universell verwendbar und kombinierbar sind. Dadurch wird der Austausch und die Verarbeitung der Daten zwischen einzelnen Akteur:innen innerhalb des Gesundheitswesens, unabhängig vom genutzten Softwaresystem, ermöglicht. Für das MIO Laborbefund wurden bewährte Konzepte aus aktuell genutzten Qualitätsstandards und Richtlinien sowie Vorarbeiten von Fachgremien berücksichtigt, um eine breite fachübergreifende als auch sektorenübergreifende Akzeptanz zu erwirken. Die öffentliche Kommentierungsphase bezüglich der Speziﬁkation hat zum Ziel, das externe Feedback durch die Mitglieder von Verbänden und Organisationen als Vertreter:innen zukünftiger Verwender:innen oder Hersteller:innen von MIOs einzuholen. Im Anschluss erfolgt die Benehmensherstellung zur offziellen Festlegung. eA80 German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 eA80 Die semantische Interoperabilität für konkrete Laboruntersuchungen wird ganz wesentlich durch die LOINC®-Codierung geschaffen, weil ein einzelner LOINC®-Code umfänglich die Eigenschaften einer Laboruntersuchung beschreibt. Eine SNOMED CT® Codierung kann für qualitative Labor-Ergebnisse verwendet werden. Zur Veranschaulichung wird kurz ein Beispiel präsentiert. Die Perspektive für die syntaktische Interoperabilität beim digitalen Labordatenaustausch ist, ein ein- heitliches Format zu haben (HL7 FHIR®), damit das MIO von sämtlichen Software-Systemen unmittelbar gelesen und angezeigt werden kann. Die semantische Interoperabilität für konkrete Laboruntersuchungen wird ganz wesentlich durch die LOINC®-Codierung geschaffen, weil ein einzelner LOINC®-Code umfänglich die Eigenschaften einer Laboruntersuchung beschreibt. Eine SNOMED CT® Codierung kann für qualitative Labor-Ergebnisse verwendet werden. Zur Veranschaulichung wird kurz ein Beispiel präsentiert. Die Perspektive für die syntaktische Interoperabilität beim digitalen Labordatenaustausch ist, ein ein- heitliches Format zu haben (HL7 FHIR®), damit das MIO von sämtlichen Software-Systemen unmittelbar gelesen und angezeigt werden kann. Thomas Streichert Thomas Streichert Universitätsklinikum Köln, Institut für Klinsiche Chemie, Köln, Germany Für Laboratorien ergibt sich aus dem erweiterten Geltungsbereich der IVDR eine neue Anforderung: Die IVDR deﬁniert nicht nur ein Reagenz, Reagenzprodukt, Kalibrator, Kontrollmaterial, Kit, Instrument, Apparat, Gerät sondern auch eine Software oder ein Softwaresystem als Medizinprodukt, sofern es vom Hersteller zur in-vitro-Untersuchung von aus dem menschlichen Körper stammenden Proben, einschließlich Blut- und Gewebespenden, bestimmt ist. So kann eine Soft- ware durchaus ein IVD im Sinne der IVDR werden. Viele Laboratorien haben im Laufe der Zeit Software entwickelt, beginnend vom einfachen Excel-Tool, über Algorithmen bis hin zu komplexen Lösungen zur Abbildung von diag- nostischen Pfaden sowie der Unterstützung oder sogar vollautomatisierten Befundung oft eingebettet in die jeweiligen LIS. Dabei werden Laboratorien ggfs. (Software-)Hersteller im Sinne der IVDR. Dieser Vortrag versucht die kritischen und unkritischen Aspekte zu Software im Lichte der IVDR zu beleuchten. Walter Fierz Walter Fierz Walter Fierz Walter Fierz Heiligenschwend, Switzerland Heiligenschwend, Switzerland The problems in standardizing immunological tests are mainly based on the individuality of the immune system of the patient and the variability of antigen presentation in different tests. The individual way the immune system recognizes antigens partly depends on its previous encounters with similar antigens, a phenomenon that is called “original antigenic sin”. On the antigen selection and presentation side in the test, variabilities might be based on the particular expression system used, the glycosylation status of the antigens, the 3D structure of the antigens, and the accessibility of hidden epitopes, as e.g. with anti-b2-GPI antibodies. A third variability might come from the different avidities of the detected antibodies, dependent on the detection system, that may correlate with different clinical phenomena as e.g. seen with anti-dsDNA antibodies. Furthermore, the characteristics of the relation between a biomarker and a clinical diagnosis, like sensitivity and speciﬁcity, might depend on the prevalence of the underlying cause responsible for the biomarker on one side and for the disease on the other side. Due to these standardization difficulties results from different test systems and therefore their clinical relevance cannot be directly compared. One way to improve the situation is to calculate the likelihood ratio of a particular result of a particular test that gives the relation between the occurrence of the result in patients versus controls and with it its diagnostic relevance. German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 eA81 Alexander Tolios Medizinische Universität Wien, Universitätsklinik für Blutgruppenserologie und Transfusionsmedizin, Wien, Austria Machine learning-Algorithmen erlauben es in den Lebenswissenschaften, komplexe Muster in Daten zu finden, welche anschließend für klinische Vorhersagen genutzt werden können. Aber der Preis für die Verwendung von solchen komplexen Methoden ist die eingeschränkte Interpretierbarkeit der algorithmischen Entscheidungsﬁndung. Insbesondere im medizinischen Setting ist es jedoch wichtig, zu verstehen, warum ein Algorithmus zu einem bestimmten Ergebnis kommt, da ja die behandelnden Ärzte in der Lage sein müssen, die Entscheidung zu verstehen und ggf. zu hinterfragen. Durch die Anwendung von "eXplainable Artificial Intelligence" (XAI)-Methoden besteht die Möglichkeit, schwierig zu interpretierbaren "black box"-Algorithmen (wie neuronale Netzwerke oder random forests) unter die Motorhaube zu schauen. Im Rahmen dieses Vortrags sollen die gängigsten Methoden vorgestellt und deren Vor- und Nachteile gegenübergestellt werden. EA-10 Alexander Tolios Jakob Adler Medical Laboratory for Clinical Chemistry, Microbiology, Infectious Diseases and Genetics "Prof. Schenk/Dr. Ansorge & Colleagues", Magdeburg, Germany Digitalisierung. Kaum ein Wort hört man heute in allen Bereich unseres Lebens häuﬁger. Wir leben in einer Zeit, die geprägt ist von der nächsten großen gesellschaftlichen Veränderung seit der Industriellen Revolution: der „Digitalen Transformation“. Doch was bedeutet das konkret für die Laboratoriumsmedizin? Scheinbar fernab vom (zukünftigen) Alltag aus elektronischer Patientenakte, MIO Laborbefund und Telemedizin gibt es eine enorm vielfältig Entwicklung im Bereich Digital Health. Welche Rolle spielen Big Tech Unternehmen wie Alphabet/Google, Amazon, Apple oder Intel? Gelten die auf uns einprasselnden Buzzwords wie Künstliche Intelligenz, Metaverse, Web 3.0, Industrie 4.0, Brave New Work, usw. auch für unsere Profession? Was gibt es bereits an routinetauglichen Anwendungen der künstlichen Intelligenz in der Labormedizin? Welche Gefahren können von solche Algorithmen ausgehen? Dieser Vortrag will einen nie- drigschwelligen Einstieg in die Thematik Digitalisierung und Künstliche Intelligenz bieten und dabei den aktuellen Stand der Entwicklungen weltweit auch fernab des klassischen Gesundheitswesens beleuchten. Hype or hope Wieviel KI braucht die Labormedizin? Explainable Artificial Intelligence - Anwendungsbeispiele für labormedizinische Daten Explainable Artificial Intelligence - Anwendungsbeispiele für labormedizinische Date St. Bernward Krankenhaus GmbH, Zentrum für Labordiagnostik, Hildesheim, Germany Business Intelligence ist ein Begriff aus dem Bereich der Wirtschaftsinformatik. Er beschreibt einen Prozess der unter Zuhilfenahme digitaler Algorithmen große Datenmengen, auch aus mehreren Quellsystemen, erfassen, konsolidieren und verständlich darstellen soll. Die größte Herausforderung in diesem Prozess ist es die Integrität und Konsistenz der Daten sicherzustellen, auf deren Basis die weitere Prozessierung und letztendlich auch die Ableitung von Maßnahmen beruht. Im Bereich der Laboratoriumsmedizin gibt es vielfältige Anwendungsfälle für diesen Analysenprozess und zuletzt auch einen wachsenden Bedarf an leicht bedienbaren Werkzeugen. Neben kommerziellen Anwendungen gibt es viele indi- viduelle Wege diesen Analysenprozess zu verwirklichen, um sich einen schnellen und verlässlichen Überblick über die Kennzahlen des eigenen Labors zu verschaffen. Anhand von Anwendungsbeispielen aus dem Krankenhauslabor werden hier die Möglichkeiten und Vorteile, aber auch die Grenzen und Hürden dieser Methodik, sowie der unterschiedlichen Herangehensweisen vorgestellt. Aus-, Fort- bzw. Weiterbildungsqualität bei nicht-ärztlichen Laborspezialisten - Symposium der Sektion Repetitorium Digitale Kompetenz - "Hot-Skills" für die Führungskräfte der digitalen Zukunft Auto-Antikörper-Diagnostik in der Diabetologie - Aktueller Stand der Analytik und klinische Anwendung in Deutschland Auto-Antikörper-Diagnostik in der Diabetologie - Aktueller Stand der Analytik und klinische Anwendung in Deutschland eA82 St. Bernward Krankenhaus GmbH, Zentrum für Labordiagnostik, Hildesheim, Germany EA-12 Erwin Schleicher1; Markus Thaler2 Erwin Schleicher1; Markus Thaler2 1Universitätsklinikum der Eberhard-Krals-Universität Tübingen, Medizinsche Klinik IV, Tübingen, Germany; 2Klinikum rechts der Isar der TU München, Institut für Klinische Chemie und Pathobiochemie, München, Germany Autoantibodies against islet cells (IAAB), i.e. autoantibodies against glutamate decarboxylase, insulinoma antigen 2, zinc transporter -8, and insulin, develop early in type 1 diabetes mellitus (DM) when metabolic changes and clinical symptoms are still absent. Presence of IAAB therefore characterizes the first stage of the disease while stage 2 and 3 show dysglycemia and chronic hyperglycemia and overt clinical diabetes, respectively. Being positive years before clinical onset of the disease, IAAB are determined in individuals at high risk for type 1 DM. IAAB are positive in up to 85% of the newly diagnosed patients with type 1 DM indicating that every 6th patient with type 1 DM fails to show IAAB at the time of diagnosis. Novel studies indicate that screening for IAAB in early childhood may predict the disease years before manifestation. In adults, IAAB may serve to classify the DM as type 1 or type 2 / maturity-onset diabetes of the young (MODY) as well as patients as “autoimmune insulin deficient” or “severe insulin deficient”– when clinical and metabolic characteristics are ambiguous. A multitude of methods are employed to determine IAAB in the clinical laboratory. Methods allowing formation of the immune-complexes partially or totally in liquid phase are to be preferred. This is due to the fact that assay formats with solid-phase immobilized antigens seem not to reach sufficient sensitivities and speci- ficities. As with other autoantibodies, comparability of different IAAB assay results is poor. Laboratories are therefore strongly encouraged to establish their own cut-off values in order to enable proper clinical interpretation of the test results. Ring trials for all IAAB mentioned above are currently available in Germany and contribute to analytical quality. Beyond that, the “Islet Autoantibody Standardization Program” as internationally coordinated endeavor aims to improve quality and comparability of IAAB assays. EA-11 Martin Christmann eA82 German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 EA-13 Jakob Adler German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 eA eA83 Medical Laboratory for Clinical Chemistry, Microbiology, Infectious Diseases and Genetics , Clinical Chemistry, Mag- deburg, Germany Medical Laboratory for Clinical Chemistry, Microbiology, Infectious Diseases and Genetics , Clinical Chemistry, Mag- deburg, Germany Wir leben in der Zeit der „Digitalen Transformation“. Wir merken immer wieder, dass viele unserer Arbeitsmittel und -abläufe den sich ständig ändernden Gegebenheiten scheinbar nicht mehr gewachsen sind. Doch was tun? Welche Fähigkeiten braucht der labormedizinische Nachwuchs, um in einer Welt aus Digital Health, Künstlicher Intelligenz und Metaverse den Aufgaben und Anforderungen an die eigene Profession gerecht zu werden? Was sind die „Hot Skills“ der Zukunft? Wie wird man zum „Digital Talent“? Im 21. Jahrhundert brauchen wir neben Lesen, Schreiben und Rechnen eine neue Grundfertigkeit: Programmieren. In einer immer digitaler werdenden Welt braucht es ein umfassendes technisches und informatisches Verständnis, um mit Trends wie Blockchain, Metaverse und Künstlicher Intelligenz sowie Notwendigkeiten wie der Cyber-Security umgehen zu können. Es braucht ein „algorithmisches Denken“, um die Welt um uns herum zu verstehen und sie aktiv mitzugestalten. Wenn wir als Ärzt:innen nicht aktiv am digitalen Gesundheitswesen der Zukunft bauen, werden es andere tun. Big Tech Unternehmen wie Google, Amazon oder Apple stehen bereit und investieren Milliarden in die Entwicklung von Digital Health Anwendungen. Die Sektion Junges Labor hat für sich erkannt, dass es eine neue Kompetenz erwerben muss: Digitale Kompetenz. Dieser Vortrag möchte die aktuellen Gegebenheiten beleuchten, die zur Gründung der Arbeitsgruppe Digitale Kompetenz geführt haben und einen Einblick in die Arbeitsweise und Inhalte der AG geben. eA84 Vergleichbarkeit hergestellt werden und ein Orientierungswert für den Ct eingeführt werden, der eine grobe Abschätzung der Kontagiosität der Patienten ermöglicht. Das RKI propagiert darüber die Entisolierung von Inﬁzierten zu steuern. Damit ist für das Labor ein Grad der Referenzierung erreicht, der das Arbeiten in der SARS-CoV-2 Diagnostik erleichtert. Es ist jedoch nicht abzusehen, wann oder ob überhaupt die SARS-CoV-2 Pandemie ein Ende hat, deshalb werden hohe Anforderungen an das Labor bestehen bleiben. EA-14 Burkhard Brandt UKSH, Institut für Klinische Chemie, Kiel, Germany Mit der Pandemie des SARS-CoV-2 Erregers kam eine bisher nicht dagewesene Flut von Aufträgen für eine PCR in die klinisch-chemischen Labore. Neben ihrer großen Anzahl kamen Probenmaterialein ins Labor, die bislang in den Rou- tineabläufen, nur einen geringen Arbeitsaufwand darstellten, deren Entnahmequalität jedoch das Analysenergebnis wesentlich beeinﬂusst. Die dritte neue Herausforderung stellte die Notwendigkeit zur Steigerung der Bearbeitungska- pazität in kürzester Zeit dar, da die Pandemie sich schnell in der Bevölkerung ausbreitete. Die Industrie reagierte schnell mit Assay- und Geräteentwicklungen, so dass 3 – 4 Monate nach den ersten Fällen halbautomatische und wenig später vollautomatische Systeme zur Verfügung standen. Dafür mussten Räume vorbereitet werden, die die dafür erforderlichen Medien (Strom, Wasser, Klimatisierung, Belüftung) enthielten, insbesondere dem Infektions- und Kontaminationsschutz genügten. Die Labore mussten die Assays ohne etabliertes Referenzmaterial validieren, den Probentransfer und die -vorbereitung unter dem besonderen Schutz der Mitarbeiter bewältigen und die Datensicherheit über ihr LIS für Eins- chleusung, Identiﬁkation, Material und Befund einschl. Kommentierung und Übermittlung, auch an die Überwa- chungsstellen (GSA, RKI), gewährleisten. Darüber hinaus wurden auch PCR-Schnelltests bereitgestellt, die in die bestehenden Eilfallstrukturen zu integrieren waren. Die Versorgung mit Entnahmesystemen war im ganzen ersten Jahr der Pandemie knapp. Ein häuﬁger Herstellerwechsel erforderte Auswertungsanpassungen. Inzwischen ist die Versorgung stabil und mit der Einführung von inaktivierenden Medien im primären Probenröhrchen könnten Infek- tionsschutzmaßnahmen reduziert werden. Die entscheidende Einﬂussgröße bleibt trotz aller Validationen im Labor und im Gegensatz zur standardisierten venösen Blutentnahme die Entnahmequalität in Nase und Rachen. Klinisch erfor- derliche Entnahmen als BAL oder Bronchialabstrich/Sputum erfordern eine speziﬁsche Probenaufbereitung. Nach wie vor schwierig, insb. in der Befundkommunikation, bleibt der Messwert der PCR, der Ct-Wert (Cycle Threshold). Er stellt anders als in der Klinischen Chemie verbreitet nicht direkt eine Stoffmenge dar. Die Wertelage bleibt darüber hinaus vom Assayhersteller und der individuellen Auswertung des qualitativen PCR-Assays abhängig. Mithilfe der inzwischen eta- blierten Referenzmaterialien von Referenzmaterialherstellern und des RKI konnte für die PCR-Methoden eine A84 German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 eA85 (continued) Name, Vorname Paper Codes Fischer, Andre P-- Fischer, Andreas P--, P--, P--, P--, P--, P--, P--, P--, P-- Fischer, Bastian P--, P--, P--, P--/ PV- Flek, Lucie P-- Fleming, Thomas P-- Flieder, Tobias P--, P--, P-- Föger, Niko P-- Franzenburg, Jeanette P-- Freckmann, Guido P--, P-- Friedrich, Nele P--, P--/ FV- Fritsche, Andreas P--, P--/ PV- Fritsche, Louise P--/ PV- Froese, Vera P-- Frölich, Matthias P--, P-- Gadi, Ihsan P--/ PV- Ganzenmueller, Tina P-- Gaudl, Alexander P--, P--, P--/ FV- Gauß, Friederike P--, P--, P--, P--, P--/ FV-, FV- Gawinecka, Joana P-- Gebauer, Julian P--, P-- Geilenkeuser, Wolf J. P--/ FV- Gerhards, Catharina P-- Gräff, Ingo P-- Grasmeier, Melina P-- Graul, Katja P-- Gröber, Konstantin P-- Groß, Stefan P-- Grossmann, Kirsten P-- Grüter, Thomas P-- Gupta, Anubhuti P--, P--, P--/ PV- Gupta, Dheerendra P--, P-- Habler, Katharina P-- Hafke, Angelika P-- Halimeh, Susan P-- Hammer, Stefanie P--/ FV- Hannig, Jürgen P-- Häring, Hans-Ulrich P--/ PV- Hartleb, Sophie P-- Haselmann, Verena P--, P--, P--, P--, P--, P--/ FV- Hasenfuß, Gerd P-- Haug, Cornelia P--, P-- Hebest, Marc P-- Hedtke, Maren P--, P-- Heilig, Niclas P-- Hendig, Doris P--, P--, P--, P-- Heni, Martin P--/ PV- Henning, Ann-Kristin P--, P--, P--/ FV-, P-- Herold, Susanne P-- Hertel, Alexander P-- Herzog Silva P-- (continued) Name, Vorname Paper Codes Hiepler, Marla P--/ FV- Hiemke, C. EA- Hilti, Dominique P-- Hoene, Miriam P-- Hoffmann, Christina P-- Hoffmann, Georg P--, P-- Hoffmann, Georg F. Autorenverzeichnis Name, Vorname Paper Codes Abdullah, Mohammed P-- Abratis, Anna P-- Adler, Jakob P--, P--, EA-, EA- Aeschbacher, Stefanie P-- Ahmad-Nejad, Parviz P--/FV- Aida, Sihem P-- Alberts, Anika P--/ PV- Althaus, Karina FV-/ P--, P-- Ambreen, Saira P--, P--, P--/ PV- Ammer, Tatjana P--/FV-, EA- Anker, Sophie P-- Arneth, Borros P--, P-- Ast, Volker P--, P--, P-- Ayzenberg, Ilya P-- Backes, Viktoria P-- Bahls, Martin P-- Bakchoul, Tamam FV-/ P--, P--, P-- Bartz, Jürgen P-- Bauer, Michael P--/ PV- Baumstark, Annette P--, P-- Bechmann, Ingo P-- Begcevic Brkovic, Ilijana P--, P-- Beil, Alexandra P--, P-- Besler, Christian P-- Bessert, Angela P-- Biemann, Ronald P--, P--, P--, P--, P--/ FV- Bigler, Susanna P-- Binder, Lutz P--, P-- Birgin, Emrullah P-- Birkenfeld, Andreas P--, P--/ PV- Birschmann, Ingvild P--, P--, P-- Biskup, Dirk P-- Boch, Tobias P-- Bode, Anna P-- Bodmer, Thomas P--, P-- Brand, Korbinian P--/ PV- Brandenburger, Christina P-- Brandhorst, Gunnar P--, P--/ FV- Brandt, Burkhard P--, EA- Braun, Valentin P-- Brings, Sebastian P-- Brockmann-Honig, Christine P-- (continued) Name, Vorname Paper Codes Brune, Maik P-- Budde, Kathrin P--, P--, P--, P--/ FV- Carl, Angelika P--, P--/ FV- Ceglarek, Uta P--, P--, P--, P--, P--/ FV- Chang, Johannes P-- Chey, Soroth P-- Christmann, M. EA- Conen, David P-- Costina, Victor P-- Danckwardt, Sven P--, P-- Dargvainiene, Justina P--, P-- Demant, Thomas P-- Dihazi, Gry Helene P--, P-- Dittrich, Julia P-- Dittrich, Marcus P-- Dolscheid-Pommerich, Ramona Christina P--, P-- Dörr, Marcus P-- Drees, Christoph P-- Duda, Angelika P--, P--, P--/ FV- Ebert, Matthias P-- Eckelt, Felix P-- Edelmann, Frank P-- Egli, Adrian P-- Ehrhardt, Sandra P-- Eichner, Romy P--, P--, P--, P--/ FV- Eidizadeh, Abass P--, P--, P-- Eisenhauer, Anton P-- Elezagic, Dzemal P-- Elwakiel, Ahmed P--, P--/ PV- Enkel, Sigrid FV- Erden, Tanja P-- Erdmann, Jürgen P-- Esser, Daniela P-- Fatima, Sameen P--, P--, P--/ PV- Faust-Hinse, Isabel P--, P--, P--, P--, P--, P--/ PV- Fenzlaff, Marc P-- Feyh, Patrik P--/ FV- Fierz, Walter EA- German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 eA German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 eA P--/ FV- Hoffmann, Markus P-- Hoffmann, Peter P-- Hoffmeier, Nicole P-- Hofheinz, Ralf P-- Holdenrieder, Stefan P-- Hollerbach, Anne P-- Hopf, Carsten P-- Hörber, Sebastian P--, P-- Horneffer-van der Sluis, Verena P-- Hörning, André P-- Hörster, Friederike P--/ FV- Hu, Chunxiu P-- Hummel, Julia P--/ PV- Isermann, Berend P--, P--/ PV-, P--, P--, P--, P--, P--, P--/ FV- Jaffal, Hisham P-- Jahic, Amir P-- Janda, Joachim P--/ FV- Jiang, Shihai P-- Joly, Philippe P-- Junker, Ralf P--/ FV-, P--, P-- Kabrodt, Kathrin P-- Kacprowski, Tim P-- Kaiser, Thorsten P-- Kappert, Günther P-- Kappert, Kai P-- Karakuyu, Aleyna P-- Keil, Norbert P-- Keller, Thomas P-- Keller, Ulrich P-- Kertzscher, Ulrich P-- Khan, Essak P-- Khan, Essak .S P-- Khawaja, Hamzah P--, P--/ PV- Kiehntopf, Michael P--, P--/ PV- Kießling, Peggy P-- Kihm, Lars P-- Kirchhoff, Jan P--, P--/ FV- Kittel, Maximilian P--, P-- Klawitter, Sandra P-- Klawonn, Frank P--, P-- Kleine, Anika P--, P--, P--/ PV- Kleinert, Moritz P-- Kliemank, Elisabeth P-- Knabbe, Cornelius P--, P--, P--, P--, P--, P--, P--, P--, P--/ PV-P-- (continued) eA86 German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 (continued) Name, Vorname Paper Codes Koerber, Ruth-Miriam P-- Kohli, Shrey P--, P--, P--, P--/ PV- Kolevica, Ana P-- Kramer, Achim P-- Krätzner, Ralph P-- Kratzsch, Juergen P--/ FV- Kraus, Max-Joseph P-- Krause, Daniela S. P-- Kreutzig, Berit P-- Krieger, David P-- Krishnan, Shruthi P--, P--, P--/ PV- Krnac, Dusan P-- Krolik, Michal P-- Kuhn, Joachim P--, P-- Kunze-Szikszay, Nils P-- Kutschker, Christoph P-- Kyeong-Hee, Lee P-- Lackner, Karl J. P-- Lange, Phil P--/ PV- Lasch, Daniel P-- Laufs, Ulrich P--, P-- Lbik, Dawid P-- Leha, Andreas P-- Lehmann, Rainer P-- Lehmann, Thomas P--/ PV- Leypoldt, Frank P--, P-- Liebig, Sven P-- Lindenkamp, Christopher P--, P--, P-- Link, Manuela P-- Loges, Sonja P-- Luppa, Peter P-- Luppa, Peter B. P-- Ly, Thanh-Diep P--, P--, P--/ PV- Machann, Jürgen P--, P--/ PV- Mangova, Gyulten P-- Manoharan, Jayakumar P--, P-- Marini, Irene P--, P-- Markewitz, Robert P--, P--/ FV- Markovic, Ivana P--, P--, P-- Markus, Marcello R.P. P-- Martens, Uwe P-- Marx, Alexander P-- Mathew, Akash P--, P--, P-- Matysik, Silke P-- Merz, Immanuel P-- Methew, Akash P--/ PV- Meyer, Anne P-- Michaelis, Julia P-- Mieritz, Anne P-- Mihov, Klara P-- Mirbach, Laura P--, P--, P--, P--/ FV- (continued) Name, Vorname Paper Codes Müller, Daniel P-- Müller, Robert P-- Müller-Calleja, Nadine P-- Mütze, Ulrike P--/ FV- Mykhailiuk, Kristina P-- Nauck, Matthias P--, P--, P--, P--/ FV-, P--, P-- Naumann, Michael P-- Neeb, Heiko P-- Nennstiel, Uta EA- Neubert, Antje P-- Neugebauer, Sophie P--/ PV-, P-- Neumaier, Michael P--, P--, P--, P-- Neumann, Konstantin P--/ PV- Neumann, Sascha P--/ FV- Noppes, Katharina P-- Nourse, Jamie P--, P-- Nowak-Harnau, Stefanie P--/ FV- Oemüller, Uwe EA- Ohmes, Justus Maximilian P-- Okun, Jürgen G. P--/ FV- Orth, Matthias P-- Ostendorf, Norbert P-- Osterhage, Michel Robin P--, P--, P-- Paal, Michael P-- Panagiotou, Gianni P-- Parulan Holzhueter, Evadne P-- Paulheim, Heiko P-- Pechermeyer, Detlef P--/ FV- Peitzsch, Mirko P-- Pelzl, Lisann P--, P-- Peter, Andreas P--, P--, P--, P--/ PV- Peter, Antonia Sophia P-- Petersmann, Astrid P--, P--, P--, P--, P--, P--/ FV-, FV- Plepi, Joan P-- Pleus, Stefan P--, P-- Plümers, Ricarda P--, P--, P-- Praktiknjo, Michael P-- Prantz, Anja P--, P-- Prokosch, Hans-Ulrich P--/ FV- Prpic, Monika P-- Puls, Miriam P-- Rahbari, Nuh P-- Ramallo Guevara, Carina P-- Rana, Rajiv P-- Rank, Christopher P--/ FV- Rauh, Manfred P--, P--, P--/ FV- Reck, Jakob P-- Regensburger, Adrian P-- Reinicke, Madlen P-- Relker, Lasse P--/ PV- German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 eA87 German Congress for Laboratory Medicine, Mannheim, October 13–14, 2022 eA87 eA87 (continued) Name, Vorname Paper Codes Risch, Martin P--, P--, P-- Roditscheff, Anna P-- Rolinski, Boris P--, P-- Rosenkranz, Daniel P--, P--, P--/ FV-, FV- Sender, Martina EA- Schäfer, Sarah P--/ FV- Schäffer, Tilman E. P-- Schaible, Samuel P-- Schanz, Julie P-- Schauer, Sebastian P-- Schellenberg, Ingo P--, P--, P-- Schick, Fritz P--/ PV- Schlack, Katrin P-- Schleicher, Erwin EA- Schmidt, Maria P-- Schmidt, Stefan P-- Schmidt, Vanessa P--, P--/ PV-, P-- Schmidt-Heck, Wolfgang P-- Schneider, Franziska P-- Schnelle, Moritz P--, P--, P--, P-- Schnitzler, Sebastian Uwe P-- Scholand, Sören P-- Scholz, Markus P-- Schönberg, Stefan P-- Schöpfel, Juliane P-- Schützenmeister, André P--/ FV- Schwartz, Stefan P-- Seger, Christoph P-- Semjonow, Axel P-- Shahzad, Khurrum P--, P--,P--, P--/ PV- Shaverskyi, Anton P-- Siebolts, Udo P-- Siekmeier, Rüdiger P-- Simon, David P-- Singh, Anurag P-- Singh, Kunal P--, P--, P--/ PV- Skevaki, Chrysanthi P--, P-- Stefan, Norbert P--, P--/ PV- Steﬁ, Jonathan P-- Stoffel-Wagner, Birgit P--, P-- Strasser, Erwin P-- Streichert, Thomas EA- Streit, Frank P--, P--, EA- Stumpe, Florian P-- Stürner, Klarissa Hanja P-- Szendrödi, Julia P-- Tamamushi, Yoko P-- Teichmann, Lino P-- Tesorero, Rafael P--/ FV- Teufel, Andreas P-- Thaler, Markus P--, EA- (continued) Name, Vorname Paper Codes Thevis, Mario P-- Thiel, Manfred P-- Toischer, Karl P-- Tolios, Alexander P--, EA- Toll, Luisa P-- Tönjes, Anke P-- Trauth, Janina P-- Überla, Klaus P-- Uzun, Günalp FV- Verlage, Sarah P-- Vierbaum, Laura P--, P--, P-- Vogeser, Michael P-- Voitz, Thomas P-- Vollmer, Anne-Kathrin P-- Völzke, Henry P-- von Bargen, Katharina P--, P--, P-- Wachter, Felix P-- Wachter, Rolf P-- Wagner, Alexandra P-- Wagner, Robert P--/ PV- Waldenmaier, Delia P-- Wallbach, Manuel P-- Wandinger, Klaus-Peter P--, P--, P--/ FV- Wang, Qingqing P-- Wanner, Yvonne FV- Weber, Matthias P--, P-- Weber, Stephan P-- Weber, Susanne P-- Wehrli, Faina P-- Weideli, Ornella C. P-- Weis, Cleo P-- Wellmann, Axel P-- Wenzel, Folker P-- Wessig, Anne Kathrin P--/ PV- Wiederhold, Mechthild P-- Winning, Johannes P--/ PV- Wittekind, Dirk P--, P-- Wohlwend, Nadia P--, P-- Wohlwend, Niklas P-- Wojtalewicz, Nathalie P--, P--, P-- Wölﬂe, Joachim P-- Wolny, Monika P--, P--, P-- Xu, Guowang P-- Younis, Ruaa P-- Zechmeister, Bozena P--, P-- Zeidler, Robert P--/ FV- Zeisberg, Elisabeth P-- Zemva, Johanna P-- Zierk, Jakob P--/ FV- Zimmermann, Silke P--, P--, P-- Zlamal, Jan P-- Zorn, Markus P-- Zylla, Stephanie P--
W2160452077.txt	https://molecular-cancer.biomedcentral.com/counter/pdf/10.1186/1476-4598-8-38	en		Equivalent benefit of mTORC1 blockade and combined PI3K-mTOR blockade in a mouse model of tuberous sclerosis	Molecular cancer	2,009	cc-by	5,352	Molecular Cancer BioMed Central Open Access Research Equivalent benefit of mTORC1 blockade and combined PI3K-mTOR blockade in a mouse model of tuberous sclerosis Kristen Pollizzi1, Izabela Malinowska-Kolodziej1, Michael Stumm2, Heidi Lane2 and David Kwiatkowski1 Address: 1Translational Medicine Division, Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts 02115, USA and 2Novartis Institutes For BioMedical Research, Oncology Basel, Novartis Pharma AG, Switzerland Email: Kristen Pollizzi - pollizzi@gmail.com; Izabela Malinowska-Kolodziej - imalinowska@rics.bwh.harvard.edu; Michael Stumm - michael.stumm@roche.com; Heidi Lane - Heidi.Lane@basilea.com; David Kwiatkowski - dk@rics.bwh.harvard.edu * Corresponding author Published: 15 June 2009 Molecular Cancer 2009, 8:38 doi:10.1186/1476-4598-8-38 Received: 23 January 2009 Accepted: 15 June 2009 This article is available from: http://www.molecular-cancer.com/content/8/1/38 © 2009 Pollizzi et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: Tuberous sclerosis (TSC) is a hamartoma syndrome in which renal and lung tumors cause the greatest morbidity. Loss of either TSC1 or TSC2 in TSC hamartomas leads to activation of mTORC1 and suppression of AKT. Recent studies indicate that inhibition of mTORC1 with RAD001 (everolimus) leads to rebound activation of AKT, which could protect tumors from druginduced cell death. Here we examine the potential benefit of inhibition of both mTOR and AKT signaling in a mouse model of TSC, using a dual pan class I PI3K/mTOR catalytic small molecule inhibitor NVP-BEZ235. Results: Using ENU to enhance Tsc2+- kidney tumor development, both RAD001 (10 mg/kg PO 5 d/week) and NVP-BEZ235 (45 mg/kg PO QD) had equivalent effects in suppressing tumor development during a 4 week treatment period, with a 99% reduction in tumor cell mass. Marked reduction in activation of mTORC1, induction of cell cycle arrest, and absence of apoptotic cell death was seen in mice treated with either drug. However, when either was discontinued, there was prompt recovery of tumor growth, with extensive proliferation. Conclusion: Both mTORC1 blockade alone and combined PI3K-mTOR blockade lead to suppression of tumor development but not tumor elimination in this TSC model. Background Tuberous sclerosis (TSC) is an autosomal dominant tumor suppressor gene syndrome, in which involvement of the brain, kidneys, and lungs cause the greatest clinical problems [1]. Seizures, mental retardation, developmental delay, and autistic features are common during childhood in these patients, and in many patients these clinical issues persist into adulthood. However, after puberty, additional major clinical problems in TSC are the progres- sive development of renal angiomyolipoma (AML) and pulmonary lymphangioleiomyomatosis (LAM) [2,3]. Both of these lesions are made up of unusual smooth muscle-like and other cells which express both melanosomal markers and VEGF-D. Although progression of these lesions is quite variable from patient to patient, loss of renal function due to either progressive growth of AMLs and/or conversion into a malignant renal tumor is seen in about 5% of TSC patients. Pulmonary LAM is seen nearly Page 1 of 9 (page number not for citation purposes) Molecular Cancer 2009, 8:38 exclusively in female TSC patients, and leads to progressive respiratory limitation and failure also in about 5%. LAM is also seen independent of the TSC syndrome, typically in a more severe and progressive form. Tsc mouse models have been generated consisting of both knock out and conditional alleles of Tsc1 and Tsc2 [4,5]. None of these replicates the typical features of AML/LAM, though renal epithelial tumors are a consistent development in both Tsc1+- and Tsc2+- mice, which progress over a period of many months from pure cysts to papillary adenomas to renal carcinoma. The TSC1 and TSC2 proteins form a tight complex, which functions in a conserved signaling pathway to regulate the kinase activity of mTORC1, through regulation of the state of GTP loading of Rheb [6,7]. Several studies have shown the benefit of rapamycin and other mTORC1 inhibitors in the treatment and prevention of renal tumors in Tsc mouse models [8,9]. Loss of TSC1/TSC2 in TSC hamartomas leads to both activation of mTORC1, as well as feedback inhibition of AKT, through downregulation of IRS and PDGFR expression and other mechanisms [10,11]. This has led to speculation that treatment of TSC hamartomas with mTORC1 inhibitors might lead to restoration of AKT activation, as seen in vitro with treatment of TSC1/TSC2 null cells, and in some patients with malignant disease [12], which may compromise clinical benefit. Here, we explore the potential benefit of the mTORC1 inhibitor RAD001 (everolimus) in comparison to a dual pan-class I PI3K/ mTOR catalytic inhibitor NVP-BEZ-235 [13] in the therapy of Tsc2 mouse kidney tumors. Methods Mouse procedures Tsc2+- mice, originally generated in this laboratory [4], were serially crossed with C57BL/6J mice for over 5 generations, and were then mated with pure 129S1/SvImJ mice to generate Tsc2+- mixed strain C57BL/6J:129S1/SvImJ mice. These mixed strain mice were used in all experiments. All procedures were carried out in accordance with the Guide for the Humane Use and Care of Laboratory Animals, and the study was approved by the Animal Care and Use Committee of Children's Hospital, Boston. Nethyl-N-nitrosourea (ENU, Sigma-Aldrich) was prepared in ethanol at 200 mg/ml, diluted in phosphate-citrate buffer, and administered by intraperitoneal (IP) injection at 60 mg/kg. Standard histology and tumor assessment Standard histology sections were prepared from mouse kidneys after 10% formalin fixation and cutting into five 1–2 mm sections. Both gross and microscopic kidney http://www.molecular-cancer.com/content/8/1/38 pathology was read by a blinded observer (KP) and scored according to a modification of a formula used previously [14]. The kidney tumor score for kidney cystadenomas was determined as a summed score for all lesions in a kidney, scoring each individual tumor grossly as follows: 1 for tumors <1 mm; 2 for 1 to 1.5 mm; 5 for 1.5 to 2 mm; 10 for > 2 mm. Microscopic kidney tumor scores were determined similarly, except that the score for each lesion was multiplied by 2 if the tumor had a papillary component, and by 4 if it was a solid adenoma. The percent cellularity of cystadenomas was determined as the percent of the tumor that contained proliferating cells as opposed to cyst cavity; pure cysts had a score of 0% cellularity while solid adenomas had a score of 100% cellularity. Comparison between sets of mice for tumor measurements were made using the non-parametric Kruskal-Wallis test. Antibodies and immunohistochemistry Antibodies used were: pS6(S235/236) clone 91B2, active caspase-3 clone 5A1 from Cell Signaling Technology, Bedford, MA; pS6(S240) clone DAK-S6-240 M7300, pAKT(S473) clone 14–5, Ki-67 clone TEC-3 M7249, and pMAPK(pTpY202/4) clone 24-2-2 from DAKO S/A, Denmark; PCNA (PC10) from Santa Cruz Biotechnology. For immunohistochemistry, kidneys were rapidly removed, sliced in thirds and fixed overnight at 4°C in 10% formalin. Paraffin sections were cut and stained by the immunoperoxidase technique, following standard methods of deparaffinization, antigen retrieval using Dako Target Retrieval solution, DAB incubation, and counterstaining with hematoxylin. The percent Ki-67 or PCNA labeling, or percent of cells expressing active caspase 3, within a lesion was determined by direct counting of at least 300 cells by a blinded observer (IM). Drug handling and administration RAD001 was provided by Novartis in a proprietary vehicle at 20 mg/ml. Prior to each administration, RAD001 was diluted in water to 0.5–2 mg/ml, and was given at 10 mg/ kg by gavage every day 5 days per week. NVP-BEZ235 was provided by Novartis as a powder, and was mixed in 10% 1-Methyl-2-pyrrolidone and 90% PEG-300 at 8 mg/ml. It was prepared fresh prior to each administration, and was given at either 15 mg/kg or 45 mg/kg by gavage every day. In the first treatment cohort, mice were treated with placebo (10% 1-Methyl-2-pyrrolidone and 90% PEG-300) by gavage 5 days per week. This was not done in subsequent cohorts. Results ENU acts as a carcinogen to enhance renal tumor growth in Tsc2+- mice Kidney tumor growth in Tsc2+- mice follows a variable though predictable pattern with an effect of strain on tumor severity [4], (unpublished). In most strains, age 12 Page 2 of 9 (page number not for citation purposes) Molecular Cancer 2009, 8:38 months is the earliest at which significant kidney tumors are seen. To accelerate the rate of development of renal tumors, we treated Tsc2+- mice with ENU, an alkylating agent which causes point mutations. We explored the effects of administration of a single IP dose of ENU at several different times during mouse development, evaluating tumor severity at 6 months of age [see Additional file 1]. To minimize effects of genetic background, we studied mice that were heterozygous for the C57BL/6J and 129S1/ SvImJ strains. We found that ENU given at ages from E13 to P21 (administered to the pregnant dam in the first case) was effective at increasing the incidence and severity of kidney cystadenomas in the Tsc2+- mice, as assessed by gross evaluation [see Additional file 1]. ENU treatment at these ages in wild type mice of the same strain mix led to rare renal tumors (e.g. only one < 1 mm tumor in 10 mice treated with ENU at P9) at age 6 months. Microscopic assessment of the ENU-treated Tsc2+- mice confirmed this enhancement in tumor development (data not shown). Administration of ENU at any of P9, E19, or E13 appeared to have similar outcomes, with nearly identical kidney tumor scores (see Methods for details) [see Additional file 1]. We chose to use P9 administration for simplicity in the following studies. Evaluation of RAD001 alone and in combination with NVP-BEZ235 in the ENU-accelerated Tsc2+- kidney tumor model We explored the potential benefit of mTORC1 inhibition with RAD001 in the ENU-accelerated Tsc2+- kidney tumor model. RAD001 was highly effective in reducing the gross tumor score, microscopic tumor score, and percent solid tumor (Figure 1A–C) in these mice, after a 4-week period of treatment at 10 mg PO QD 5 days out of seven each week, beginning at age 20 weeks. Combining the reduction in overall tumor size with reduction in cellularity indicates that there was an approximate 99% reduction in tumor development. In addition, the residual lesions seen in the RAD001-treated mice generally had a flattened epithelium, in contrast to the enlarged columnar-like epithelial cells seen in untreated mice [see Additional file 2]. We also examined the acute effects of treatment with RAD001 in this model. Tumor analysis 3–5 days after initiation of therapy demonstrated that RAD001 markedly reduced expression of pS6(S240) and pS6(S235/236), consistent with mTORC1 blockade [see Additional file 3]. In addition, the Ki-67 labeling index in the short-term treated tumors was reduced from an average of 6% to an average of 1% (Figure 1D) [see Additional file 3]. However, there was no indication of induction of apoptosis or necrosis in the tumors, as expression of activated caspase-3 was very low in the treated tumors (0–1% cells) similar to that seen in untreated tumors [see Additional file 4A] (in contrast to control nude mouse tumor xenograft samples, data not shown). In addition, there was no consistent effect on MAPK signaling, as assessed by staining for http://www.molecular-cancer.com/content/8/1/38 pMAPK(pTpY202/4) (data not shown), in the treated tumors. However, RAD001 treatment caused an increase in pAKT(S473) levels in the tumors, which were very low in tumors from untreated mice [see Additional file 5]. Total S6 and AKT protein levels were similar in normal kidney and in the tumors, and did not appear to change significantly with treatment with either compound [see Additional file 5]. Four week treatment with RAD001 also did not lead to significant apoptosis in these kidney tumors, though it did cause continued suppression of proliferation [see Additional file 4]. Simultaneously, we evaluated the potential benefit of combining RAD001 inhibition of mTORC1 with PI3KmTOR inhibition using NVP-BEZ235. NVP-BEZ235 was also given alone (without RAD001), as a control, and at relatively low dosage (15 mg/kg PO QD) appeared to have significant therapeutic effect in the ENU-treated Tsc2+- mice (Figure 1A–C). Improvement was seen in both gross and microscopic kidney tumor scores, with a more modest change in tumor cellularity. These observations are probably due to the activity of NVP-BEZ235 as a direct mTOR inhibitor, affecting both mTORC1 and mTORC2, in addition to its PI3K inhibition activity [13]. Consistent with this effect, NVP-BEZ235 inhibits phosphorylation of S6 at the S235/236 sites in Tsc2 null murine embryo fibroblast cell lines at 10–100 nM, and has a potent anti-proliferative effect on these cells with an IC50 of 3 nM [15]. Comparison of RAD001 and NVP-BEZ235 as therapy for the ENU-accelerated Tsc2+- kidney tumor model Since NVP-BEZ235 had effects in inhibiting mTOR, and at low doses could reduce tumor development in this model, we treated a cohort of ENU-treated Tsc2+- mice with NVP-BEZ235 at full dosage, 45 mg/kg PO QD [13], and compared outcome with RAD001 treatment. NVPBEZ235 had similar effects to RAD001 in reducing both gross and microscopic kidney tumor scores by about 80%, with most residual lesions being simple cysts (Figure 2A– C). Tumor cellularity also appeared reduced in general, though the presence of a single solid adenoma in an NVPBEZ235-treated mouse ran against this trend. Tumor cell size was reduced in NVP-BEZ235-treated mice [see Additional file 2]. In short term as well as 4 week treatment trials, NVP-BEZ235 stopped cell proliferation, with complete loss of Ki-67 or PCNA staining in the treated tumors (Figure 2D) [see Additional files 3, 4]. Similar to RAD001, NVP-BEZ235 did not appear to cause apoptosis in the tumor cells [see Additional file 4A]. pS6(S240) and pS6(S235/236) expression was markedly reduced in the 5 day NVP-BEZ235-treated mouse kidney tumors [see Additional file 3]. pAKT(S473) levels were low in the NVPBEZ235-treated mouse tumors, similar to untreated mice, but in contrast to RAD001-treated mice [see Additional file 5]. There was no statistically significant difference Page 3 of 9 (page number not for citation purposes) Molecular Cancer 2009, 8:38 http://www.molecular-cancer.com/content/8/1/38 +model Figure 1 Renal tumor development blocked by administration of RAD001 with or without NVP-BEZ235 in the ENU-treated Renal tumor development blocked by administration of RAD001 with or without NVP-BEZ235 in the ENUtreated Tsc2+- model. A-C. Dot plot analysis of gross kidney tumor score (A), microscopic kidney tumor score (B), and per+- mice at age 24 weeks that received either 1) placebo, 2) RAD001 10 cent cellularity of each lesion (C) in ENU-treated mg/kg PO 5 d/week, 3) NVP-BEZ235 15 mg/kg PO QD, or 4) a combination of RAD001 and NVP-BEZ235 at the same dosage; all for four weeks from age 20 – 24 weeks. A, B: Each dot represents a mouse kidney. C: Each dot represents a mouse kidney cystadenoma. A, p < .001; B, p = .03; C, p NS; by Kruskal-Wallis test. D. Dot plot analysis of percent Ki-67+ cells in kidney tumors of mice with genotype and treatment as in A-C, but that were analyzed 3 or 5 days after initiation of treatment with RAD001 10 mg/kg PO QD at age 24 weeks. Each dot represents a mouse kidney cystadenoma. p < .0001 Kruskal-Wallis test. between the kidney tumor scores or cellularity of the tumors seen in these mice after RAD001 or NVP-BEZ235 treatment. Comparison of RAD001 and NVP-BEZ235 as therapy for the ENU-accelerated Tsc2+- kidney tumor model with long-term follow-up Since RAD001 and NVP-BEZ235 had similar effects in arresting the growth of the kidney tumor epithelial cells during a four week period of treatment, we asked whether one or the other treatment might be more beneficial in terms of lasting effects on tumor growth in mice treated transiently. To explore this question, mice were treated with either drug for a period of 4 weeks, age 20 – 24 weeks, and then were taken off drug for 8 weeks and sacrificed for examination. Kidney tumors in mice treated with either drug showed robust growth with development of relatively large papillary and solid tumors, and rePage 4 of 9 (page number not for citation purposes) Molecular Cancer 2009, 8:38 http://www.molecular-cancer.com/content/8/1/38 +- model Figure 2 Renal tumor development blocked by administration of either RAD001 or NVP-BEZ235 in the ENU-treated Renal tumor development blocked by administration of either RAD001 or NVP-BEZ235 in the ENU-treated Tsc2+- model. A-C. Dot plot analysis of gross kidney tumor score (A), microscopic kidney tumor score (B), and percent cellu+- mice at age 24 weeks that received either 1) no treatment, 2) RAD001 10 mg/kg larity of each lesion (C) in ENU-treated PO 5 d/week, or 3) NVP-BEZ235 45 mg/kg PO QD; all for four weeks from age 20 – 24 weeks. A, B: Each dot represents a mouse kidney. C: Each dot represents a mouse kidney cystadenoma. A, p < .001; B, p = .03; C, p NS; by Kruskal-Wallis test. D. Dot plot analysis of percent Ki-67+ cells in kidney tumors of mice with genotype and treatment as in A-C, but that were analyzed 3 or 5 days after initiation of treatment with NVP-BEZ235 45 mg/kg PO QD at age 24 weeks. Each dot represents a mouse kidney cystadenoma. p < .0001 Kruskal-Wallis test. expression of PCNA [see Additional file 4B]. Gross tumor scores were significantly reduced in mice treated with either drug in comparison to never-treated mice; however, there was no significant difference in microscopic tumor scores or percent cellularity (Figure 3) [see Additional file 2]. Gross and microscopic kidney tumor scores, percent papillary and solid tumors, and general histologic characteristics of the tumors in these mice did not differ according to the drug treatment received. Thus, both RAD001 and NVP-BEZ235 had major effects on tumor growth during the treatment period, but resumption of brisk tumor growth occurred upon cessation of treatment. Page 5 of 9 (page number not for citation purposes) Molecular Cancer 2009, 8:38 Discussion Tuberous sclerosis affects an estimated 40,000 individuals in the United States, and about 10% of these individuals are projected to sustain significant morbidity and often mortality due to development and progression of renal AML, pulmonary LAM, other less common tumors of the http://www.molecular-cancer.com/content/8/1/38 retroperitoneum (lymphangioma) or liver (angiomyolipoma), PEComas arising in various sites, and neuroendocrine tumors [1]. The major understanding of the function of the TSC1/TSC2 protein complex as a critical regulator of the Rheb GTPase and thereby mTORC1 activity in recent years has led to considerable enthusiasm about the Figuretumor Renal 3 recurrence in mice treated with either RAD001 or NVP-BEZ235 8 weeks after coming off drug Renal tumor recurrence in mice treated with either RAD001 or NVP-BEZ235 8 weeks after coming off drug. A-C. Dot plot analysis of gross kidney tumor score (A), microscopic kidney tumor score (B), and percent cellularity of each +- mice at age 32 weeks that received either 1) no treatment, 2) RAD001 10 mg/kg PO 5 d/ lesion (C) in ENU-treated week, or 3) NVP-BEZ235 45 mg/kg PO QD; all for four weeks from age 20 – 24 weeks. A, B: Each dot represents a mouse kidney. C: Each dot represents a mouse kidney cystadenoma. A, p = .002; B and C, p NS; by Kruskal-Wallis test. Page 6 of 9 (page number not for citation purposes) Molecular Cancer 2009, 8:38 potential efficiency of rapamycin and other mTORC1 inhibitors as therapy for these TSC tumors [7]. Two phase I-II trials of rapamycin therapy for TSC renal AML and/or pulmonary LAM have been published [16,17]. The larger trial reported that among the 80% patients staying on drug, those with renal AML responded to rapamycin with an average 47% reduction in tumor volume. Although this was gratifying, the reduction in size was largely reversed, returning to 86% of starting volume on average, when follow-up for a year off drug was performed. Since no biopsies were performed in these patients, there is a lack of understanding of precisely what happened to these tumors both when the patients were treated and when the drug was stopped. Here we demonstrate, similar to previous reports using rapamycin [8,9], that RAD001 is highly effective in suppressing the growth of Tsc mouse kidney cystadenomas, with an average 99% reduction in tumor cell burden in this ENU-accelerated Tsc2+- model. In addition, we demonstrate that there is very effective suppression of cell growth within these lesions, as assessed by reduction in Ki-67 positivity, with a lack of apoptosis or tumor cell death. Pathway inhibition with reduction in markers of mTORC1 activation, pS6(S240) and pS6(S235/236), was also seen, consistent with the expected mechanism of action of the drug. However, marked tumor regrowth occurred by 8 weeks after discontinuation of RAD001, though some persistent reduction in overall tumor burden could be appreciated in comparison to never-treated mice (Figure 3A). We find these observations to be in striking parallel to those made in AML patients, taking into account the markedly different growth rates of renal AMLs in patients in comparison to renal cystadenomas in this model. They suggest that rapamycin/RAD001 has a cytostatic effect, effectively blocking the growth of the renal AMLs and likely reducing cell size, but without tumor cell death. Upon drug removal, there is regrowth of tumors. Further, they suggest the possibility that continued mTORC1 inhibition may have benefit for maintaining growth suppression of these tumors. It is well-recognized that the mTOR signaling pathway in which TSC1/TSC2 participate is more complex than first thought, with a number of feedback inhibitory effects of loss of the TSC protein complex on AKT activation [10,11,18]. In addition, the related mTORC2 complex has a major if not sole role in phosphorylating and enhancing activation of AKT at Serine 473 [19]. In most tumor cell lines, including Tsc1 null and Tsc2 null MEF cell lines, treatment with rapamycin or RAD001 leads to phosphorylation of AKT at the S473 site, and increased activation [11,12,20]. There has been concern that this effect could mitigate any positive treatment effect of mTORC1 inhibitors on tumor growth. Here we assessed the possibility http://www.molecular-cancer.com/content/8/1/38 that combined PI3K-AKT-mTOR blockade with NVPBEZ235 could lead to a better therapeutic outcome in this Tsc2+- kidney tumor model. We found that the short-term effects of NVP-BEZ235 were similar to those of RAD001 with a major reduction in cell proliferation, lack of apoptosis or cell death, and reduction in markers of mTORC1 activation. NVP-BEZ235 has PI3K inhibitory activity at low nM levels in vitro for all PI3Kα including mutant forms, and has been shown to reduce pAKT(S473) levels in xenograft models [13]. As expected, pAKT(S473) levels were low in the kidney tumors from untreated Tsc2+- mice, and were increased by treatment with RAD001, but not NVP-BEZ235. In addition, in vitro studies demonstrate that in full serum pAKT(S473) levels are low in Tsc2 null MEF lines, are increased somewhat with RAD001 treatment, and reduced somewhat by NVP-BEZ235 treatment [15]. Despite the short-term effects of treatment with NVP-BEZ235, we found that in both the 4 week course of drug, and 4-week course with 8-week off drug follow-up, that RAD001 and NVP-BEZ235 had indistinguishable effects, with marked regrowth of tumor following treatment cessation. Thus, these observations suggest that the reactivation of mTORC1 in TSC-related neoplasms that might occur with rapamycin/RAD001 treatment has no significant clinical effect, at least in this Tsc model tumor. Conclusion We have conducted a trial of the pure mTORC1 inhibitor RAD001 and the combined PI3K/mTOR inhibitor NVPBEZ235 in a mouse model of TSC in which the mice develop renal cystadenomas. Both drugs were highly effective at tumor growth suppression, and there was no difference between combined PI3K-mTOR blockade in comparison to mTORC1 inhibition alone. When treatment was discontinued, rapid tumor regrowth was seen after each drug. In this model, both drugs appear to have a primarily cytostatic effect. Abbreviations (TSC): Tuberous sclerosis; (AML): angiomyolipoma; (LAM): lymphangioleiomyomatosis; (ENU): N-ethyl-Nnitrosourea. Competing interests This work was partially supported by Novartis Pharma AG. Authors' contributions KP performed most of the experimentation in this project, including mouse treatment, pathology preparation, and tumor scoring. MS and IM performed the immunohistochemical studies and their interpretation. HL assisted in the conception of the study and monitoring during its execution. DJK conceived of the study, monitored the mice and treatment, and drafted the manuscript. All authors Page 7 of 9 (page number not for citation purposes) Molecular Cancer 2009, 8:38 http://www.molecular-cancer.com/content/8/1/38 contributed to the editing of the manuscript, and approved the final version. Additional file 5 Signaling pathway analysis by IHC in Tsc2+-mice. A single representative image is shown for untreated mice (top row), mice treated for 5 days with RAD001 10 mg/kg PO (middle row), and mice treated for 5 days with NVP-BEZ235 45 mg/kg PO (bottom row). All mice were ENUtreated Tsc2+- mice of age 24 weeks. Columns are: pS6(S235/236); S6 (total); pAKT(S473); AKT (total). Adjacent sections from each tumor are shown. All images are taken at 100×. Click here for file [http://www.biomedcentral.com/content/supplementary/14764598-8-38-S5.pdf] Additional material Additional file 1 Effects of ENU on kidney tumor development in Tsc2+- mice. A dot plot graph is shown of the gross kidney tumor scores in Tsc2+- mice at 6 months of age. E13, E19, P9, P21 indicates different ages of treatment with a single dose of ENU at 60 mg/kg IP. Each dot represents a single mouse. The differences among the 5 groups are statistically significant at p = 0.02; for the four ENU treatment groups, p = 0.035; for the three ENU treatment groups of E13, E19, and P9, p is not significant (0.79); all done by the Kruskall-Wallis test. Click here for file [http://www.biomedcentral.com/content/supplementary/14764598-8-38-S1.pdf] Additional file 2 Morphology of RAD001- and NVP-BEZ235-treated Tsc2+- kidney lesions. Multiple tumor H&E stained images are shown from ENUtreated Tsc2+- kidneys. All scale bars are 100 microns. Each image is taken from a different mouse. Light black lines delineate tumor (T) or cyst (C) vs. normal kidney (N). Row A- Mice of age 24 weeks; inset shows hyperplastic columnar-type epithelium lining the cyst; Row B- Mice of age 24 weeks that were treated with RAD001 5 days per week, from age 20– 24 weeks; inset shows flat epithelium lining the cyst: Row C- Mice of age 24 weeks that were treated with NVP-BEZ235 45 mg/kg daily from age 20–24 weeks; Row D- Mice of age 32 weeks; Row E- Mice of age 32 weeks that were treated with RAD001 5 days per week, from age 20–24 weeks; Row F- Mice of age 32 weeks that were treated with NVP-BEZ235 45 mg/ kg daily from age 20–24 weeks. Click here for file [http://www.biomedcentral.com/content/supplementary/14764598-8-38-S2.pdf] Acknowledgements For expert histotechnical assistance we would like to thank Maja R. Walker and Noelle Hanoteau. This work was supported by NIH/NCI 1P01CA120964, and Novartis Pharma AG. Neither funding source had any role in study design, data collection and interpretation, writing of the manuscript, or decision as to where to submit it for publication. References 1. 2. 3. 4. 5. 6. Additional file 3 Signaling pathway and growth analysis by IHC in Tsc2+- mice. A single representative image is shown for untreated mice (top row), mice treated for 5 days with RAD001 10 mg/kg PO (middle row), and mice treated for 5 days with NVP-BEZ235 45 mg/kg PO (bottom row). All mice were ENU-treated Tsc2+- mice of age 24 weeks. Columns are: pS6(S240), pS6(S235/236), Ki-67. Adjacent sections from each tumor are shown. All images are taken at 100×. Note that the third row has a background pink/orange shade, which is present in all cells on this section, and distinct from the dark brown stain seen in the top row. Click here for file [http://www.biomedcentral.com/content/supplementary/14764598-8-38-S3.pdf] 7. 8. 9. 10. 11. Additional file 4 Apoptosis and proliferation in Tsc2+- mice. A. The percent cleaved caspase 3 positive cells are shown for Tsc2+- mice, treated for either 5 days ('SHORT') or for 4 weeks ('CHRONIC'). Each dot represents a different renal cystadenoma for which the percent positive cells was determined by a blinded observer (IM). B. The percent PCNA positive cells are shown for Tsc2+- mice, treated with either RAD001 or NVP-BEZ235, or treated with these drugs and then taken off drug for 8 weeks ('ON/OFF'). Each dot represents a different renal cystadenoma. Click here for file [http://www.biomedcentral.com/content/supplementary/14764598-8-38-S4.pdf] 12. 13. 14. Crino PB, Nathanson KL, Henske EP: The tuberous sclerosis complex. N Engl J Med 2006, 355:1345-56. Bissler JJ, Kingswood JC: Renal angiomyolipomata. Kidney Int 2004, 66:924-34. McCormack FX: Lymphangioleiomyomatosis: a clinical update. Chest 2008, 133:507-16. Onda H, Lueck A, Marks PW, Warren HB, Kwiatkowski DJ: Tsc2(+/ -) mice develop tumors in multiple sites that express gelsolin and are influenced by genetic background. J Clin Invest 1999, 104:687-95. Kwiatkowski DJ, Zhang H, Bandura JL, Heiberger KM, Glogauer M, elHashemite N, Onda H: A mouse model of TSC1 reveals sexdependent lethality from liver hemangiomas, and up-regulation of p70S6 kinase activity in Tsc1 null cells. Hum Mol Genet 2002, 11:525-34. Inoki K, Corradetti MN, Guan KL: Dysregulation of the TSCmTOR pathway in human disease. Nat Genet 2005, 37:19-24. Guertin DA, Sabatini DM: Defining the role of mTOR in cancer. Cancer Cell 2007, 12:9-22. Kenerson H, Dundon TA, Yeung RS: Effects of rapamycin in the Eker rat model of tuberous sclerosis complex. Pediatr Res 2005, 57:67-75. Lee L, Sudentas P, Donohue B, Asrican K, Worku A, Walker V, Sun Y, Schmidt K, Albert MS, El-Hashemite N, Lader AS, Onda H, Zhang H, Kwiatkowski DJ, Dabora SL: Efficacy of a rapamycin analog (CCI-779) and IFN-gamma in tuberous sclerosis mouse models. Genes Chromosomes Cancer 2005, 42:213-27. Harrington LS, Findlay GM, Gray A, Tolkacheva T, Wigfield S, Rebholz H, Barnett J, Leslie NR, Cheng S, Shepherd PR, Gout I, Downes CP, Lamb RF: The TSC1-2 tumor suppressor controls insulin-PI3K signaling via regulation of IRS proteins. J Cell Biol 2004, 166:213-23. Zhang H, Cicchetti G, Onda H, Koon HB, Asrican K, Bajraszewski N, Vazquez F, Carpenter CL, Kwiatkowski DJ: Loss of Tsc1/Tsc2 activates mTOR and disrupts PI3K-Akt signaling through downregulation of PDGFR. J Clin Invest 2003, 112:1223-33. O'Reilly KE, Rojo F, She QB, Solit D, Mills GB, Smith D, Lane H, Hofmann F, Hicklin DJ, Ludwig DL, Baselga J, Rosen N: mTOR inhibition induces upstream receptor tyrosine kinase signaling and activates Akt. Cancer Res 2006, 66:1500-8. Maira S, Stauffer F, Brueggen J, Furet P, Schnell C, Fritsch C, Brachmann S, Chène P, De Pover A, Schoemaker K, Fabbro D, Gabriel D, Simonen M, Murphy L, Finan P, Sellers W, García-Echeverría C: Identification and characterization of NVP-BEZ235, a new orally available dual PI3K/mTOR inhibitor with potent in vivo antitumor activity. Mol Cancer Ther 2008, 7:1851-63. El-Hashemite N, Walker V, Kwiatkowski DJ: Estrogen enhances whereas tamoxifen retards development of Tsc mouse liver Page 8 of 9 (page number not for citation purposes) Molecular Cancer 2009, 8:38 15. 16. 17. 18. 19. 20. http://www.molecular-cancer.com/content/8/1/38 hemangioma: a tumor related to renal angiomyolipoma and pulmonary lymphangioleiomyomatosis. Cancer Res 2005, 65:2474-81. Breuleux M, Klopfenstein M, Stephan C, Doughty CA, Barys L, Maira SM, Kwiatkowski D, Lane HA: Increased AKT S473 phosphorylation after mTORC1 inhibition is rictor dependent and does not predict tumor cell response to PI3K/mTOR inhibition. Mol Cancer Ther 2009, 8:742-53. Bissler JJ, McCormack FX, Young LR, Elwing JM, Chuck G, Leonard JM, Schmithorst VJ, Laor T, Brody AS, Bean J, Salisbury S, Franz DN: Sirolimus for angiomyolipoma in tuberous sclerosis complex or lymphangioleiomyomatosis. N Engl J Med 2008, 358:140-51. Davies DM, Johnson SR, Tattersfield AE, Kingswood JC, Cox JA, McCartney DL, Doyle T, Elmslie F, Saggar A, de Vries PJ, Sampson JR: Sirolimus therapy in tuberous sclerosis or sporadic lymphangioleiomyomatosis. N Engl J Med 2008, 358:200-3. Manning BD, Logsdon MN, Lipovsky AI, Abbott D, Kwiatkowski DJ, Cantley LC: Feedback inhibition of Akt signaling limits the growth of tumors lacking Tsc2. Genes Dev 2005, 19:1773-8. Sarbassov DD, Guertin DA, Ali SM, Sabatini DM: Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex. Science 2005, 307:1098-101. Sarbassov DD, Ali SM, Sengupta S, Sheen JH, Hsu PP, Bagley AF, Markhard AL, Sabatini DM: Prolonged rapamycin treatment inhibits mTORC2 assembly and Akt/PKB. Mol Cell 2006, 22:159-68. Publish with Bio Med Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical researc h in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright BioMedcentral Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp Page 9 of 9 (page number not for citation purposes)
https://openalex.org/W2062376907	https://www.scielo.br/j/rbz/a/rY3ndPzxWkVXqm38ZsF3hwH/?lang=pt&format=pdf	Portuguese	null	Bagaço de cana-de-açúcar tratado com hidróxido de sódio	Revista Brasileira de Zootecnia	2,006	cc-by	3,819	Aureliano José Vieira Pires1, Ricardo Andrade Reis2, Gleidson Giordano Pinto de Carvalho3, Gustavo Rezende Siqueira4, Thiago Fernandes Bernardes5 1 Departamento de Tecnologia Rural e Animal, UESB, Itapetinga, BA. p 3 Mestrando em Zootecnia, UFV, Viçosa, MG. 4 Mestrando em Zootecnia, UNESP, Jaboticabal, SP. 5 Doutorando em Zootecnia, UNESP, Jaboticabal, SP RESUMO - O experimento foi conduzido com o objetivo de avaliar a composição química e a digestibilidade in vitro da matéria seca (MS) do bagaço de cana-de-açúcar contendo 60% de MS submetido a doses crescentes de hidróxido de sódio (NaOH) (0; 2,5; 5 e 7,5% de uma solução 2:1 de água:NaOH na MS) em diferentes períodos de tratamento (1, 3, 5 e 7 dias). Foram utilizados baldes plásticos com capacidade de 10 L, mantidos em uma câmara climática à temperatura constante de 25oC. Não foi verificado efeito dos tratamentos (dose de NaOH e dias de tratamento) sobre os teores de PB, que apresentaram valor médio de 1,6%. A MS aumentou com os dias de tratamento, não sendo observadas alterações para essa variável em relação às doses crescentes de NaOH. Foi observada redução das frações de FDN, FDA, celulose (CEL), hemicelulose (HEM) e lignina (LIG). A digestibilidade in vitro da matéria seca (DIVMS) e o teor de sódio aumentaram quando o bagaço de cana foi submetido a doses crescentes de NaOH, mas não foi observado efeito do período de tratamento sobre essas variáveis. O valor nutritivo do bagaço de cana é melhorado com a adição de NaOH, comprovado pela redução nos constituintes da parede celular e pelo aumento na DIVMS. Palavras-chave: digestibilidade, parede celular, resíduo Correspondências devem ser enviadas para: aureliano@uesb.br Key Words: cell wall, digestibility, residue Sugarcane bagasse treated with sodium hydroxide ABSTRACT - The experiment was conducted with the objective of evaluating chemical composition and in vitro dry matter digestibility of sugar-cane bagasse containing 60% dry matter submited to increasing sodium hydroxide concentrations (0, 2.5, 5, and 7.5% NaOH on a dry matter basis). NaOH was applied as a 2:1 water:NaOH solution, over different treatment times (1, 3, 5, and 7 days). Plastic pails with 10 L capacity were utilized and stocked in climatic chamber at 25oC constant temperature. The experimental treatments had no effect on the crude protein content of the sugar cane bagasse which averaged 1.6% CP. The dry matter content of the bagasse increased over of the days of treatment, without any effect of the NaOH solution concentration. Mean values for the bagasse neutral detergent fiber, acid detergent fiber, cellulose, hemicellulose and lignin contents declined in response to NAOH solution concentration. NaOH treatment improved the in vitro dry matter digestibility (IVDMD) and sodium content of the bagasse. Time of treatment had no effect on these variables. The treatment of sugar cane bagasse with the NaOH solution improved its nutritive value in terms of its reduced cell wall constituents and increased IVDMD. Key Words: cell wall, digestibility, residue Palavras-chave: digestibilidade, parede celular, resíduo Revista Brasileira de Zootecnia © 2006 Sociedade Brasileira de Zootecnia ISSN impresso: 1516-3598 ISSN on-line: 1806-9290 www.sbz.org.br Revista Brasileira de Zootecnia © 2006 Sociedade Brasileira de Zootecnia ISSN impresso: 1516-3598 ISSN on-line: 1806-9290 www.sbz.org.br R. Bras. Zootec., v.35, n.3, p.953-957, 2006 (supl.) Introdução representa a fração menos digestível dos alimentos. A fração fibrosa dilui a energia do alimento e reduz o consumo voluntário, pelo efeito do enchimento ruminal e pela satu- ração da capacidade de ruminação do animal. O bagaço de cana de cana-de-açúcar tem sido utilizado como fonte alternativa na suplementação animal no período de escassez de forragens. Porém, uma vez que, como outros alimentos fibrosos, é constituído de celulose, hemicelulose e lignina, sua utilização tem sido minimizada, por ser consi- derado volumoso de baixa qualidade. Alguns tratamentos são realizados com o intuito de melhorar a utilização do bagaço de cana-de-açúcar e de outros subprodutos vegetais, que podem ser físicos e/ou químicos. Entre os tratamentos físicos, destacam-se a moagem e o tratamento térmico, e, entre os químicos, a uréia, a amônia anidra e o hidróxido de sódio (NaOH), produtos Segundo Ferreira et al. (2000), o teor de fibra tem sido utilizado como índice negativo de qualidade, uma vez que Pires et al. 954 alcalinos que normalmente promovem redução da FDN, podendo influenciar positivamente o consumo de MS do alimento. constante sobre uma lona. Após a homogeneização, o material foi armazenado nos baldes correspondentes a cada repetição, os quais permaneceram abertos nos respectivos períodos de tratamento (1, 3, 5 e 7 dias). Amostras foram coletadas, pré-secas em estufa (a 65oC por 72 horas), pro- cessadas em moinho tipo Willey e armazenadas para poste- riores análises de MS, nitrogênio total (NT), FDN, FDA, celulose, hemicelulose, lignina e digestibilidade in vitro da matéria seca (DIVMS), conforme procedimentos descritos por Silva & Queiroz (2002). O tratamento químico de alimentos volumosos tem crescido bastante nos últimos anos e várias pesquisas têm evidenciado que o valor nutritivo de diferentes volumosos pode ser melhorado com a utilização de produtos químicos (Cândido et al., 1999; Reis et al., 2001; Santos et al., 2004), entre eles, o tratamento de volumosos com amônia anidra, uréia e/ou NaOH (Souza et al., 2002). Segundo Al-Masri & Guenther (1999), o tratamento químico com NaOH tem sido utilizado para quebrar o composto lignocelulósico das forragens e aumentar pro- porcionalmente seus componentes nutritivos. Conforme Pereira Filho et al. (2003), as palhadas, os restolhos de culturas, o bagaço de cana-de-açúcar e os fenos de gramíneas de baixo valor nutritivo estão entre os alimentos mais submetidos ao tratamento químico com NaOH, que, normalmente, proporciona ganhos de 20 a 50% na DIMS. Resultados e Discussão Não foi observado efeito (P>0,05) da interação doses de NaOH × dias de tratamento para nenhuma das variáveis estudadas. Verificou-se efeito (P<0,05) dos dias de trata- mento sobre o teor de MS, que apresentou aumento (Figura 1) pelo fato de o bagaço ficar armazenado conforme os dias de tratamento em uma câmara climática com temperatura cons- tante de 25oC, o que fez com que perdesse umidade com o passar dos dias. Não foi observado efeito (P>0,05) para essa variável em função das doses de NaOH. Santos et al. (2004) relataram que a associação de NaOH mais uréia no tratamento de resíduo de lixadeira de algodão melhoraram a degradabilidade da MS e da FDN, melhorando seu valor nutritivo. Realizou-se este experimento com o objetivo de avaliar o efeito de doses de NaOH sobre a composição química e a digestibilidade in vitro da matéria seca (DIVMS) do bagaço de cana-de-açúcar em diferentes períodos de tratamento. Não houve efeito (P>0,05) das doses de NaOH e dos dias de tratamento sobre o teor de PB, calculado pelo teor de NT multiplicado por 6,25, registrando-se valor médio de PB de 1,59%, que pode ser considerado comum, visto que não foi adicionada fonte externa de nitrogênio. Introdução Os dados foram analizados por meio de regressão, utilizando-se o programa SAEG versão 8.0 (UFV, 1998). Material e Métodos Também não houve efeito (P>0,05) dos dias de trata- mento sobre a parede celular e seus constituintes, porém, verificou-se efeito significativo (P<0,05) das doses de NaOH, O experimento foi conduzido no setor de Forragicultura da FCAV/UNESP, Campus de Jaboticabal, SP. Foi utilizado o bagaço de cana-de-açúcar proveniente da indústria de álcool. © S Figura 1 - Efeito dos dias de tratamento com hidróxido de sódio (NaOH) sobre o teor de MS do bagaço de cana-de- açúcar. Figure 1 - DM content of sugarcane bagasse as a function of sodium hydroxide (NaOH) days of treatment. 55 60 65 70 75 1 3 5 7 = 58,413 + 1,71277 x Dia r2 = 95,6 Dia de tratamento Day of treatment Teor de MS (%) DM content (%) = 58,413 + 1,71277 x Dia r2 = 95,6 Utilizou-se o esquema de parcelas subdivididas, con- siderando-se parcelas as quatro doses (0; 2,5; 5 e 7,5% base da MS) de uma solução 2:1 (peso:peso) água:hidróxido de sódio (NaOH) e, como subparcelas, os períodos de trata- mento (1, 3, 5 e 7 dias), com três repetições, totalizando 48 unidades amostrais. O bagaço utilizado estava triturado e não necessitou ser picado. O material apresentou 60% de MS e, mesmo contendo alta umidade, não evidenciava sinais de fungos. O bagaço foi acondicionado em baldes plásticos com capacidade de 10 L e permaneceu em câmara climática à temperatura constante de 25oC. A solução de NaOH foi adicionada ao bagaço nas respectivas dosagens de 0; 2,5; 5 e 7,5% na MS por meio de um pulverizador, sendo adicionada ao bagaço aos poucos, com homogeneização Dia de tratamento Day of treatment Figura 1 - Efeito dos dias de tratamento com hidróxido de sódio (NaOH) sobre o teor de MS do bagaço de cana-de- açúcar. Figure 1 - DM content of sugarcane bagasse as a function of sodium hydroxide (NaOH) days of treatment. Figure 1 - DM content of sugarcane bagasse as a function of sodium hydroxide (NaOH) days of treatment. © 2006 Sociedade Brasileira de Zootecnia © 2006 Sociedade Brasileira de Zootecnia Bagaço de cana-de-açúcar tratado com hidróxido de sódio 955 ocorrendo redução nos teores de FDN, FDA, celulose (Figura 2), lignina e hemicelulose (Figura 3). Material e Métodos A redução na FDN observada neste estudo resultou da solubilização parcial dos constituintes da parede celular, pois o efeito de produtos alcalinos sobre volumosos de baixa qualidade normalmente ocorre pela solubilização par- cial da hemicelulose e pela expansão da celulose, o que facilita o ataque dos microrganismos do rúmen à parede celular (Van Soest, 1994). Semelhantemente, Reis et al. (1990) observaram decréscimo nos teores de FDN (81,0 para 70,5%) para o feno de Brachiaria decumbens não-tratado ou tratado com amônia anidra (3,0% da MS), respectivamente, o que corresponde à redução de 12,96%. Ao avaliarem resíduo de lixadeira de algodão submetido a tratamentos físicos e químicos, Santos et al. (2004) encon- traram valores de 91,51; 90,17; 86,23; 87,85 e 82,89% de FDA, respectivamente, para os tratamentos controle (sem trata- mento), pressão e vapor, NaOH, uréia, e NaOH mais uréia. Segundo os autores, a combinação de tratamentos pode ser uma alternativa para melhorar o valor nutritivo de resíduos e subprodutos agroindustriais. Pires et al. (2004) avaliaram o tratamento do bagaço de cana-de-açúcar com amônia anidra e/ou, sulfeto de sódio e constataram que a amônia anidra (4% na MS) melhorou a degradabilidade da MS, da FDN e da FDA, enquanto o sulfeto de sódio (2,5% na MS) não foi eficiente no trata- mento do bagaço de cana-de-açúcar, mesmo quando asso- ciado à amônia anidra (2,5% de Na2S + 4% de NH3 da MS). 55 60 65 70 75 80 85 90 95 100 0 2,5 5 7,5 ( ) FDN NDF FDA ADF Celulose Cellulose YFDA = 81,078 – 1,0543 x (NaOH) + 0,0325221 x (NaOH)2 R2 = 94,5 Dose de NaOH (% MS) NaOH level (% DM) Teor (%) Content (%) FDN = 97,4481 - 1,15098 x (NaOH); R2 = 97,8 FDA = 81,078 - 1,0543 x (NaOH) + 0,0325221 x (NaOH)2; R2 = 94,5 celulose = 65,6725 - 0,513756 x (NaOH); R2 = 78,6 Figura 2 - Teores de FDN, FDA e celulose do bagaço de cana- de-açúcar submetido a doses crescentes de hidróxido de sódio (NaOH). Figure 2 - NDF, ADF and cellulose content of sugarcane bagasse submitted to increasing levels of sodium hydroxide (NaOH). Material e Métodos 55 60 65 70 75 80 85 90 95 100 0 2,5 5 7,5 ( ) FDN NDF FDA ADF Celulose Cellulose YFDA = 81,078 – 1,0543 x (NaOH) + 0,0325221 x (NaOH)2 R2 = 94,5 Dose de NaOH (% MS) NaOH level (% DM) Teor (%) Content (%) FDN = 97,4481 - 1,15098 x (NaOH); R2 = 97,8 FDA = 81,078 - 1,0543 x (NaOH) + 0,0325221 x (NaOH)2; R2 = 94,5 celulose = 65,6725 - 0,513756 x (NaOH); R2 = 78,6 A redução da hemicelulose é comum em gramíneas ou resíduos e subprodutos de gramíneas tratados com produ- tos alcalinos, como resultado de sua solubilização parcial. Houve efeito quadrático crescente (P<0,05) das doses de NaOH (Figura 4) sobre a DIVMS. O aumento na DIVMS de materiais tratados com produtos alcalinos tem sido realatado na literatura (Pires et al., 1999; Sarmento et al., 1999), como resultado da solubilização parcial da hemicelulose e da expansão da celulose, o que facilita o ataque de microrganismos à parede celular. 7,5 Dose de NaOH (% MS) NaOH level (% DM) Figura 2 - Teores de FDN, FDA e celulose do bagaço de cana- de-açúcar submetido a doses crescentes de hidróxido de sódio (NaOH). ( ) Figure 2 - NDF, ADF and cellulose content of sugarcane bagasse submitted to increasing levels of sodium hydroxide (NaOH). Manzano et al. (2000) encontraram valores de 32,4; 48,7 e 62,4% para DIVMS do bagaço de cana-de-açúcar sem tratamento, com 2% de Na2S + 3% de NaOH e 4% de Na2S + 4% de NaOH, respectivamente. Embora os autores tenham utilizado uma fonte sulfurosa associada ao NaOH, tudo indica que o NaOH exerceu influência sobre o bagaço, melhorando sua digestibilidade. Os aumentos na DIVMS de volumosos tratados com produtos alcalinos normalmente estão relacionados ao aumento do consumo e do desempenho de animais alimentados com esses volumosos, que podem, às vezes, apresentar resultados de desempenho semelhantes em comparação a dietas de melhor qualidade. Figura 3 - Teores de hemicelulose e lignina do bagaço de cana- de-açúcar submetido a doses crescentes de hidróxido de sódio (NaOH). Figure 3 - Hemicellulose and lignin contents of sugarcane bagasse submitted to increasing levels of sodium hydroxide (NaOH). Literatura Citada AL-MASRI, M.R.; GUENTHER, K.D. Changes in digestibility and cell-wall constituents of some agricultural by-products and urea AL-MASRI, M.R.; GUENTHER, K.D. Changes in digestibility and cell-wall constituents of some agricultural by-products and urea treatments. Radiation Physics and Chemistry, v.55, n.3, p.324-329, 1999. Figura 5 - Teor de sódio (Na) do bagaço de cana-de-açúcar submetido a doses crescentes de hidróxido de sódio (NaOH). Figure 5 - Sodium (Na) content of sugarcane bagasse submitted to increasing levels of sodium hydroxide (NaOH). 0 0,5 1 1,5 2 2,5 0 2,5 5 7,5 Y = 0,03583 +0,276615 x (NaOH) r2 = 98,0 Dose de NaOH (% MS) NaOH level (% DM) Teor de Na (%) Na content (%) = 0,03583 + 0,276615 x (NaOH); R2 = 98,0 0 0,5 1 1,5 2 2,5 0 2,5 5 7,5 Y = 0,03583 +0,276615 x (NaOH) r2 = 98,0 Dose de NaOH (% MS) NaOH level (% DM) Teor de Na (%) Na content (%) = 0,03583 + 0,276615 x (NaOH); R2 = 98,0 CÂNDIDO, M.J.D.; NEIVA, J.N.M.; PIMENTEL, J.C.M. et al. Avaliação do valor nutritivo do bagaço de cana-de-açúcar amonizado com uréia. Revista Brasileira de Zootecnia, v.28, n.5, p.928-935, 1999. amonizado com uréia. Revista Brasileira de Zootecnia, v.28, n.5, p.928-935, 1999. D´ARCE, R.D.; HADDAD, C.M. Bagaço de cana tratado com hidróxido de sódio na terminação de bovinos em confinamento. In: REUNIÃO ANUAL DA SOCIEDADE BRASILERA DE ZOOTECNIA, 26., 1989, Porto Alegre. Anais... Porto Alegre: Sociedade Brasileira de Zootecnia, 1989. p.155. FERREIRA, M.A.; SANTOS, D.C.; LIRA, M.A. et al. Associação da palma forrageira (Opuntia fícus indica Mill) com diferentes fontes de fibra na alimentação de vacas 5/8 Holandês/Zebu em lactação. Revista Brasileira de Zootecnia, v.29, n.7, p.2128- 2134, 2000. Dose de NaOH (% MS) NaOH level (% DM) MANZANO, R.P.; FUKUSHIMA, R.S.; GOMES, J.D.F. et al. Digestibilidade do bagaço de cana-de-açúcar tratado com reagentes químicos e pressão de vapor. Revista Brasileira de Zootecnia, v.29, n.4, p.1196-1204, 2000. MARTIN, L.C.T. Bovinos: volumosos suplementares. São Paulo: Nobel, 1997. 143p. MANZANO, R.P.; FUKUSHIMA, R.S.; GOMES, J.D.F. et al. Digestibilidade do bagaço de cana-de-açúcar tratado com reagentes químicos e pressão de vapor. Revista Brasileira de Zootecnia v 29 n 4 p 1196-1204 2000 Zootecnia, v.29, n.4, p.1196-1204, 2000. MARTIN, L.C.T. Bovinos: volumosos suplementares. São Paulo: Nobel, 1997. 143p. Figura 5 - Teor de sódio (Na) do bagaço de cana-de-açúcar submetido a doses crescentes de hidróxido de sódio (NaOH). Material e Métodos Dose de NaOH (% MS) NaOH level (% DM) DIVMS (%) IVDMD (%) 20 25 30 35 40 45 0 2,5 5 7,5 = 25,5695 + 1,29723** x (NaOH) + 0,0977757* x (NaOH)2; R2 = 91,4 Dose de NaOH (% MS) NaOH level (% DM) DIVMS (%) IVDMD (%) 20 25 30 35 40 45 0 2,5 5 7,5 = 25,5695 + 1,29723** x (NaOH) + 0,0977757* x (NaOH)2; R2 = 91,4 tratado é importante para ajustar a quantidade de NaCl a ser adicionada à mistura mineral suficiente para atender às exigências dos animais, uma vez que elevados teores de Na nas dietas podem provocar a redução do consumo da mistura e, conseqüentemente, diminuir a ingestão de macro e microminerais. Conclusões O bagaço de cana-de-açúcar tratado com hidróxido de sódio apresenta melhor valor nutritivo, sendo, portanto, uma alternativa para utilização como volumoso na alimen- tação de ruminantes. Figura 4 - Digestibilidade in vitro da matéria seca (DIVMS) do bagaço de cana-de-açúcar submetido a doses cres- centes de hidróxido de sódio (NaOH). Figure 4 - Dry matter in vitro digestibility (IVDMD) of sugarcane bagasse submitted to increasing levels of sodium hydroxide (NaOH). Figura 4 - Digestibilidade in vitro da matéria seca (DIVMS) do bagaço de cana-de-açúcar submetido a doses cres- centes de hidróxido de sódio (NaOH). Figure 4 - Dry matter in vitro digestibility (IVDMD) of sugarcane bagasse submitted to increasing levels of sodium hydroxide (NaOH). Material e Métodos 10 11 12 13 14 15 16 17 0 2,5 5 7,5 Hemicelulose Hemicellulose Lignina Lignin Dose de NaOH (% MS) NaOH level (% DM) Teor (%) Content (%) hemicelulose = 16,3132 - 0,0283163 x (NaOH) - 0,0416372 x (NaOH)2; R2 = 77,8 lignina = 15,484 - 0,4842 x (NaOH) + 0,01929* x (NaOH)2; R2 = 75,5 Lignina Lignin Segundo Neto & Ferreira (1984), citados por Martin (1997), a DIVMS do bagaço de cana-de-açúcar aumentou gradativamente, com valores de 25,9; 46,6 e 77,0% para os tratamentos com 3, 6 e 14% de NaOH. D’arce & Haddad (1989) estudaram a utilização do bagaço de cana-de-açúcar tratado com NaOH (3,6% da MS) na terminação de bovinos de corte em confinamento, divi- didos ao acaso em dois grupos: Tratamento A – dieta com 60% de bagaço tratado + 5% de cana integral + 35% de Figura 3 - Teores de hemicelulose e lignina do bagaço de cana- de-açúcar submetido a doses crescentes de hidróxido de sódio (NaOH). Figura 3 - Teores de hemicelulose e lignina do bagaço de cana- de-açúcar submetido a doses crescentes de hidróxido de sódio (NaOH). ( ) Figure 3 - Hemicellulose and lignin contents of sugarcane bagasse submitted to increasing levels of sodium hydroxide (NaOH). ( ) Figure 3 - Hemicellulose and lignin contents of sugarcane bagasse submitted to increasing levels of sodium hydroxide (NaOH). © 2006 Sociedade Brasileira de Zootecnia Pires et al. 956 concentrado à base de milho, cama de frango e farelo de Figura 5 - Teor de sódio (Na) do bagaço de cana-de-açúcar submetido a doses crescentes de hidróxido de sódio (NaOH). Figure 5 - Sodium (Na) content of sugarcane bagasse submitted to increasing levels of sodium hydroxide (NaOH). 0 0,5 1 1,5 2 2,5 0 2,5 5 7,5 Y = 0,03583 +0,276615 x (NaOH) r2 = 98,0 Dose de NaOH (% MS) NaOH level (% DM) Teor de Na (%) Na content (%) = 0,03583 + 0,276615 x (NaOH); R2 = 98,0 Figura 4 - Digestibilidade in vitro da matéria seca (DIVMS) do bagaço de cana-de-açúcar submetido a doses cres- centes de hidróxido de sódio (NaOH). Figure 4 - Dry matter in vitro digestibility (IVDMD) of sugarcane bagasse submitted to increasing levels of sodium hydroxide (NaOH). Literatura Citada PIRES, A.J.V.; GARCIA, R.; CECON, P.R. et al. Amonização da quirera de milho com alta umidade. Revista Brasileira de Zootecnia, v.28, n.6, p.1186-1193, 1999. Figure 5 - Sodium (Na) content of sugarcane bagasse submitted to increasing levels of sodium hydroxide (NaOH). PIRES, A.J.V.; GARCIA, R.; VALADARES FILHO, S.C. et al. Degradabilidade do bagaço de cana-de-açúcar tratado com amônia anidra e, ou, sulfeto de sódio. Revista Brasileira de Zootecnia, v.33, n.4, p.1071-1077, 2004. concentrado à base de milho, cama de frango e farelo de algodão; e Tratamento B – dieta com 65% de cana integral + 35% do mesmo concentrado. Os autores concluíram que não houve diferença entre os tratamentos quanto ao ganho médio de peso, ao consumo médio diário e à conversão alimentar e sugeriram a utilização do bagaço de cana tratado com NaOH como volumoso em dietas de terminação, desde que verificada sua viabilidade econômica. Zootecnia, v.33, n.4, p.1071-1077, 2004. REIS, R.A.; GARCIA, R.; SILVA, D.J. et al. Efeitos da aplicação de amônia anidra sobre a digestibilidade do feno de capim-brachiaria (Bracharia decumbens Stapf). Revista Brasileira de Zootecnia, v.33, n.4, p.1071 1077, 2004. REIS, R.A.; GARCIA, R.; SILVA, D.J. et al. Efeitos da aplicação de amônia anidra sobre a digestibilidade do feno de capim-brachiaria (Bracharia decumbens Stapf). Revista Brasileira de Zootecnia, v.19, n.3, p.201-208, 1990. (Bracharia decumbens Stapf). Revista Brasileira de Zootecnia, v.19, n.3, p.201-208, 1990. REIS, R.A.; RODRIGUES, R.L.A.; RESENDE, K.T. et al. Avaliação de fontes de amônia para o tratamento de fenos de gramíneas tropicais. 1. constituintes da parede celular, poder tampão e atividade ureática. Revista Brasileira de Zootecnia, v.30, n.3, p.674-681, 2001. ROSA, B.; REIS, R.A.; RESENDE, K.T. et al. Valor nutritivo do feno de Brachiaria decumbens Stapf cv. Basilisk submetido a tratamento com amônia anidra ou uréia. Revista Brasileira de Zootecnia, v.27, n.4, p.815-822, 1998. O teor de sódio (Na) no bagaço (Figura 5) aumentou linearmente (P<0,05) com o aumento das doses de NaOH, provavelmente pelo fato de o hidróxido apresentar signifi- cativo teor de Na. A determinação do teor de Na no bagaço p ROSA, B.; SOUZA, H.; RODRIGUES, K.F. Composição química do feno de Brachiaria brizantha cv. Marandu tratado com diferentes © 2006 Sociedade Brasileira de Zootecnia Bagaço de cana-de-açúcar tratado com hidróxido de sódio 957 proporções de uréia e de água. Ciência Animal Brasileira, v.1, n.2, p.107-113, 2000. proporções de uréia e de água. Ciência Animal Brasileira, v.1, n.2, p.107-113, 2000. SOUZA, A.L.; GARCIA, R.; PEREIRA, O.G. et al. Valor nutritivo da casca de café tratada com amônia anidra. Revista Ceres, v.26, n.286, p.669-681, 2002. UNIVERSIDADE FEDERAL DE VIÇOSA - UFV. Sistema de análises estatísticas e genéticas - SAEG. Versão 8.0. Viçosa, MG: 1998. 150p. (Manual do usuário). Van SOEST, P.J. Nutritional ecology of the ruminant. 2.ed. Ithaca: Cornell University Press, 1994. 476p. © 2006 Sociedade Brasileira de Zootecnia Literatura Citada SANTOS, J.; CASTRO, A.L.A.; PAIVA, P.C.A. et al. Efeito dos tratamentos físicos e químicos no resíduo de lixadeira do algodão. Ciência Agrotecnologia, v.28, n.4, p.919-923, 2004. SARMENTO, P.; GARCIA, R.; PIRES, A.J.V. et al. Tratamento do bagaço de cana-de-açúcar com uréia. Revista Brasileira de Zootecnia, v.28, n.6, p.1203-1208, 1999. SILVA, D.J.; QUEIROZ, A.C. Análise de alimentos (métodos químicos e biológicos). Viçosa, MG: Universidade Federal de Viçosa, 2002. 235p. SOUZA, A.L.; GARCIA, R.; PEREIRA, O.G. et al. Valor nutritivo da casca de café tratada com amônia anidra. Revista Ceres, v.26, n.286, p.669-681, 2002. Recebido: 14/06/05 Aprovado:18/11/05 Recebido: 14/06/05 Aprovado:18/11/05 © 2006 Sociedade Brasileira de Zootecnia
https://openalex.org/W2964879393	https://www.problecon.com/export_pdf/problems-of-economy-2019-2_0-pages-5_14.pdf	Ukrainian	null	Influence of Globalization of the Economy on the Formation of Land Relations in Agriculture	Problemi ekonomìki	2,019	cc-by-sa	7,371	Q ; Данкевич В. Є., Данкевич Є. М., Шегеда О. В. Вплив глобалізації економіки на формування земельних відносин у сільському господарстві Данкевич В. Є., Данкевич Є. М., Шегеда О. В. Вплив глобалізації економіки на формування земельних відносин у сільському господарст Метою статті є вивчення впливу глобалізації економіки на формування земельних відносин у сільському господарстві. Обґрунтовано, що глобаліза ція економіки є багатовимірним процесом, який охоплює усі сфери суспільної життєдіяльності, докорінно змінює принципи та характер виробничої діяльності, а також специфіку землекористування. Доведено, що вплив глобальних викликів на господарсько-технологічні й економічні особливості використання сільськогосподарських земель є не лише вагомим, але й має тенденцію до посилення. Прояв глобалізації в аграрному секторі економіки найбільшою мірою здійснюється через інтенсифікацію виробництва, розвиток біоекономіки, запровадження елементів точного землеробства, ви користання геоінформаційних систем, капіталізацію сільськогосподарського виробництва і монополізацію орендних відносин, запровадження вах тового методу при здійсненні основних господарських процесів. Моніторинг використання сільськогосподарських земель на прикладі п’яти регіонів світу (Австралії, Азії, Африки, Європи, Північної та Південної Америки) дозволив виокремити основні сучасні тенденції розвитку земельних відносин: інтенсифікацію землекористування, збільшення площ сільськогосподарських земель, що використовуються під технічні енергомісткі культури, зміщення географічних меж сільськогосподарських зон, розширення регіонів із зрошувальним землеробством, використання зернових та олійних культур для енергетичних потреб, нарощування земельних банків аграрними холдингами, запровадження інноваційних систем управління станом посівів. Запропоновано практичні рекомендації щодо адаптації землекористувачів до сучасних глобальних викликів. Обґрунтовано, що вирішення проблем повноцінного забезпечення населення продуктами харчування і промисловості сировиною, відповідно положень глобалізаційної теорії роз витку земельних відносин, можливе як за рахунок внутрішніх трансформацій наявних земельних ресурсів, так і через процес купівлі-продажу сіль ськогосподарських земель на світових ринках. Ключові слова: глобалізація, сільськогосподарські землі, ринок земель, ефективність, продовольча безпека, інтенсифікація. Данкевич Віталій Євгенович – доктор економічних наук, кафедра міжнародних економічних відносин та європейської інтеграції, Житомирський на ціональний агроекологічний університет (Старий бульвар, 7, Житомир, 10008, Україна) Данкевич Віталій Євгенович – доктор економічних наук, кафедра міжнародних економічних від ціональний агроекологічний університет (Старий бульвар, 7, Житомир, 10008, Україна) Researcher ID: http://www.researcherid.com/E-3739-2017 Данкевич Євген Михайлович – доктор економічних наук, професор кафедри екологічної безпеки та економіки природокористування, Житомирський національний агроекологічний університет (Старий бульвар, 7, Житомир, 10008, Україна) Шегеда Олександр Васильович – здобувач, Житомирський національний агроекологічний університет (Старий бульвар, 7, Житомир, 10008, Україна) E-mail: avs0310@ukr.net УДК 332.3: 502.35(477):339.9 JEL Classification: Q13; R52 УДК 332.3: 502.35(477):339.9 JEL Classification: Q13; R52 Данкевич В. Є., Данкевич Є. М., Шегеда О. В. i Вплив глобалізації економіки на формування земельних відносин у сільському господарстві  2019 Данкевич В. Є., Данкевич Є. М., Шегеда О. В. Dankevych V. Y., Dankevych Y. M., Sheheda A. V. Influence of Globalization of the Economy on the Formation of Land Relations in Agriculture Dankevych V. Y., Dankevych Y. M., Sheheda A. V. Influence of Globalization of the Economy on the Formation of Land Relations in Agriculture The aim of the article is to study the influence of economic globalization on the formation of land relations in agriculture. It is substantiated that global ization of the economy is a multidimensional process which covers all spheres of public life and fundamentally changes the principles and nature of produc tion as well as the specifics of land use. It is proved that the influence of global challenges on the economic, technological and economic features of the use of agricultural land is not only significant now but also has a tendency to increase. Globalization is manifested in the agrarian sector of the economy to the greatest extent through the intensification of production, develop ment of a bioeconomy, introduction of elements of precision agriculture, use of geographic information systems, capitalization of agricultural production Целью статьи является изучение влияния глобализации экономики на формирование земельных отношений в сельском хозяйстве. Обосно вано, что глобализация экономики является многомерным процессом, который охватывает все сферы общественной жизнедеятельности, в корне меняет принципы и характер производственной деятельно сти, а также специфику землепользования. Доказано, что влияние глобальных вызовов на хозяйственно-технологические и экономиче ские особенности использования сельскохозяйственных земель явля ется не только весомым, но и имеет тенденцию к усилению. Проявле ние глобализации в аграрном секторе экономики в наибольшей степе ни осуществляется через интенсификацию производства, развитие 5 Проблеми економіки № 2 (40), 2019 Світова економіка та міжнародні відносини биоэкономики, внедрение элементов точного земледелия, использова ние геоинформационных систем, капитализацию сельскохозяйствен ного производства и монополизацию арендных отношений, внедрение вахтового метода при осуществлении основных хозяйственных про цессов. Мониторинг использования сельскохозяйственных земель на примере пяти регионов мира (Австралии, Азии, Африки, Европы, Се верной и Южной Америки) позволил выделить основные современные тенденции развития земельных отношений: интенсификацию зем лепользования, увеличение площадей сельскохозяйственных земель, используемых под технические энергоемкие культуры, смещение гео графических границ сельскохозяйственных зон, расширение регионов с оросительным земледелием, использование зерновых и масличных культур для энергетических нужд, увеличение земельных банков аграрными холдингами, внедрение инновационных систем управле ния состоянием посевов. Предложены практические рекомендации по адаптации землепользователей к современным глобальным вызовам. Обосновано, что решение проблем полноценного обеспечения насе ления продуктами питания и промышленности сырьем, в соответ ствии с положениями глобализационной теории развития земельных отношений, возможно как за счет внутренних трансформаций имею щихся земельных ресурсов, так и через процесс купли-продажи сель скохозяйственных земель на мировых рынках. Fig.: 2. Tabl.: 5. Bibl.: 21. Dankevych Vitalii Ye. – Doctor of Sciences (Economics), Department of Inter national Economic Relations and European Integration, Zhytomyr National Agroecological University (7 Staryi Blvd., Zhytomyr, 10008, Ukraine) Ключевые слова: глобализация, сельскохозяйственные земли, рынок земель, эффективность, продовольственная безопасность, интен сификация. ORCID: http://orcid.org/0000-0002-0522-2927 t Researcher ID: http://www.researcherid.com/E-3739-2017 Dankevych Yevgen M. – Doctor of Sciences (Economics), Professor of the De partment of Environmental Safety and Environmental Economics, Zhytomyr National Agroecological University (7 Staryi Blvd., Zhytomyr, 10008, Ukraine) E-mail: dankevych2020@gmail.com Рис.: 2. Табл.: 5. Библ.: 21. Dankevych V. Y., Dankevych Y. M., Sheheda A. V. Influence of Globalization of the Economy on the Formation of Land Relations in Agriculture and monopolization of rental relations, application of a rotational system in the implementation of basic business processes. Monitoring the use of agri cultural land, based on the example of five regions of the world (Australia, Asia, Africa, Europe, North and South America), made it possible to identify the main modern trends in the development of land relations: intensification of land use, increase in the area of agricultural land used for technically chal lenging energy-intensive crops, shift in the geographical boundaries of agri cultural areas, expansion of regions with irrigated agriculture, use of grains and oilseeds as alternative energy sources, increase of land banks by agrarian holdings, introduction of innovative systems for managing the state of crops. Practical recommendations on the adaptation of land users to modern global challenges are proposed. It is substantiated that, in accordance with the pro visions of the globalization theory of land relations, solving the problems of providing the population with food and industry with raw materials is pos sible both through internal transformations of the existing land resources and the process of buying and selling agricultural land on world markets. биоэкономики, внедрение элементов точного земледелия, использова ние геоинформационных систем, капитализацию сельскохозяйствен ного производства и монополизацию арендных отношений, внедрение вахтового метода при осуществлении основных хозяйственных про цессов. Мониторинг использования сельскохозяйственных земель на примере пяти регионов мира (Австралии, Азии, Африки, Европы, Се верной и Южной Америки) позволил выделить основные современные тенденции развития земельных отношений: интенсификацию зем лепользования, увеличение площадей сельскохозяйственных земель, используемых под технические энергоемкие культуры, смещение гео графических границ сельскохозяйственных зон, расширение регионов с оросительным земледелием, использование зерновых и масличных культур для энергетических нужд, увеличение земельных банков аграрными холдингами, внедрение инновационных систем управле ния состоянием посевов. Предложены практические рекомендации по адаптации землепользователей к современным глобальным вызовам. Обосновано, что решение проблем полноценного обеспечения насе ления продуктами питания и промышленности сырьем, в соответ ствии с положениями глобализационной теории развития земельных отношений, возможно как за счет внутренних трансформаций имею щихся земельных ресурсов, так и через процесс купли-продажи сель скохозяйственных земель на мировых рынках. Keywords: globalization, agricultural land, land market, efficiency, food se curity, intensification. Рис.: 2. Табл.: 5. Библ.: 21. Данкевич Виталий Евгеньевич – доктор экономических наук, кафедра международных экономических отношений и европейской интегра ции, Житомирский национальный агроэкологический университет (Старый бульвар, 7, Житомир, 10008, Украина) ORCID: http://orcid.org/0000-0002-0522-2927 Sheheda Alexander V. – Applicant, Zhytomyr National Agroecological Univer sity (7 Staryi Blvd., Zhytomyr, 10008, Ukraine) E-mail: avs0310@ukr.net Sheheda Alexander V. – Applicant, Zhytomyr National Agroecological Univer sity (7 Staryi Blvd., Zhytomyr, 10008, Ukraine) Sheheda Alexander V. – Applicant, Zhytomyr National Agroecological Univer sity (7 Staryi Blvd., Zhytomyr, 10008, Ukraine) E-mail: avs0310@ukr.net Researcher ID: http://www.researcherid.com/E-3739-2017 Данкевич Евгений Михайлович – доктор экономических наук, профес сор кафедры экологической безопасности и экономики природопользо вания, Житомирский национальный агроэкологический университет (Старый бульвар, 7, Житомир, 10008, Украина) E-mail: dankevych2020@gmail.com Шегеда Александр Васильевич – соискатель, Житомирский нацио нальный агроэкологический университет (Старый бульвар, 7, Жито мир, 10008, Украина) Проблеми економіки № 2 (40), 2019 E-mail: avs0310@ukr.net E-mail: avs0310@ukr.net країнами та країнами, що розвиваються, формується сис тема нерівноправних відносин, коли основна частка додат кового продукту привласнюється країнами, які постачають технології та обладнання, а не тими, на території яких від бувається виробництво. Згідно з положеннями теорії за лежності, країни, що розвиваються, бідніють за рахунок того, що їхні ресурси і капітал переміщуються в розвинені країни. Бідність країн, що розвиваються, існує не тому, що вони не інтегровані у світовий ринок або слабоінтегровані, а навпаки, тому, що є частиною його. Постановка проблеми. Глобалізація економіки є ба гатовимірним процесом, який охоплює усі сфери суспільної життєдіяльності, докорінно змінює принципи і характер виробничої діяльності. Вона передбачає інтернаціоналіза цію виробництва та формування господарських зв’язків у межах діяльності корпорацій, а також розширення фі нансових операцій у світовому масштабі. Основними ха рактеристиками глобалізації є зближення інфраструктури, виробництва і споживання, принципів маркетингу різних країн світу, а також процес об’єднання ринків капіталу та робочої сили. Методологія. Теоретико-методичною основою до слідження є діалектичний метод пізнання економічних процесів, фундаментальні положення сучасної економічної теорії, наукові праці вітчизняних і зарубіжних учених щодо впливу глобалізації економіки на формування земельних відносин у сільському господарстві. Методологічною осно вою дослідження стали наукові методи пізнання на осно ві системного підходу. Для досягнення поставленої мети Слід зауважити, що у процесах глобалізації можуть проявлятися як позитивні, так і негативні наслідки для економічного, екологічного та соціального розвитку окре мих країн світу. У теорії залежності Р. Пребиш та А. Франк виокремлюють можливі оптимістичні та песимістичні на слідки глобалізації. За песимістичним варіантом, у резуль таті створення «ланцюга залежності» між розвинутими 6 Проблеми економіки № 2 (40), 2019 Світова економіка та міжнародні відносини забезпечення продовольчої безпеки у світі. І. Казакова, до сліджуючи вплив глобальних змін на ґрунтові ресурси та сільськогосподарське виробництво, зазначає, що демогра фічні проблеми, зміна клімату та зростання конкуренції за земельні ресурси в умовах відсутності достатньої кількості продовольства спричиняють зростання незахищеності на селення, перш за все, у країнах Африки та Азії [9]. Завдання забезпечення кожного жителя планети достатньою кіль кістю продовольства ще ніколи не було настільки масш табним. використано низку загальних і спеціальних методів до слідження: системний аналіз; абстрактно-логічний; метод аналогій. Аналіз останніх досліджень і публікацій. Пробле ми використання ресурсного потенціалу в умовах глобалі зації набули важливого значення у наукових дослідженнях видатних вітчизняних учених. Зокрема, у працях О. Боро діної, М. Орлатого, Т. Сидорченко, В. E-mail: avs0310@ukr.net Сайко розроблено нові підходи щодо розвитку економіки в умовах сучасних глобальних викликів, в основу яких покладено як власні наукові здобутки, так і оцінку зарубіжного досвіду теорії та практики здійснення аграрної політики [4; 5]. Обґрун туванню науково-методичних засад землекористування в умовах зміни світової кон’юнктури ринку присвячені праці таких науковців: Г. Дудича, В. Заяця, М. Зось-Кіора, О. Коваліва та ін. [1; 6]. Тенденції зменшення земельних ре сурсів, придатних до використання, зростання споживання продуктів харчування, підвищення цін на продовольство в умовах глобалізації наведено у працях зарубіжних уче них: P. Ciaian, d’Artis Kancs, J. Swinnen, K. Herck, L. Vranken, C. Runge та багатьох інших [11; 12; 17; 19]. Food and Agriculture Organization (FAO) наводить дані, що до 2050 р. чисельність населення збільшиться при близно до 9 млрд осіб. До цього часу з метою вирішення продовольчих проблем буде необхідно щорічно виробляти додатково 1 млрд т зернових і 200 млн т продукції тварин ництва. Завдання збільшення сільськогосподарського ви робництва найгостріше стосується країн, що розвивають ся, в яких проблема полягає не тільки у тому, щоб виро бляти продовольство, а й у тому, щоб забезпечити до нього доступ населення [2]. У XXI ст., незважаючи на технологічний прогрес, майже 1 млрд людей не мають достатньо коштів для ку півлі продуктів харчування, перш за все, в країнах Африки, на південь від Сахари (239 млн осіб), та Азії (578 млн осіб). Навіть за умови зростання сільськогосподарського вироб ництва до 2050 р. у два рази, загроза недоїдання буде збе рігатися для кожного двадцятого жителя, що еквівалентно 370 млн голодуючих [3]. Для поліпшення ситуації з про довольчим забезпеченням і досягнення успіхів у боротьбі з недоїданням необхідно, щоб зростання обсягів сільсько господарського виробництва випереджало зростання чи сельності населення. Вирішити це завдання можливо за рахунок інтенсифікації землекористування, що дозволить мінімізувати наявні продовольчі та соціальні проблеми. Існують проблеми, які мають першочергове значення не лише для окремих країн, а і для певних регіонів світу. Для визначення значущості тієї або іншої проблеми необхідний системний підхід. Г. С. Хозін критерії глобальності зводить до двох чинників: комплексність і складний внутрішній взаємозв’язок цих проблем, а також їх високий динамізм. Водночас незмінною залишається проблема, пов’язана із використанням природних ресурсів при екстенсифікації та інтенсифікації господарської діяльності. Систематизація наукових напрацювань Т. Зінчук, Т. Сидорченка, М. Зось-Кіора, K. Herck, L. Vranken у галузі міжнародної економіки дозволяє стверджувати, що сучас ні глобалізаційні процеси спричиняють вагомий вплив на специфіку використання природних ресурсів, особливо земельних [7; 18]. E-mail: avs0310@ukr.net Тенденції розвитку сільського господарства в умовах глобалізації різке зростання цін на продукти харчування; скорочення запасів продовольства розширення площ, зайнятих під сільськогосподарським виробництвом, інтенсифікація господарської діяльності коливання цін на продукцію; невизначеність динаміки економічного розвитку збільшення площ сільськогосподарських земель, що викорстовуються під технічні енергомісткі культури (соя, соняшник) Основні характеристики Прояви впливу на земельні відносини дефіцит водних ресурсів як важливої умови виробництва; скорочення сільськогоспо- дарських площ, зокрема ріллі посилення розорюваності, екстенсифікація земле- користування за рахунок розширення задіяних в обробіток земель, деградація та виснаження грунту зміщення географічних меж сільськогоспо- дарських зон, зниження урожайності та зменшення обсягів виробництва розширення регіонів із зрошувальним землероб- ством, нестача води, зміна природно-кліматичних зон вирощування сільськогосподарських культур використання зернових та олійних культур для енергетичних потреб; скорочення пропозиції продуктів харчування на світовому ринку розширення земельних площ під енергетичними культурами; посилення конкуренції за земельні ресурси між виробниками біопалива та продовольства інвестування коштів в аграрний сектор, запровадження вахтового методу господар- ської діяльності, монополізація ринку нарощування земельних банків аграрними холдингами; інтенсифікація виробництва на основі інноваційних технологій землекористування, монокультуризація Агфляція Зміна кон’юнктури ринку Тенденція Екстенсивне використання ресурсів Стрімке потепління Розвиток виробництва біопалива Корпоратизація аграрного бізнесу Рис. 1. Тенденції розвитку сільського господарства в умовах глобалізації збільшення площ сільськогосподарських земель, що викорстовуються під технічні енергомісткі культури (соя, соняшник) посилення розорюваності, екстенсифікація земле- користування за рахунок розширення задіяних в обробіток земель, деградація та виснаження грунту зміщення географічних меж сільськогоспо- дарських зон, зниження урожайності та зменшення обсягів виробництва розширення регіонів із зрошувальним землероб- ством, нестача води, зміна природно-кліматичних зон вирощування сільськогосподарських культур розширення регіонів із зрошувальним землероб- ством, нестача води, зміна природно-кліматичних зон вирощування сільськогосподарських культур використання зернових та олійних культур для енергетичних потреб; скорочення пропозиції продуктів харчування на світовому ринку розширення земельних площ під енергетичними культурами; посилення конкуренції за земельні ресурси між виробниками біопалива та продовольства Розвиток виробництва біопалива нарощування земельних банків аграрними холдингами; інтенсифікація виробництва на основі інноваційних технологій землекористування, монокультуризація Рис. 1. Тенденції розвитку сільського господарства в умовах глобалізації Джерело: авторська розробка нім доходом ВНП на душу населення у межах від 726 до 8995 дол. США; до третьої групи («high-income countries») відносяться країни, в яких ВНП на душу населення стано вить 8995 дол. США і вище [15]. Кожна з груп країн має свої природно-кліматичні особливості, різні площі земель, при датні для сільськогосподарського виробництва (табл. 1). ція. E-mail: avs0310@ukr.net Вагомі дослідження у сфері сільськогосподарського землекористування на рівні окремих регіонів світу про водять Світовий банк і Міжнародна фінансова корпора Земельні та водні ресурси, а також способи їх вико ристання відіграють вагому роль при вирішенні завдань Проблеми економіки № 2 (40), 2019 7 Світова економіка та міжнародні відносини р різке зростання цін на продукти харчування; скорочення запасів продовольства розширення площ, зайнятих під сільськогосподарським виробництвом, інтенсифікація господарської діяльності коливання цін на продукцію; невизначеність динаміки економічного розвитку збільшення площ сільськогосподарських земель, що викорстовуються під технічні енергомісткі культури (соя, соняшник) Основні характеристики Прояви впливу на земельні відносини дефіцит водних ресурсів як важливої умови виробництва; скорочення сільськогоспо- дарських площ, зокрема ріллі посилення розорюваності, екстенсифікація земле- користування за рахунок розширення задіяних в обробіток земель, деградація та виснаження грунту зміщення географічних меж сільськогоспо- дарських зон, зниження урожайності та зменшення обсягів виробництва розширення регіонів із зрошувальним землероб- ством, нестача води, зміна природно-кліматичних зон вирощування сільськогосподарських культур використання зернових та олійних культур для енергетичних потреб; скорочення пропозиції продуктів харчування на світовому ринку розширення земельних площ під енергетичними культурами; посилення конкуренції за земельні ресурси між виробниками біопалива та продовольства інвестування коштів в аграрний сектор, запровадження вахтового методу господар- ської діяльності, монополізація ринку нарощування земельних банків аграрними холдингами; інтенсифікація виробництва на основі інноваційних технологій землекористування, монокультуризація Агфляція Зміна кон’юнктури ринку Тенденція Екстенсивне використання ресурсів Стрімке потепління Розвиток виробництва біопалива Корпоратизація аграрного бізнесу Рис. 1. E-mail: avs0310@ukr.net Іншим чинником, який суттєво впливає на сільсько господарське землекористування, є зміна клімату. Викиди парникових газів від сільського господарства і пов’язані з ним вирубки лісу, становлять майже третину всіх антро погенних викидів газів. Водночас, за прогнозами FAO, змі на клімату матиме значний вплив на сільськогосподарське земле- і водокористування, що особливо буде проявлятися для країн Африки та Азії [15]. Враховуючи зазначене, фі нансування заходів щодо адаптації сільськогосподарських систем до змін клімату є нагальним завданням для країн, що спеціалізуються на виробництві продовольства. Виклад основного матеріалу дослідження. При розвитку земельних відносин глобалізаційні зміни про являються у такому: агфляція – зростання цін на агропро довольчу продукцію, що здебільшого відбувається у най бідніших країнах світу; різка зміна кон’юнктури аграрного ринку, яка характеризується такими рисами, як нестійкість цін, їх непередбачені злети та падіння; інтенсивне вико ристання природних ресурсів, яке наближається у деяких країнах до абсолютної межі; дефіцит водних ресурсів як важливої умови виробництва продовольства; скорочення сільськогосподарських площ, зокрема ріллі, у результаті індустріалізації; стрімке потепління, яке здатне суттєво вплинути на сільське господарство, викликавши зниження урожайності та зменшення обсягів виробництва продукції, зміщення меж сільськогосподарських зон; швидкий розви ток виробництва біопалива із зернових та олійних культур, що зумовило скорочення пропозиції продуктів харчування на світовому ринку [8; 10; 13; 14]. Логічно виявити кожен із зазначених сучасних глобальних викликів і розробити від повідний методологічний апарат дослідження їх впливу на розвиток земельних відносин (рис. 1). Основна маса продуктів харчування у XXI ст. отри мується лише на 9 % поверхні Землі (рілля, сади, планта ції, луки, пасовища). Згідно із прогнозами FAO, у світовому сільському господарстві може бути використано до 3,2– 3,5 млрд га (20 %), з яких лише 0,45 млрд га після освоєння можуть стати високопродуктивними сільськогосподар ськими землями [15]. При цьому з усіх придатних під ріллю земель може бути зрошено тільки 1,1 млрд га. Дестабілізу ючим чинником є те, що лише на частині угідь, зайнятих під сільськогосподарським виробництвом, використову ються інтенсивні технології землекористування. Таблиця 2 Частка оброблюваних земель, придатних для вирощування продукції рослинництва у світі Група країн Оброблювані землі по регіонах, млн га Населення, млн осіб Площа земель в обробітку на 1 особу, га Богарні землі (%) високої якості середньої якості деградовані З низьким рівнем доходів 441 2651 0,17 28 50 22 З середнім рівнем доходів 735 3223 0,23 27 55 18 З високим рівнем доходів 380 1031 0,37 32 50 19 Всього 1556 6905 0,23 29 52 19 Джерело: розраховано за даними [15; 16] Частка оброблюваних земель, придатних для вирощування продукції рослинництва у світі Джерело: розраховано за даними [15; 16] модифікованої сільськогосподарської продукції. У 2015 р. у світі використовувалося близько 200 млн га земельних угідь для цих цілей. Комерційне виробництво модифіко ваної бавовни зосереджено переважно в африканському та азіатському регіонах, у той час як у країнах Латинської Америки зосереджені основні площі під генетично модифі кованою соєю [16]. Незважаючи на зазначену спеціалізацію у виробництві цих культур, можна стверджувати, що вона пов’язується не стільки з потребами внутрішнього ринку цих країн, скільки обумовлюється зростанням світового попиту, створюючи тим самим для країн-виробників кон курентні переваги. мату підвищує ризик і непередбачуваність господарської діяльності для товаровиробників у результаті потепління і пов’язаної з ним посушливості, зміни сезонності опадів і збільшення числа екстремальних погодних явищ. Ферме ри та підприємства, що спеціалізуються на сільськогоспо дарському виробництві, у країнах із низьким доходом насе лення найбільш уразливі і без належної державної підтрим ки найменше здатні адаптуватися до сучасних глобальних викликів [20]. Характерною тенденцією в умовах глобалізації еко номіки є те, що збільшення ціни на газ і вугілля, нафту і на фтопродукти та поступове виснаження викопних паливних ресурсів змушують країни переходити на альтернативний вид енергоносіїв – біологічне паливо. З 2005 р. у країнах з високим рівнем доходів (США, Німеччина, Франція, Ні дерланди) спостерігається стрімкий розвиток біоенергети ки [15]. Починаючи з цього періоду, зростання конкуренції за земельні ресурси стає все більш очевидним, інвестори активно починають орендувати сільськогосподарські угід дя для енергетичних цілей. На значних площах оброблю ваних земель здійснюється заготівля сировини для біопа лива. Біоенергетика починає конкурувати з виробництвом продовольства. Значні інвестиції країни з високим рівнем доходів спрямовують на дослідження у галузі генетики. Відповід но до розрахунків International Service for the Acquisition of Agri-biotech Applications (ISAAA), у 2016 р. площа, яку за ймають трансгенні культури у світі, становила 150 млн га і збільшилася порівняно з 2009 р. на 14 млн га. E-mail: avs0310@ukr.net Відповідно до аналітичної доповіді Doing Business за останні 50 років площа оброблюваних земель нетто у світі збільшилася на 12 %, в основному – за рахунок скорочення лісових, водно-болотистих і лугових угідь [16; 21]. Водно час площа зрошуваних територій подвоїлася, розподіл зе мельних ресурсів і стан їх використання у різних країнах неоднаковий, що чітко прослідковується залежно від рівня розвитку країн. Регіональний розподіл країн дозволяє стверджува ти, що лише в окремих районах, а саме у країнах з низь ким рівнем доходів, все ще є можливості для нарощування оброблюваних земель. Сприятливими є можливості щодо розширення площі ріллі у країнах Африки і Північної Аме рики, потенційно родючі землі країн Азії освоєні на 90 %, а Європи – на 97 %. Водночас наявність сільськогосподар ських земель не є вирішальним чинником продовольчого забезпечення. Важлива роль в умовах глобалізації та інду стріалізації виробництва належить інноваційному техно логічному оснащенню товаровиробників, які здійснюють сільськогосподарську діяльність і надання їм доступу до кредитних ресурсів. Одним із найпоширеніших методів класифікації кра їн у світовій економіці є об’єднання їх у групи за показни ком валового національного продукту на душу населення. Цей економічний параметр прийнятий головним критері єм Світового банку для економічного аналізу стану роз витку економіки. Зарахування будь-якої країни до тієї чи іншої групи цієї класифікації дає їй можливість отримува ти, за необхідності, пільгові кредити від інвесторів або без коштовну допомогу від розвинених країн та міжнародних організацій. За рівнем доходу країни класифікуються на еконо міки з низьким, середнім і високим доходом. Класифіка ція Світового банку охоплює 209 країн: до першої групи («low-income countries») відносяться держави, у яких ВНП на душу населення становить 725 дол. Джерело: розраховано за даними [15; 16] Потреби в освоєнні нових земель для країн з низь ким і середнім рівнями доходів обумовлені низкою причин: збільшенням кількості населення, підвищенням потреб у продуктах харчування і матеріалах біологічного похо дження, розвитком біоенергетики. Водночас зміна клі Таблиця 2 Америка (37 %) [3]. У середньому для країн із високим до ходом, частка родючих земель складає 32 % (табл. 2). Ґрунти у країнах із низьким доходом часто менш родючі, і тільки 28 % всіх оброблюваних площ класифікуються як землі ви сокої якості. E-mail: avs0310@ukr.net США або менше; до другої групи («middle-income countries») – країни з серед Аналіз розподілу основних категорій земель за група ми країн дозволяє стверджувати, що найвища регіональна частка оброблюваних земель сконцентрована у Централь ній Америці і країнах Карибського басейну (42 %), за ними розміщені Західна і Центральна Європа (38 %) й Північна 8 Проблеми економіки № 2 (40), 2019 Світова економіка та міжнародні відносини Таблиця 1 Таблиця 1 Таблиця 1 Регіональний розподіл основних категорій земель Група країн Частка території в світовому масштабі, % Оброблювані землі Землі під лісами Землі під пасовищами Низькородючі землі Землі під об’єктами інфраструктури млн га % млн га % млн га % млн га % млн га % З низьким рівнем доходів 22 441 15 564 20 1002 36 744 26 52 1,8 З середнім рівнем доходів 53 735 11 2285 33 2266 33 1422 21 69 1 З високим рівнем доходів 25 380 12 880 27 1299 39 592 18 31 1 Джерело: розраховано за даними [15; 16] Регіональний розподіл основних категорій земель Джерело: розраховано за даними [15; 16] Таблиця 3 Країни світу, які використовують сільськогосподарські угіддя для вирощування ГМ-культур Країна Площа під ГМ-культурами, млн га Сільськогосподарські культури США 66,8 соя, кукурудза, бавовник, ріпак Бразилія 25,4 соя, кукурудза, бавовник Аргентина 22,9 соя, кукурудза, бавовник Індія 9,4 бавовник Канада 8,8 соя, кукурудза, ріпак Китай 3,5 бавовник Пакистан 2,4 бавовник Уругвай 2,2 соя, кукурудза, бавовник ПАР 1,1 соя, кукурудза Джерело: розраховано за даними [15; 16] Країни світу, які використовують сільськогосподарські угіддя для вирощування ГМ-культур К ї П і ГМ Сі і Країни світу, які використовують сільськогосподарські угіддя для вирощування ГМ-культур Джерело: розраховано за даними [15; 16] Протилежною тенденцією до вирощування генетич но модифікованих культур є розвиток органічного земле робства. Зацікавленість споживачів органічної продукції продовжує зростати з кожним роком. У розвинутих кра їнах (Японія, США, Німеччина, Італія, Велика Британія) понад 30 % населення споживають органічну продукцію, водночас 37 % із них вживають ці продукти більше одного разу на день, 39 % – принаймні, раз на тиждень і лише 24 % вживають органічні продукти нерегулярно [16]. Органічне землеробство не допускає використання синтетичних за собів сільськогосподарського виробництва, зберігає ґрунт і воду від надмірного забруднення. Воно передбачає систе му комплексного управління, в рамках якої мінімізується застосування або взагалі виключається використання пес тицидів і генетично модифікованих організмів. Основними агротехнічними заходами при органічному землеробстві є такі: сівозміна і поліпшена різноманітність сільськогоспо дарських культур; різні комбінації галузей тваринництва і рослинництва; біологічна боротьба зі шкідниками та бур’янами. швидкими темпами нарощують земельні банки, інтенсифі кують виробничий процес, змінюють технології господа рювання. Зазначені процеси спричиняють суттєвий вплив і на специфіку землекористування. Водночас виникає необхідність розробки нових методологічних підходів до землеволодіння, користування та розпорядження в інте грованих формуваннях холдингового типу. Для характеристики особливостей прояву сучасних глобальних викликів на специфіку використання сільсько господарських земель розглянуто п’ять основних регіонів світу: Азію, Європу, Північну та Південну Америку, Афри ку та Австралію (табл. 4). Кожний з досліджуваних регіонів має свої ресурсні, економічні, соціально-політичні, еколо гічні та природно-кліматичні особливості, які впливають на специфіку землекористування (рис. 2). Так, перспек тиви розвитку сільського господарства Азії нерозривно пов’язані з освоєнням нових площ ріллі, запровадженням зрошуваного землеробства. Водночас більша частина регі ону зайнята горами і пустелями, тому придатних для ве дення сільського господарства земельних ресурсів обме жена кількість. Органічне землеробство має значні комерційні пер спективи через зростання попиту на екологічну продукцію з боку споживачів. В органічному землеробстві у світі за йнято 1,2 млн осіб та близько 32 млн га. Таблиця 2 Лідерами у вирощуванні генетично модифікованих культур, як і рані ше, залишаються США (45 % світових посівів ГМ-культур), Бразилія (17 %) і Аргентина (15 %) (табл. 3). Основні тран сгенні культури – соя (81 % від усіх посівів цієї культури), бавовник (64 %), кукурудза (29 %) та ріпак (23 %). Суттєвого впливу на специфіку землекористуван ня спричиняє також і активізація виробництва генетично Розширення площ під генетично модифіковані куль тури є предметом дискусій багатьох науковців, адже сучасні 9 Проблеми економіки № 2 (40), 2019 Світова економіка та міжнародні відносини культур зростає урожайність, зменшується кількість засо бів захисту рослин, які необхідно використовувати, підви щується ефективність сільськогосподарського виробни цтва. З усіх типів трансгенних рослин найпоширеніші саме невитривалі до гербіцидів. Причому компанія-виробник продає одночасно рослину та засоби її захисту. пізнання в біології, екології та природознавстві не настіль ки вичерпні, аби повністю оцінити наслідки вирощування й використання трансгенних рослин як для організму лю дини, так і для навколишнього природнього середовища. Водночас представники біотехнологічних компаній ствер джують, що при вирощуванні генетично модифікованих Таблиця 3 с.-г. угіддя 1139,6 1135,6 1167,2 1176,1 1172,9 102,9 рілля 199,7 198,6 216,4 224,4 225,9 113,1 Світова економіка та міжнародні відносини Азія Північна та Південна Америка Австралія Африка Європа Регіон 4500 Млн га 4000 3500 3000 2500 2000 1500 1000 500 луки і пасовища 0 3087 3889 1648 1203 2965 2206 1006 906 455 276 171 357 807 794 1173 395 378 226 49 27 0 1092 481 75 30 рілля в т. ч. сільськогосподарські угіддя багаторічні насадження з них сільськогосподарські органічні угілля загальна земельна площа Рис. 2. Склад земельних ресурсів за регіонами світу Джерело: побудовано за даними [15; 16]. Таблиця 3 Найбільш поши реним органічне землеробство є у фермерських господар ствах з площею угідь 50–70 га, які мають змогу отримувати рентабельність виробництва 40–50 %. Ринок органічної продукції у світі постійно зростає. Значна кількість країн (Франція, Італія, Нідерланди, США) мають вже повністю сформовані ринки із широким асортиментом органічної продукції та постійними споживачами. Склад земельних площ Азії за досліджуваний період (1990–2016 рр.) не зазнав суттєвих змін. Спостерігається негативна тенденція щодо скорочення площ сільсько господарських угідь на 1,7 % (28,2 млн га), яка пов’язана з природно-географічними умовами регіону, екстенсифі кацією господарської діяльності та змінами клімату. Осно вними причинами зменшення площ земель сільськогоспо дарського призначення та ріллі є виведення їх із виробни чого обороту внаслідок погіршення якості земель, деграда ції (ерозійні процеси, осушення, утворення солончакових пустель), забруднення ґрунтів хімікатами, недотримання системи сівозмін, ґрунтозахисних технологій, недостат нім внесенням поживних речовин та іншими чинниками як технологічного, так і організаційно-економічного ха Важливою тенденцією в умовах зростаючого попиту на продукти харчування у світі й інтенсифікації виробни цтва є корпоратизації агробізнесу та формування на цій основі інтегрованих структур. Інтегровані формування 10 Проблеми економіки № 2 (40), 2019 11 Проблеми економіки № 2 (40), 2019 Світова економіка та міжнародні відносини Азія Північна та Південна Америка Австралія Африка Європа Регіон 4500 Млн га 4000 3500 3000 2500 2000 1500 1000 500 луки і пасовища 0 3087 3889 1648 1203 2965 2206 1006 906 455 276 171 357 807 794 1173 395 378 226 49 27 0 1092 481 75 30 рілля в т. ч. сільськогосподарські угіддя багаторічні насадження з них сільськогосподарські органічні угілля загальна земельна площа Рис. 2. Склад земельних ресурсів за регіонами світу Джерело: побудовано за даними [15; 16]. Таблиця 4 Динаміка площ сільськогосподарських земель за регіонами світу, млн га Вид угідь Рік 2016 р. до 1990 р., % 1990 2000 2005 2010 2016 1 2 3 4 5 6 7 Азія Загальна земельна площа 3097,1 3094,2 3093,7 3093,5 3087,2 99,7 в т. ч. с.-г. угіддя 1660,4 1666,8 1640,4 1638,8 1648,2 99,3 рілля 481,3 485,4 483 473,2 481,2 100,0 Північна та Південна Америка Загальна земельна площа 3890,7 3890,7 3890,6 3887,8 3889,2 100,0 в т. ч. с.-г. угіддя 1188,4 1193,9 1193,7 1202,7 1203,3 101,3 рілля 359,1 363,7 357,7 357,4 356,8 99,4 Австралія Загальна земельна площа 796,5 794,5 794,5 794,5 794,1 99,7 в т. ч. с.-г. угіддя 474,9 473,7 472 436,1 395,3 83,2 рілля 49,7 49,1 50,2 48 49,4 99,4 Африка Загальна земельна площа 2972,1 2964,3 2964,2 2965,1 2964,6 99,7 в т. ч. Проблеми економіки № 2 (40), 2019 Таблиця 4 Таблиця 4 Динаміка площ сільськогосподарських земель за регіонами світу, млн га Вид угідь Рік 2016 р. до 1990 р., % 1990 2000 2005 2010 2016 1 2 3 4 5 6 7 Азія Загальна земельна площа 3097,1 3094,2 3093,7 3093,5 3087,2 99,7 в т. ч. с.-г. угіддя 1660,4 1666,8 1640,4 1638,8 1648,2 99,3 рілля 481,3 485,4 483 473,2 481,2 100,0 Північна та Південна Америка Загальна земельна площа 3890,7 3890,7 3890,6 3887,8 3889,2 100,0 в т. ч. с.-г. угіддя 1188,4 1193,9 1193,7 1202,7 1203,3 101,3 рілля 359,1 363,7 357,7 357,4 356,8 99,4 Австралія Загальна земельна площа 796,5 794,5 794,5 794,5 794,1 99,7 в т. ч. с.-г. угіддя 474,9 473,7 472 436,1 395,3 83,2 рілля 49,7 49,1 50,2 48 49,4 99,4 Африка Загальна земельна площа 2972,1 2964,3 2964,2 2965,1 2964,6 99,7 в т. ч. с.-г. угіддя 1139,6 1135,6 1167,2 1176,1 1172,9 102,9 рілля 199,7 198,6 216,4 224,4 225,9 113,1 Динаміка площ сільськогосподарських земель за регіонами світу, млн га Проблеми економіки № 2 (40), 2019 11 Світова економіка та міжнародні відносини 1 2 3 4 5 6 7 Європа Загальна земельна площа 2209,4 2208 2208 2207,3 2206,2 99,9 в т. ч. с.-г. угіддя 469,9 468,5 462,5 464,1 455,4 96,9 рілля 287,5 287,5 278,5 277,9 276,2 96,1 Джерело: розраховано за даними [15; 16] Закінчення табл. 4 аграрних регіонів світу. Внутрішні потреби країни повніс тю задовольняються власним виробництвом. Австралія є лідером Кернської групи країн-виробників сільськогос подарської продукції (на частку країн цієї групи припадає 20 % світового експорту). Ефективність австралійського землекористування пояснюється тісною співпрацею уря ду країни з національними асоціаціями виробників сіль ськогосподарської продукції. Держава ініціює наукові до слідження, консультативні й освітні послуги, організовує служби міжнародного маркетингу, регулює рівень цін, надає фінансову підтримку фермерам у періоди засух і по веней, стимулює розвиток екологічно безпечних систем землеробства. аграрних регіонів світу. Внутрішні потреби країни повніс тю задовольняються власним виробництвом. Австралія є лідером Кернської групи країн-виробників сільськогос подарської продукції (на частку країн цієї групи припадає 20 % світового експорту). Ефективність австралійського землекористування пояснюється тісною співпрацею уря ду країни з національними асоціаціями виробників сіль ськогосподарської продукції. Держава ініціює наукові до слідження, консультативні й освітні послуги, організовує служби міжнародного маркетингу, регулює рівень цін, надає фінансову підтримку фермерам у періоди засух і по веней, стимулює розвиток екологічно безпечних систем землеробства. рактеру. Проблемою землекористування досліджуваного регіону є надмірна хімізація виробництва: 10 із 30 млн га сільськогосподарських угідь потребують виведення з ко ристування й реабілітації. Таблиця 4 Регіони Північної та Південної Америки достатньо забезпечені земельними ресурсами, які завдяки своїм властивостям дозволяють вирощувати більшість сільсько господарських культур. Загальний земельний фонд регіону становить 3889 млн га. Сільськогосподарські угіддя займа ють 1194 млн га, з яких рілля – 30 %. Характерною тенденцією у 2005–2015 рр. є еколо гізація землекористування, понад 9,2 млн га сільськогос подарських угідь, що використовуються для органічного землеробства. Найбільші площі з них знаходяться у США (2,0 млн га) та Бразилії (1,8 млн га), вони постійно розши рюються. Суттєві особливості землекористування має Афри канський континент, де зосереджені значні ґрунтові та рос линні ресурси. Водночас наявні сільськогосподарські землі використовуються вкрай неефективно. З низки об’єктивних та суб’єктивних причин у багатьох країнах Африки спосте рігається продовольча криза. Аналізуючи використання земельних ресурсів Африки у 1990–2015 рр., необхідно зазначити їх значне виснаження, що є результатом примі тивного ведення сільськогосподарського виробництва при швидкому рості населення, запровадження монокультури, розорювання нових малопродуктивних земель, переванта ження у структурі угідь пасовищ. Екстенсивні методи ве дення землеробства прискорюють деградацію ґрунтів і їх виснаження. Завдяки сприятливому природному середовищу й ефективному використанню земельних ресурсів США повністю забезпечує власні продовольчі потреби і є най більшим у світі експортером сільськогосподарської про дукції. Для потреб аграрного сектора у країні використову ється понад 190 млн га орних земель і 220 млн га пасовищ, близько 20 млн га земель зрошується. Головна перевага сільського господарства США – його висока продуктив ність. За вартістю продукції, виробленої на одного зайня того, з США можуть конкурувати лише Канада, Австралія та Нова Зеландія. Значна кількість земель використовуєть ся для енергетичних потреб – виробництва альтернатив них видів палива. Деградація сільськогосподарських земель спричи няється і перетворенням їх в антропогенні ландшафти. За значені процеси суттєво зменшують площі резервних для освоєння земель у світі (табл. 5). Важливим гравцем на аграрному ринку світу є Ав стралія, яка має значні земельні ресурси, що ефективно ви користовуються. За розмірами оброблюваної поверхні на одного мешканця Австралія значно випереджає більшість Загальною тенденцією для усіх досліджених регіонів (Азії, Європи, Північної та Південної Америки, Африки та Проблеми економіки № 2 (40), 2019 Джерело: розраховано за даними [15; 16] ЛІТЕРАТУРА 1. Заяць В. М. Розвиток ринку сільськогосподарських зе мель : монографія. Київ : ННЦ ІАЕ, 2011. 390 с. 1. Заяць В. М. Розвиток ринку сільськогосподарських зе мель : монографія. Київ : ННЦ ІАЕ, 2011. 390 с. 2. Звіт про наявність земель та розподіл їх за власниками землі, землекористувачами, угіддями та видами економічної діяльності на 01.01.2015 р. / Державна служба статистики Украї ни. Київ, 2015. 12 с. 2. Звіт про наявність земель та розподіл їх за власниками землі, землекористувачами, угіддями та видами економічної діяльності на 01.01.2015 р. / Державна служба статистики Украї ни. Київ, 2015. 12 с. Таблиця 5 Таблиця 5 Резервні площі сільськогосподарських земель, придатних для освоєння у світі Регіон Орнопридатні землі, млн га Освоєні в землеробстві, млн га Ступінь освоєності, % Резерви для освоєння, млн га Частка у світовому резерві, % Європа 160 140 88 20 2 Азія 600 460 77 140 14 Африка 430 185 43 245 25 Північна Америка 380 275 72 105 11 Південна Америка 410 140 34 270 27 Австралія 130 50 38 80 8 Увесь світ 2470 1480 60 990 100 Джерело: розраховано за даними [15; 16] Резервні площі сільськогосподарських земель, придатних для освоєння у світі Проблеми економіки № 2 (40), 2019 12 Світова економіка та міжнародні відносини 12. Cheshire P. C. Land market regulation: market versus policy failures. Journal of Property Research. 2013. Vol. 30 (3). Р. 170–188. 12. Cheshire P. C. Land market regulation: market versus policy failures. Journal of Property Research. 2013. Vol. 30 (3). Р. 170–188. Австралії) є процес деградації сільськогосподарських зе мель. Так, внаслідок ерозії, із обороту щорічно виводиться 6–7 млн га, а через заболочування і засолення – ще 1,5 млн га. Серйозну загрозу земельному фондові у країнах Афри ки та Азії становить опустелювання раніше оброблюваних земель. 13. Dankevych V., Dankevych Y., Pyvovar P. Clustering of the international agricultural trade between Ukraine and the EU. Management Theory and Studies for Rural Business and Infrastructure Development. 2018. Vol. 40. No. 3. Р. 307–319. Висновки. Результати проведених досліджень до зволяють констатувати, що в умовах глобалізації економі ки ризики у землекористуванні почали проявлятися у всіх регіонах світу з новою силою. Низька ефективність засто сування традиційних антикризових заходів і механізмів прийняття рішень щодо оптимізації землекористування свідчать про їх невідповідність новим реаліям. Глобальні ризики, особливо в аграрній сфері, не піддаються прогно зуванню та потребують значних затрат на мінімізацію їх негативного впливу. 14. Dankevych Y., Dankevych V., Chaikin O. Ukraine agricultural land market formation preconditions. Acta Univ. Agric. Silvic. Mendelianae Brun. 2017. No. 65. P. 259–271. 15. European statistics. Eurostat : database // European Comission. Brusseles, 2015. URL: http://ec.europa.eu/eurostat/ data/database 16. FAOSTAT. URL: http://faostat.fao.org/site 17. Fedets I. Demand and Supply for Agricultural Land / Institute for Economic Research and Policy Consulting, Land Reform Barometer. 2012. № 5. URL: http://www.ier.com.ua/ua/ land_reform_barometer/project_publications При екстенсифікації господарської діяльності значні масиви земель знаходяться під загрозою поступового зни ження свого виробничого потенціалу із причини надмір ного демографічного навантаження і нестійкості методів ведення сільського господарства. 18. Institutional Transformation of Ukraine's Agricultural Sector / T. Zinchuk, N. Kutsmus, V. Таблиця 5 Dankevych [et al.]. Review of Economic Perspectives. 2017. Vol. 17, iss. 1. P. 57–80. fl 19. Sagoff M. Environmental Economics and the Conflation of Value and Benefit. Environmental Science & Technology. 2000. No. 34:8. Р. 1426–1432. До фізичних обмежень доступності земельних ресур сів у окремих країнах додаються і зовнішні чинники: зміна клімату, конкуренція з іншими галузями та соціально- економічні зміни. 20. Statistical Yearbook «Land Governance Monitoring in Ukraine: 2016–2017». URL: http://www.kse.org.ua/en/research- policy/land/governance-monitoring/yearbook-2016-2017/ 21. Wenbing T., Xuemei L. Guidance of the coordination theory of man-land relationship to land exploitation and utilization // Industrial Economics System and Industrial Security Engineering (IEIS'2017), 2017 4th International Conference on. IEEE, 2017. Р. 1–5. REFERENCES Cheshire, P. C. “Land market regulation: market versus policy failures“. Journal of Property Research, vol. 30 (3) (2013): 170-188. Cheshire, P. C. “Land market regulation: market versus policy failures“. Journal of Property Research, vol. 30 (3) (2013): 170-188. 3. Звіт ФАО «The State of Food Insecurity in the World». URL: http://www.fao.org/docrep/014/i2330e/i2330 3. Звіт ФАО «The State of Food Insecurity in the World». URL: http://www.fao.org/docrep/014/i2330e/i2330 Dankevych, V., Dankevych, Y., and Pyvovar, P. “Clustering of the international agricultural trade between Ukraine and the EU“. Management Theory and Studies for Rural Business and Infrastructure Development, vol. 40, no. 3 (2018): 307-319. 4. Земельна реформа в Україні в контексті розвитку аграрної економіки та розвитку сільських територій : інформ.- просвітн. вид. / Ін-т розвитку аграр. ринків. Київ, 2013. 22 с. 4. Земельна реформа в Україні в контексті розвитку аграрної економіки та розвитку сільських територій : інформ.- просвітн. вид. / Ін-т розвитку аграр. ринків. Київ, 2013. 22 с. Dankevych, Y., Dankevych, V., and Chaikin, O. “Ukraine ag ricultural land market formation preconditions“. Acta Univ. Agric. Silvic. Mendelianae Brun, no. 65 (2017): 259-271. 5. Земельна реформа на регіональному рівні (на прикла ді Київської області за 1991–2011 рр.) / Й. М. Дорош, С. О. Осип чук, М. П. Стецюк, О. С. Дорош. Київ : ВІПОЛ, 2011. 182 с. Dorosh, Y. M. et al. Zemelna reforma na rehionalnomu rivni (na prykladi Kyivskoi oblasti za 1991-2011 rr.) [Land reform at the regional level (for example, the Kiev region for 1991-2011)]. Kyiv: VIPOL, 2011. Dorosh, Y. M. et al. Zemelna reforma na rehionalnomu rivni (na prykladi Kyivskoi oblasti za 1991-2011 rr.) [Land reform at the regional level (for example, the Kiev region for 1991-2011)]. Kyiv: VIPOL, 2011. 6. Земельна реформа: заходи, що мають передувати її завершенню / Я. М. Гадзало та ін. Вісник аграрної науки. 2015. № 9. С. 5–11. 7. Зінчук Т. О., Данкевич В. Є. Європейський досвід фор мування ринку сільськогосподарських земель. Економіка АПК. 2016. № 12. С. 84–92. “European statistics. Eurostat : database“. European Comis sion Brusseles 2015 http://ec europa eu/eurostat/data/database sion. Brusseles, 2015. http://ec.europa.eu/eurostat/data/database FAOSTAT. http://faostat.fao.org/site sion. Brusseles, 2015. http://ec.europa.eu/euro FAOSTAT. http://faostat.fao.org/site 8. Національна доповідь щодо завершення земельної реформи / [Я. М. Гадзало, С. О. Балюк та ін.] ; за ред. Л. Я. Нова ковського. Київ : Аграрна наука, 2015. 48 с. Fedets, I. “Demand and Supply for Agricultural Land“. Insti tute for Economic Research and Policy Consulting, Land Reform Barometer. 2012. http://www.ier.com.ua/ua/land_reform_barom eter/project_publications Fedets, I. REFERENCES “Demand and Supply for Agricultural Land“. Insti tute for Economic Research and Policy Consulting, Land Reform Barometer. 2012. http://www.ier.com.ua/ua/land_reform_barom eter/project_publications 9. Результати опитування щодо моделей обігу земель сільськогосподарського призначення в Україні / авт. кол. Ю. О. Лупенко, О. В. Ходаківська, О. М. Шпичак та ін. Київ : ННЦ «ІАЕ», 2018. 52 с. Hadzalo, Ya. M. et al. “Zemelna reforma: zakhody, shcho mai ut pereduvaty yii zavershenniu“ [Land reform: measures that must precede its completion]. Visnyk ahrarnoi nauky, no. 9 (2015): 5-11. Hadzalo, Ya. M. et al. Natsionalna dopovid shchodo zaversh ennia zemelnoi reformy [National report on completion of land re form]. Kyiv: Ahrarna nauka, 2015. Hadzalo, Ya. M. et al. “Zemelna reforma: zakhody, shcho mai ut pereduvaty yii zavershenniu“ [Land reform: measures that must d i l i ] Vi k h i k 9 (20 ) 10. Розвиток земельних відносин в аграрній сфері : мо нографія / за ред. Ю. О. Лупенка, О. В. Ходаківської. Київ : ННЦ «ІАЕ», 2016. 430 с. p ennia zemelnoi reformy [National report on completion of land re form]. Kyiv: Ahrarna nauka, 2015. 11. Смит А. Исследование о природе и причинах богат ства народов. М. : Наука, 1962. 684 с. Lupenko, Yu. O. et al. Rezultaty opytuvannia shchodo modelei obihu zemel silskohospodarskoho pryznachennia v Ukraini [Survey Проблеми економіки № 2 (40), 2019 13 Світова економіка та міжнародні відносини results on agricultural land use patterns in Ukraine]. Kyiv: NNTs «IAE», 2018. Rozvytok zemelnykh vidnosyn v ahrarnii sferi [Devel opment of land relations in the agrarian sector]. Kyiv: NNTs «IAE», 2016. Zaiats, V. M. Rozvytok rynku silskohospodarskykh zemel [De velopment of agricultural land market]. Kyiv: NNTs IAE, 2011. Zaiats, V. M. Rozvytok rynku silskohospodarskykh zemel [De velopment of agricultural land market]. Kyiv: NNTs IAE, 2011. Zemelna reforma v Ukraini v konteksti rozvytku ahrarnoi ekonomiky ta rozvytku silskykh terytorii [Land reform in Ukraine in the context of the development of the agrarian economy and the development of rural areas]. Kyiv, 2013. Sagoff, M. “Environmental Economics and the Conflation of Value and Benefit“. Environmental Science & Technology, no. 34:8 (2000): 1426-1432. Zinchuk, T. et al. “Institutional Transformation of Ukraine's Agricultural Sector“. Review of Economic Perspectives, vol. 17, no. 1 (2017): 57-80. Smit, A.Issledovaniye o prirode i prichinakh bogatstva naro dov [Research on the nature and causes of the wealth of nations]. Moscow: Nauka, 1962. Zinchuk, T. O., and Dankevych, V. Ye. Стаття надійшла до редакції 17.04.2019 р. Проблеми економіки № 2 (40), 2019 REFERENCES “Yevropeiskyi dosvid formuvannia rynku silskohospodarskykh zemel“ [European experi ence in the formation of the agricultural land market]. Ekonomika APK, no. 12 (2016): 84-92. Statistical Yearbook «Land Governance Monitoring in Ukraine: 2016-2017». http://www.kse.org.ua/en/research- policy/land/governance-monitoring/yearbook-2016-2017/ TAN, Wenbing, and LI, Xuemei. “Guidance of the coordination the ory of man-land relationship to land exploitation and utilization“. Industrial Economics System and Industrial Security Engineering (IE IS'2017), 2017 4th International Conference on. IEEE, 2017. 1-5. Zvit pro naiavnist zemel ta rozpodil yikh za vlasnykamy zemli, zemlekorystuvachamy, uhiddiamy ta vydamy ekonomichnoi diialnos ti na 01.01.2015 r. [Report on the availability of land and their distri bution by land owners, land users, lands and types of economic ac tivity as of 01.01.2015]. Kyiv: Derzhavna sluzhba statystyky Ukrainy, 2015. “Zvit FAO «The State of Food Insecurity in the World»“ [FAO report The State of Food Insecurity in the World]. http://www.fao. org/docrep/014/i2330e/i2330 Стаття надійшла до редакції 17.04.2019 р. 14 Проблеми економіки № 2 (40), 2019 Проблеми економіки № 2 (40), 2019
https://openalex.org/W2119574612	https://dash.harvard.edu/bitstream/1/10594299/1/3263885.pdf	English	null	Lifestyle Counseling in Routine Care and Long-Term Glucose, Blood Pressure, and Cholesterol Control in Patients With Diabetes	Diabetes care	2,012	cc-by	6,861	Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of-use#LAA This article was downloaded from Harvard University’s DASH repository, under the terms and conditions applicable to Other Posted Material, as s nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of-use#LAA Permanent link http://nrs.harvard.edu/urn-3:HUL.InstRepos:10594299 Citation Morrison, Fritha, Maria Shubina, and Alexander Turchin. 2012. Lifestyle counseling in routine care and long-term glucose, blood pressure, and cholesterol control in patients with diabetes. Diabetes Care 35(2): 334-341. Published Version doi:10.2337/dc11-1635 Share Your Story The Harvard community has made this article openly available. Please share how this access benefits you. Submit a story . Accessibility E p i d e m i o l o g y / H e a l t h S e r v i c e s R e s e a r c h O R I G I N A L A R T I C L E CONCLUSIONSd CONCLUSIONSd Lifestyle counseling in the primary care setting is strongly associated with faster achievement of A1C, blood pressure, and LDL cholesterol control. These results conﬁrm that the ﬁndings of controlled clinical trials are applicable to the routine care setting and provide evidence to support current treatment guidelines. Diabetes Care 35:334–341, 2012 y Patients with diabetes seen by primary care physicians(PCPs)afﬁliatedwiththeBrigham and Women’s Hospital (BWH) and Massachusetts General Hospital (MGH) for at least 2 years between 1 January 2000 and 1 January 2010 were identiﬁed. Patients were included in the analysis if they were at least 18 years old, had a documented di- agnosis of diabetes or hemoglobin A1C $7.0%, and at least one instance of A1C, blood pressure, or LDL cholesterol above treatment target. Patients with missing zip codes were excluded to enable adjustment for median household income by zip code. D weight counseling with follow-up for patients with diabetes (11,12). Many short-term randomized clinical trials have shown that intensive lifestyle coun- seling interventions of up to 1 year in du- ration can lead to lower blood glucose (13–16) and blood pressure (17–21), but long-term data on the efﬁcacy of life- style counseling are lacking (22–24). Fur- thermore, clinical trials typically involve resource-intensive interventions that may not be feasible in routine care, and the D iabetes is increasingly common in the U.S. and worldwide (1,2). Ele- vated blood glucose, blood pressure, and LDL cholesterol are associated with in- creased risk for micro- and macrovascular complications, and their reduction de- creases the risk (3–8). Nevertheless, most patients with diabetes do not have A1C, blood pressure, and LDL cholesterol under control (9,10). American and European guidelines widely recommend diet, exercise, and This study was approved by the Part- ners HealthCare System institutional re- view board; the requirement for written informed consent was waived. From the 1Division of Endocrinology, Brigham and Women’s Hospital, Boston, Massachusetts; the 2Harvard Medical School, Boston, Massachusetts; and 3Clinical Performance Measurement, Partners HealthCare System, Boston, Massachusetts. From the 1Division of Endocrinology, Brigham and Women’s Hospital, Boston, Massachusetts; the 2Harvard Medical School, Boston, Massachusetts; and 3Clinical Performance Measurement, Partners HealthCare System, Boston, Massachusetts. RESEARCH DESIGN AND METHODSd RESEARCH DESIGN AND METHODSdThis retrospective cohort study of 30,897 patients with diabetes aimed to determine whether lifestyle counseling is associated with time to A1C, blood pressure, and LDL cholesterol control in patients with diabetes. Patients were included if they had at least 2 years of follow-up with primary care practices afﬁliated with two teaching hospitals in eastern Massachusetts between 1 January 2000 and 1 January 2010. RESULTSdComparing patients with face-to-face counseling rates of once or more per month versus less than once per 6 months, median time to A1C ,7.0% was 3.5 versus 22.7 months, time to blood pressure ,130/85 mmHg was 3.7 weeks versus 5.6 months, and time to LDL cholesterol ,100 mg/dL was 3.5 versus 24.7 months, respectively (P , 0.0001 for all). In multivariable analysis, one additional monthly face-to-face lifestyle counseling episode was as- sociated with hazard ratios of 1.7 for A1C control (P , 0.0001), 1.3 for blood pressure control (P , 0.0001), and 1.4 for LDL cholesterol control (P = 0.0013). care.diabetesjournals.org Lifestyle Counseling in Routine Care and Long-Term Glucose, Blood Pressure, and Cholesterol Control in Patients With Diabetes FRITHA MORRISON, MPH1 MARIA SHUBINA, SCD1 ALEXANDER TURCHIN, MD, MS1,2,3 FRITHA MORRISON, MPH1 MARIA SHUBINA, SCD1 ALEXANDER TURCHIN, MD, MS1,2,3 efﬁcacy of lifestyle counseling in everyday clinical practice remains questionable (25–27). Consequently, further evidence is needed to establish that lifestyle coun- seling as practiced in routine care im- proves the outcomes of patients with diabetes. ALEXANDER TURCHIN, MD, MS1,2,3 OBJECTIVEdIn clinical trials, diet, exercise, and weight counseling led to short-term improvements in blood glucose, blood pressure, and cholesterol levels in patients with diabetes. However, little is known about the long-term effects of lifestyle counseling on patients with diabetes in routine clinical settings. We therefore conducted a retrospec- tive study of over 30,000 patients with diabetes and hyperglycemia, hyperten- sion, and/or hyperlipidemia who received care in a primary care setting to test the hypothesis that higher rates of lifestyle counseling in routine care are associated with better diabetes control. RESEARCH DESIGN AND METHODSdWe conducted a retro- spective cohort study to determine the optimal lifestyle counseling rate for pa- tients with diabetes. We evaluated the relationship between the average counsel- ing rate and time to A1C, blood pressure, and LDL cholesterol control. Study cohort Diabetes Care 35:334–341, 2012 Study measurements As patients’ measures could ﬂuctuate above and below target multiple times dur- ing the study period, one patient could contribute more than one period to the analysis. A combined uncontrolled period started on the ﬁrst date when any of the three measures was above the treatment target and ended on the ﬁrst subsequent excluded to focus the analysis on the pri- mary care setting. Finally, hyperglycemic and hyperlipidemic periods where rate of change of A1C and LDL cholesterol, re- spectively, was greater than 3 SD from the mean were excluded to eliminate likely measurement errors from the analysis. Time to target for A1C, blood pressure, and LDL cholesterol during the respective un- controlled periods was the length of the uncontrolled period. Lifestyle counseling instances were deﬁned as distinct days when a PCP provided diet, exercise, or weight counseling during the uncontrolled period. Documentation of lifestyle coun- seling was computationally abstracted from the notes, including direct, such as “strongly encouraged more walking,” and inferred, for example “weight has gone up,” instances of lifestyle counseling, as previously described (30). We inferred lifestyle counseling if the subject was The lowest measurement on a given date was used in the analysis. Lowest blood pressure was deﬁned as the blood pressure measurement with the lowest mean arterial pressure. Transient elevations were deﬁned as periods that contained only a single elevated measurement that subsequently normalized without any medication inten- siﬁcation and were excluded from the anal- ysis. Uncontrolled periods without at least one annual encounter with a BWH/MGH PCP were excluded. Periods without any medication information available in the electronic medical record (EMR) were excluded to enable inclusion of insulin treatment as a confounder variable in the analysis. Study measurements y A single uncontrolled period served as the unit of analysis. We conducted four analyses: one for each of the three treat- ment targets (A1C, blood pressure, and LDL cholesterol) and a combined analysis This article contains Supplementary Data online at http://care.diabetesjournals.org/lookup/suppl/doi:10 .2337/dc11-1635/-/DC1. This article contains Supplementary Data online at http://care.diabetesjournals.org/lookup/suppl/doi:10 .2337/dc11-1635/-/DC1. © 2012 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for proﬁt, and the work is not altered. See http://creativecommons.org/ licenses/by-nc-nd/3.0/ for details. © 2012 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for proﬁt, and the work is not altered. See http://creativecommons.org/ licenses/by-nc-nd/3.0/ for details. DIABETES CARE, VOLUME 35, FEBRUARY 2012 334 care.diabetesjournals.org Morrison, Shubina, and Turchin that integrated all three. We used treatment goals recommended at the beginning of the study period: A1C ,7.0% (28), blood pressure ,130/85 mmHg (28,29), and LDL cholesterol ,100 mg/dL (28). For analyses of individual treatment targets, an uncontrolled period started on the day when the relevant measurement (A1C, blood pressure, or LDL cholesterol for hy- perglycemic, hypertensive, and hyperlipi- demic periods, respectively) was noted to ﬁrst be above the treatment target. The pe- riod ended on the ﬁrst subsequent date when the measurement fell below the tar- get. As patients’ measures could ﬂuctuate above and below target multiple times dur- ing the study period, one patient could contribute more than one period to the analysis. A combined uncontrolled period started on the ﬁrst date when any of the three measures was above the treatment target and ended on the ﬁrst subsequent date when all of the measures were below their targets. Last known value was carried forward if all measurements were not avail- able on the same date. that integrated all three. We used treatment goals recommended at the beginning of the study period: A1C ,7.0% (28), blood pressure ,130/85 mmHg (28,29), and LDL cholesterol ,100 mg/dL (28). For analyses of individual treatment targets, an uncontrolled period started on the day when the relevant measurement (A1C, blood pressure, or LDL cholesterol for hy- perglycemic, hypertensive, and hyperlipi- demic periods, respectively) was noted to ﬁrst be above the treatment target. The pe- riod ended on the ﬁrst subsequent date when the measurement fell below the tar- get. Study measurements Periods that contained multiple encounters with an endocrinologist were Hyperglycemic period patients Hyperlipidemic period patients Hypertensive period patients Combined uncontrolled period patients n 17,404 18,639 30,784 30,897 Age (years)* 60.1 (13.8) 58.4 (13.4) 60.4 (13.9) 59.5 (14.1) Women, n (%) 8,941 (51.4) 10,301 (55.3) 16,274 (52.9) 16,117 (52.2) Race/ethnicity, n (%) White 10,756 (61.8) 11,528 (61.9) 20,882 (67.8) 20,937 (67.8) Black 2,388 (13.7) 2,544 (13.7) 3,561 (11.6) 3,371 (10.9) Hispanic 2,494 (14.3) 2,742 (14.7) 3,619 (11.8) 3,684 (11.9) Other† 1,766 (10.1) 1,825 (9.8) 2,722 (8.8) 2,905 (9.4) English as the primary language, n (%) 14,050 (80.7) 15,112 (81.1) 25,745 (83.6) 25,686 (83.1) Health insurance, n (%) Private 6,946 (39.9) 8,128 (43.6) 12,611 (41.0) 12,885 (41.7) Medicare 8,403 (48.3) 8,362 (44.9) 15,100 (49.1) 14,921 (48.3) Medicaid 1,771 (10.2) 1,872 (10.0) 2,657 (8.6) 2,662 (8.6) None/unknown 284 (1.6) 277 (1.5) 416 (1.4) 429 (1.4) Median income by zip code ($1,000) 52.0 (20.8) 52.5 (21.4) 53.0 (20.6) 53.3 (20.7) Number of uncontrolled periods 1.6 (0.9) 1.4 (0.7) 3.7 (2.9) 2.3 (1.9) Hemoglobin A1C (%) 7.7 (1.2) 7.2 (1.3) Systolic blood pressure (mmHg) 130.5 (10.0) 129.5 (10.6) Diastolic blood pressure (mmHg) 74.7 (6.7) 74.4 (6.8) LDL cholesterol (mg/dL) 108.7 (23.2) 99.2 (27.5) BMI (kg/m2), mean (SD, % patients with measures) 32.8 (7.4, 63.2%) 32.6 (7.2, 67.2%) 32.6 (7.3, 66.3%) 32.4 (7.2, 65.1%) Charlson comorbidity index 6.2 (4.6) 5.5 (4.5) 5.6 (4.5) 5.4 (4.5) Follow-up time (months) 80.9 (28.2) 83.3 (28.1) 75.8 (29.5) 74.8 (29.4) Total time above treatment target (months) 35.4 (30.0) 37.1 (28.2) 31.2 (24.7) 52.4 (33.6) Data are mean (SD), unless otherwise indicated. *Age calculated at the start date of the ﬁrst uncontrolled period. †Includes unknown. care diabetesjournals org DIABETES CARE VOLUME 35 FEBRUARY 2012 335 DIABETES CARE, VOLUME 35, FEBRUARY 2012 335 Lifestyle counseling and diabetes control A1C, blood pressure, and LDL cholesterol targets between different counseling rates. referred to in a way that made it likely that it was discussed with the patient (e.g., not simply weight recorded in the vital signs section). The natural language processing software was previously validated and had a sensitivity and speciﬁcity that ranged between 91–97 and 88–94%, re- spectively. Weight counseling was lim- ited to periods when the patient had BMI $30 kg/m2. During the study pe- riod, none of the study practices had a program that encouraged a particular type of lifestyle counseling or monitored lifestyle counseling delivered by provid- ers. Study measurements To capture both face-to-face and re- mote interactions between patients and providers, we deﬁned any note in the EMR as an encounter and any direct or inferred mention of lifestyle counseling in the notes as lifestyle counseling. Dates on which billing data included Current Pro- cedural Terminology codes for evaluation and management were considered face- to-face lifestyle counseling encounters, whereas all other instances of lifestyle counseling were considered remote. Aver- age lifestyle counseling rate was calculated by dividing the number of instances of life- style counseling by the period length. In our analyses, we categorized counseling rates as once or more per month, as less than once per month and once or more per 6 months, and as less than once per 6 months. Mean encounter interval was de- termined by dividing the period length by the number of encounters with PCPs dur- ing that period. Medication intensiﬁcation was deﬁned as initiation of a new or an in- crease in the dose of an existing medication (31). Medication intensiﬁcation rate was deﬁned as the number of unique dates per month on which at least one medica- tion in the relevant class was intensiﬁed. The patient’s PCP was deﬁned as the phy- sician in a primary care practice who had the most encounters with the patient dur- ing the uncontrolled period. Demographic information, weight, height, blood pressure measurements, and medication and laboratory data medication intensiﬁcation rate, presence of obesity during the period, A1C and LDL cholesterol measurement rate and maximumA1C,systolicblood pressure, di- astolic blood pressure, and LDL cholesterol (where appropriate). P values were ob- tained using a type III test. targets between different counseling rates. Marginal Cox proportional-hazards model for clustered data (32) was used to estimate the association between time to target and lifestyle counseling rate while accounting for clustering within patient- provider pairs. Two models were run: one with an overall lifestyle counseling rate and one with separate rates for face- to-face and remote lifestyle counseling. The models also adjusted for demographic confounders (age, sex, race, primary lan- guage, health insurance, and median in- come by zip code) as well as a patient’s Charlson comorbidity index (33) for the period of the study, insulin administration as a marker of severity of disease (in hy- perglycemic and combined uncontrolled periods), PCP encounter frequency, All analyses were performed with SAS statistical software, version 9.2 (SAS In- stitute, Inc., Cary, NC). Study measurements RESULTSdWe identiﬁed 37,863 adults with diabetes who were regularly seen by BWH or MGH PCPs and had experienced at least one hyperglycemic, hypertensive, or hyperlipidemic period (Supplementary Fig.). We excluded 6,702 hyperglycemic, 5,760 hypertensive, and 6,428 hyper- lipidemic patients because of treatment by endocrinologists; no PCP at BWH or MGH; Table 2dUncontrolled period characteristics Table 2dUncontrolled period characteristics Table 2 Uncontrolled period characteristics Hyperglycemic periods Hyperlipidemic periods Hypertensive periods Combined uncontrolled periods Study periods, n 26,984 26,893 112,716 72,532 Period length (months) 22.8 (24.9) 25.7 (25.0) 8.5 (11.7) 22.3 (28.2) Average initial hemoglobin A1C (%) 8.1 (1.4) Average initial LDL cholesterol (mg/dL) 126.8 (25.4) Average initial systolic blood pressure (mmHg) 140.0 (12.8) Average initial diastolic blood pressure (mmHg) 78.1 (10.7) Average maximum hemoglobin A1C (%) 8.7 (1.9) 7.8 (2.0) Average maximum LDL cholesterol (mg/dL) 136.6 (30.5) 111.2 (40.4) Average maximum systolic blood pressure (mmHg) 148.4 (17.4) 149.3 (19.6) Average maximum diastolic blood pressure (mmHg) 83.8 (10.5) 84.9 (10.9) Periods where treatment target was reached, n (%) 18,526 (68.7) 20,903 (77.8) 108,737 (92.1) 52,109 (71.9) Rate of medication intensiﬁcation per month 0.09 (0.14) 0.06 (0.12) 0.22 (1.1) 0.17 (0.84) Rate of measure testing per month 0.23 (0.14) 0.17 (0.17) 1.0 (1.6) Rate of face-to-face lifestyle counseling per month 0.24 (0.26) 0.20 (0.23) 0.36 (0.97) 0.24 (0.74) Rate of remote lifestyle counseling per month 0.12 (0.20) 0.09 (0.18) 0.20 (0.69) 0.18 (0.60) Encounter interval (months) 1.9 (1.7) 2.3 (1.9) 1.5 (1.5) 1.9 (1.8) Periods with patients on insulin, n (%) 7,194 (26.7) 13,646 (18.8) Periods with patients who are obese, n (%) 15,469 (57.3) 15,608 (58.0) 63,837 (56.6) 39,483 (54.4) Data are mean (SD), unless otherwise indicated. 2 care.diabetesjournals.org Demographic information, weight, height, blood pressure measurements, and medication and laboratory data were obtained from the EMR at Partners HealthCaredan integrated health care de- livery network in eastern Massachusetts that includes BWH and MGH. Statistical analysis Summary statistics were constructed by using frequencies and proportions for cat- egorical data and using means, SDs, me- dians, and ranges for continuous variables. Log-rank test was used to compare times to 336 DIABETES CARE, VOLUME 35, FEBRUARY 2012 336 Morrison, Shubina, and Turchin uncontrolled periods was 8.1%, 140/78 mmHg, and 126.8 mg/dL (Table 2). Sub- sequently median times to reach treatment targets ranged from 19 weeks (for hyper- tensive patients) to over 16 months (for hyperlipidemic patients). Hyperglycemic patients had A1C above target a mean of 46.5% of the time, hypertensive patients had uncontrolled blood pressure 42.0% of the time, and hyperlipidemic patients had elevated LDL cholesterol 46.7% of the time. median remote lifestyle counseling rates ranged from once every 25 months for hy- perglycemic periods to never for hyperten- sive periods (Table 2). Mean times between patient encounters with a PCP were 1.9 months when hyperglycemic, 1.5 months when hypertensive, and 2.3 months when hyperlipidemic. During hyperglycemic pe- riods, A1C testing occurred on average just over once every 4 months, blood pressure was measured once every month during hypertensive periods, and LDL cholesterol was measured once every 6 months during hyperlipidemic periods. Antihyperglyce- mic medications were intensiﬁed on av- erage just over once every 11 months, no medication records; only transient ele- vations in A1C, blood pressure, and LDL cholesterol; suspected A1C or LDL cho- lesterol measurement errors; and missing demographic information. The remaining 17,404 hyperglycemic, 30,784 hyperten- sive, and 18,639 hyperlipidemic patients (a total of 30,897 unique individuals) were included in the study. Study patients (Table 1) did not have their A1C, blood pressure, or LDL choles- terol under control 71.3% of the time, and 66% of patients never achieved full control during the study period. Their mean initial A1C, blood pressure, and LDL choles- terol at the beginning of the respective Medianface-to-face lifestylecounseling rates ranged from once every 5.3 months for hyperglycemic periods to once every 8 months for hypertensive periods, whereas Figure 1dLifestyle counseling frequency and time to treatment target. Kaplan-Meier curves for time to treatment target from ﬁrst elevated A1C, blood pressure, or LDL cholesterol were plotted for different average counseling rates. Distinct uncontrolled periods (from the ﬁrst elevated to the ﬁrst normal measurement) for the same patient were analyzed separately. A: Lifestyle counseling frequency and time to A1C target. B: Lifestyle counseling frequency and time to blood pressure target. care.diabetesjournals.org Lifestyle counseling and diabetes control Lifestyle counseling and diabetes control Table 3dEffects of patient and treatment characteristics on time to treatment target antihypertensive medications once every 4.5 months, and antihyperlipidemic med- ications almost once every 17 months. Overall, patients with at least one of the measurements above target had their treatment intensiﬁed on average once ev- ery 6 months. Hazard ratio 95% Conﬁdence limits P value (x2) Hyperglycemic periods Normalized maximum A1C, per 1% increase 0.539 0.528 0.550 ,0.0001 Normalized age, per 1-year increase 0.993 0.991 0.994 ,0.0001 Female 0.881 0.851 0.911 ,0.0001 Non–English speaker 0.990 0.936 1.046 0.7098 Caucasian 1.000 Black 1.115 1.061 1.171 ,0.0001 Hispanic 1.099 1.028 1.174 0.0058 Asian 1.019 0.934 1.112 0.6658 Other/unknown 0.987 0.919 1.061 0.7310 Income, per $1,000 increase 0.999 0.999 1.000 0.1831 Nonprivate insurance 0.965 0.926 1.005 0.0853 On insulin 0.630 0.602 0.659 ,0.0001 PCP encounter interval, log(months) 0.803 0.770 0.837 ,0.0001 Obesity during period 0.887 0.856 0.920 ,0.0001 Charlson comorbidity index 1.016 1.011 1.020 ,0.0001 Rate of A1C testing, per month 29.812 19.482 45.620 ,0.0001 Rate of antihyperglycemic medication intensiﬁcation, per month 2.727 2.156 3.450 ,0.0001 Rate of face-to-face lifestyle counseling, per month 1.705 1.422 2.044 ,0.0001 Rate of remote lifestyle counseling, per month 1.699 1.488 1.941 ,0.0001 Hypertensive periods Normalized maximum systolic blood pressure, per 1- mmHg increase 0.968 0.968 0.969 ,0.0001 Normalized maximum diastolic blood pressure, per 1- mmHg increase 0.975 0.974 0.976 ,0.0001 Normalized age, per 1-year increase 0.995 0.994 0.996 ,0.0001 Female 0.938 0.921 0.954 ,0.0001 Non–English speaker 1.035 1.006 1.065 0.0179 Caucasian 1.000 Black 1.150 1.121 1.180 ,0.0001 Hispanic 1.108 1.072 1.144 ,0.0001 Asian 1.175 1.120 1.233 ,0.0001 Other/unknown 1.049 1.010 1.090 0.0128 Income, per $1,000 increase 1.000 1.000 1.000 0.9251 Nonprivate insurance 0.997 0.977 1.018 0.7828 PCP encounter interval, log(months) 0.302 0.298 0.308 ,0.0001 Obesity during period 0.895 0.879 0.910 ,0.0001 Charlson comorbidity index 1.009 1.007 1.012 ,0.0001 Rate of antihypertensive medication intensiﬁcation, per month 1.625 1.568 1.685 ,0.0001 Rate of face-to-face lifestyle counseling, per month 1.267 1.235 1.301 ,0.0001 Rate of remote lifestyle counseling, per month 1.022 0.981 1.064 0.2933 Hyperlipidemic periods Normalized maximum LDL cholesterol, per 1-mg/dL increase 0.974 0.972 0.975 ,0.0001 Normalized age, per 1-year increase 1.007 1.005 1.008 ,0.0001 Female 0.885 0.856 0.916 ,0.0001 Non–English speaker 1.088 1.034 1.144 0.0012 Caucasian 1.000 Black 1.088 1.037 1.140 0.0005 Hispanic 1.142 1.077 1.210 ,0.0001 Asian 1.150 1.062 1.245 0.0006 Other/unknown 1.057 0.968 1.153 0.2151 Statistical analysis C: Lifestyle counseling frequency and time to LDL cholesterol target. D: Lifestyle counseling frequency and time to combined target. DBP, diastolic blood pressure; LDL, LDL cholesterol; SBP, systolic blood pressure. Figure 1dLifestyle counseling frequency and time to treatment target. Kaplan-Meier curves for time to treatment target from ﬁrst elevated A1C, blood pressure, or LDL cholesterol were plotted for different average counseling rates. Distinct uncontrolled periods (from the ﬁrst elevated to the ﬁrst normal measurement) for the same patient were analyzed separately. A: Lifestyle counseling frequency and time to A1C target. B: Lifestyle counseling frequency and time to blood pressure target. C: Lifestyle counseling frequency and time to LDL cholesterol target. D: Lifestyle counseling frequency and time to combined target. DBP, diastolic blood pressure; LDL, LDL cholesterol; SBP, systolic blood pressure. DIABETES CARE, VOLUME 35, FEBRUARY 2012 337 337 care.diabetesjournals.org 338 DIABETES CARE, VOLUME 35, FEBRUARY 2012 care.diabetesjournals.org Lifestyle counseling rate and time to treatment target achievement g In alltreatmentcategories,timetotreatment target rose progressively at the less frequent rates of lifestyle counseling (Fig. 1). Com- pared with patients with mean face-to-face counseling rate of once or more per month, median times to A1C target for patients whosemean counseling rateswerebetween once per 1–6 months and less than once per 6 months were 3.5 months (95% CI 3.2–3.7) vs. 14.0 (13.6–14.5) vs. 22.7 (21.8–23.5); time to blood pressure target was 3.7 weeks (3.6–3.7) vs. 5.1 months (5.1–5.2) vs. 5.6 (5.5–5.7) months and time to LDL cholesterol target was 3.5 months (3.0–3.8) vs. 15.6 (15.2–16.0) vs. 24.7 (24.1–25.4), respectively. For all treatment targets combined, median time to target was 3.9 (3.7–4.0) weeks vs. 13.5 months (13.0–13.9) vs. 13.1 (12.9–13.5) with mean face-to-face counseling rates of once or more per month versus once per 1–6 months versus less than once per 6 months. As counseling rates decreased, the proportion of patients who never reached treatment targets rose steadily. Comparing patients with mean face-to-face counseling rates of once or more per month to be- tween once per 1–6 months and less than once per 6 months, uncontrolled periods that never reached treatment target in- creased from 11.0 to 28 to 35.9% for hy- perglycemic patients, from 5.63 to 7.2 to 8.86% for hypertensive patients, and from 15.6 to 18.2 to 25.8% for hyperlipidemic patients. For all treatment targets com- bined, the proportion of uncontrolled periods that never achieved all targets was 9.3% for counseling rates of once or more per month versus 26.0% for coun- seling rates between once per 1–6 months versus 30.5% for counseling rates of less than once per 6 months. p In multivariable Cox proportional hazards models adjusted for demographic characteristics, presence of obesity during the uncontrolled period, Charlson co- morbidity index, insulin administration (in hyperglycemic and combined uncon- trolled periods), maximum A1C, systolic blood pressure, diastolic blood pressure, and LDL cholesterol (where relevant), rate 338 DIABETES CARE, VOLUME 35, FEBRUARY 2012 338 care.diabetesjournals.org Morrison, Shubina, and Turchin CONCLUSIONSdIn this large, retro- spective study, we have demonstrated a strong association between lifestyle coun- seling and glucose, blood pressure, and LDL cholesterol control in patients with diabetes. This association was indepen- dent of other treatment processes that could colocalize with lifestyle counseling, including frequency of patient-provider encounters, medication intensiﬁcation, and rates of A1C or LDL cholesterol mea- surement. Lifestyle counseling rate and time to treatment target achievement Table 3dContinued Hazard ratio 95% Conﬁdence limits P value (x2) Income, per $1,000 increase 1.001 1.000 1.002 0.0834 Nonprivate insurance 0.992 0.952 1.032 0.6808 PCP encounter interval, log(months) 0.720 0.691 0.750 ,0.0001 Obesity during period 0.888 0.847 0.932 ,0.0001 Charlson comorbidity index 1.009 1.005 1.013 ,0.0001 Rate of LDL cholesterol testing, per month 257.64 114.99 577.26 ,0.0001 Rate of antihyperlipidemic medication intensiﬁcation, per month 3.600 1.950 6.644 ,0.0001 Rate of face-to-face lifestyle counseling, per month 1.403 1.141 1.726 0.0013 Rate of remote lifestyle counseling, per month 1.215 1.054 1.400 0.0073 Combined uncontrolled periods Normalized maximum A1C, per 1% increase 0.675 0.667 0.683 ,0.0001 Normalized maximum systolic blood pressure, per 1-mmHg increase 0.979 0.979 0.980 ,0.0001 Normalized maximum diastolic blood pressure, per 1-mmHg increase 0.978 0.976 0.979 ,0.0001 Normalized maximum LDL cholesterol, per 1-mg/dL increase 0.983 0.983 0.984 ,0.0001 Normalized age, per 1-year increase 0.997 0.996 0.998 ,0.0001 Female 0.919 0.899 0.940 ,0.0001 Non–English speaker 1.037 0.997 1.078 0.0693 Caucasian 1.000 Black 1.252 1.208 1.298 ,0.0001 Hispanic 1.187 1.133 1.244 ,0.0001 Asian 1.160 1.088 1.238 ,0.0001 Other/unknown 1.060 1.009 1.114 0.0204 Income, per $1,000 increase 1.002 1.001 1.002 ,0.0001 Nonprivate insurance 1.011 0.985 1.037 0.4155 On insulin 0.894 0.866 0.923 ,0.0001 PCP encounter interval, log(months) 0.726 0.719 0.734 ,0.0001 Obesity during period 0.962 0.941 0.984 0.0007 Charlson comorbidity index 1.037 1.034 1.040 ,0.0001 Rate of A1C testing, per month 1.113 1.069 1.159 ,0.0001 Rate of LDL cholesterol testing, per month 1.160 1.122 1.200 ,0.0001 Rate of antihyperglycemic medication intensiﬁcation, per month 1.480 1.205 1.817 0.0002 Rate of antihypertensive medication intensiﬁcation, per month 1.255 1.192 1.321 ,0.0001 Rate of antihyperlipidemic medication intensiﬁcation, per month 0.955 0.902 1.010 0.1045 Rate of face-to-face lifestyle counseling, per month 1.937 1.836 2.044 ,0.0001 Rate of remote lifestyle counseling, per month 1.615 1.560 1.673 ,0.0001 Several clinical trials have previously documented the beneﬁt of lifestyle coun- seling on control of glucose (13–16) and blood pressure (17–21), but many provid- ers question whether results of expensive and tightly controlled clinical trials apply to their practice. Thisstudy provides evidence for the efﬁcacy of lifestyle counseling as practiced in routine patient care and lends support to the current treatment guidelines for patients with diabetes. care.diabetesjournals.org Lifestyle counseling rate and time to treatment target achievement Infor- mation on the extent to which counseling followed a structured format (e.g., 5As) was not obtained because it is frequently absent in narrative documentation (40). The software we used did not distinguish between lifestyle counseling aimed to ad- dress hyperglycemia, hypertension, and hyperlipidemia severally, which may have led to an overestimate of lifestyle counseling rates associated with any one uncontrolled period. However, this lack of speciﬁcity should have biased our ﬁndings toward the null hypothesis. In our analy- sis, we did not analyze individual effects of diet, exercise, and weight counseling. However,theireffectsarelikely overlapping because both diet and exercise, for exam- ple, can lead to weight loss. Furthermore, the best approach to counseling may dif- fer depending on the individual patient and their readiness to change, rendering any statement about relative efﬁcacy of different counseling types moot. We therefore chose to combine all lifestyle counseling into a single measure to avoid this type of confounding. The retrospec- tive nature of this study does not allow us to make causal inferences about the rela- tionship between counseling rates and time to glucose, blood pressure, and LDL cholesterol control in patients with diabe- tes. It could also have led to an analytical bias. For example, when lifestyle counsel- ing is sparse, shorter uncontrolled periods 5. UK Prospective Diabetes Study (UKPDS) Group. Intensive blood-glucose control with sulphonylureas or insulin compared with conventional treatment and risk of complications in patients with type 2 di- abetes (UKPDS 33). Lancet 1998;352: 837–853 In summary, this large long-term ret- rospective study found that lifestyle coun- seling is associated with faster achievement of A1C, blood pressure, and LDL choles- terol control in routine patient care, con- sistent with ﬁndings of randomized controlled studies. Monthly lifestyle coun- seling was associated with a particularly strong effect and could be recommended for patients at particularly high risk of com- plications from uncontrolled diabetes. In- terventional studies are needed to further establish optimal type and frequency of lifestyle counseling and its effects on the micro- and macrovascular complications of diabetes. 6. Goldberg RB, Mellies MJ, Sacks FM, et al.; The Care Investigators. Cardiovascular events and their reduction with pravastatin in diabetic and glucose-intolerant myo- cardial infarction survivors with average cholesterol levels: subgroup analyses in the cholesterol and recurrent events (CARE) trial. Circulation 1998;98:2513– 2519 7. Pyorälä K, Pedersen TR, Kjekshus J, Faergeman O, Olsson AG, Thorgeirsson G. Lifestyle counseling rate and time to treatment target achievement WildS, Roglic G, Green A, Sicree R, King H. Global prevalence of diabetes: estimates for the year 2000 and projections for 2030. Diabetes Care 2004;27:1047–1053 are more likely to have had no counseling episodes. However, this bias would have predisposed against the strong inverse as- sociation between lifestyle counseling and the length of uncontrolled periods that we have found. Furthermore, most uncon- trolled periods in our study were substan- tially longer than the average observed rate of lifestyle counseling, making an artifac- tual associationbetween lifestylecounseling rate and the length of uncontrolled period unlikely. Additionally, we were unable to distinguish between patients with type 1 and type 2 diabetes; however, the majority of patients in this population have type 2 diabetes, so our ﬁndings may not be ap- plicable to patients with type 1 diabetes. role of midlevel providers, such as nurse practitioners, physician assistants, nutri- tionists, or exercise physiologists. Another option may be to implement group coun- seling sessions in order to more efﬁciently educate and address patients’ concerns. 3. The Diabetes Control and Complications Trial Research Group. The effect of intensive treatment of diabetes on the development and progression of long-term complica- tions in insulin-dependent diabetes mel- litus. N Engl J Med 1993;329:977–986 This study used advanced compu- tational technology that permitted cost- and time-efﬁcient analysis of thousands of patient records, including examination of hundreds of thousands of narrative provider notes in a matter of hours. In the future, similar technologies could also be used to monitor quality of patient care and/or supply feedback to providers. g 4. Nathan DM, Cleary PA, Backlund JY, et al.; Diabetes Control and Complica- tions Trial/Epidemiology of Diabetes In- terventions and Complications (DCCT/ EDIC) Study Research Group. Intensive diabetes treatment and cardiovascular disease in patients with type 1 diabetes. N Engl J Med 2005;353:2643–2653 Our study had several limitations. The software we used to identify docu- mentation of lifestyle counseling did not provide details on the speciﬁc counseling approach or the type of diet or exercise recommended to the patient. However, little evidence exists for superiority of any one approach over the others (24,26). It is therefore likely that multiple different counseling techniques can be successful, and speciﬁc type of counseling should be chosen in accordance with the particular patient and clinical circumstances. Lifestyle counseling rate and time to treatment target achievement Few clinical trials of lifestyle counsel- ing had follow-up longer than 12 months (21); this study, with an average length of follow-up time per patient of almost 7 years, provides evidence for long-term ef- fects of lifestyle counseling. This is partic- ularly important because some studies suggest that effects of intensive diet and exercise interventions may not be durable (24,34,35). Our ﬁndings suggest that, on the contrary, persistent lifestyle counseling has lasting effects. Our results conﬁrmed thatintensivecounselingisneededtoachieve beneﬁts: the effects of lifestyle counseling were particularly pronounced in patients who were counseled at least once a month. Studies have shown that lifestyle coun- seling in the U.S. remains inadequate (36–39). Despite the focus on lifestyle changes in many treatment guidelines, one study showed no difference in preva- lence of exercise counseling in a sample of the U.S. population in 2002 compared with 1995 (39). Further education of phy- sicians on the importance of lifestyle coun- seling and its positive impact on patient behavior and health outcomes may be nec- essary. Physicians may provide more coun- seling to underserved populations if made aware that patients with lower income, lower education level, who are male (37) and non–English speaking (38) receive life- style counseling at lower rates, compared with other equally high-risk patients. associated with a hazard ratio of 1.9 for achieving control of all treatment targets (P , 0.0001). For most measures, remote lifestyle counseling rates were also associ- ated with faster time to target, but hazard ratios were smaller than those for face-to- face lifestyle counseling. A combined face- to-face and remote lifestyle counseling rate analysis was also conducted; results for this multivariable analysis are provided in Supplementary Appendix A. of A1C and LDL cholesterol measurement (where relevant), visit frequency, and medication intensiﬁcation, one additional episode of face-to-face lifestyle counseling per month was associated with hazard ratios of 1.7 for A1C control (P , 0.0001) (Table 3), 1.3 for blood pressure control (P , 0.0001), and 1.4 for LDL cholesterol control (P = 0.0013). In multivariable analysis of combined uncontrolled peri- ods, an increase of one face-to-face life- style counseling instance per month was q y g p Lifestyle counseling is time consuming. Therefore, implementation of current guidelines may require modiﬁcation of the prevalent physician-patient treatment care model. One option may be to increase the DIABETES CARE, VOLUME 35, FEBRUARY 2012 339 339 care.diabetesjournals.org Lifestyle counseling and diabetes control 2. Lifestyle counseling rate and time to treatment target achievement Cholesterol lowering with simvastatin improves prognosis of diabetic patients with coronary heart disease. A subgroup analysis of the Scandinavian Simvastatin Survival Study (4S). Diabetes Care 1997; 20:614–620 AcknowledgmentsdThis study was supported in part by grants from the Agency for Health- care Research and Quality (5R18HS017030), the National Library of Medicine (5RC1LM010460), and the Diabetes Action Research and Education Foundation. AcknowledgmentsdThis study was supported in part by grants from the Agency for Health- care Research and Quality (5R18HS017030), the National Library of Medicine (5RC1LM010460), and the Diabetes Action Research and Education Foundation. 8. UK Prospective Diabetes Study Group. Tight blood pressure control and risk of macrovascular and microvascular com- plications in type 2 diabetes: UKPDS 38. BMJ 1998;317:703–713 No potential conﬂicts of interest relevant to the article were reported. F.M. conducted data analysis and drafted the manuscript. M.S. assisted in study design, pro- vided biostatistical support, and critically re- viewed the manuscript. A.T. designed the study, obtained funding, and critically reviewed the manuscript. F.M. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. 9. Resnick HE, Foster GL, Bardsley J, Ratner RE. Achievement of American Diabetes Association clinical practice recommen- dations among U.S. adults with diabetes, 1999-2002: the National Health and Nu- trition Examination Survey. Diabetes Care 2006;29:531–537 10. Ong KL, Cheung BM, Wong LY, Wat NM, Tan KC, Lam KS. Prevalence, treatment, and control of diagnosed diabetes in the U.S. National Health and Nutrition Ex- amination Survey 1999-2004. Ann Epi- demiol 2008;18:222–229 Parts of this study were presented in poster form at the 71st Scientiﬁc Sessions of the American Diabetes Association, San Diego, California, 24–28 June 2011. 11. Nathan DM, Buse JB, Davidson MB, et al. Management of hyperglycemia in type 2 diabetes: A consensus algorithm for the initiation and adjustment of therapy: a consensus statement from the American Diabetes Association and the European Association for the Study of Diabetes. Di- abetes Care 2006;29:1963–1972 340 DIABETES CARE, VOLUME 35, FEBRUARY 2012 References 1. Cowie CC, Rust KF, Byrd-Holt DD, et al. Prevalence of diabetes and high risk for diabetes using A1C criteria in the U.S. population in 1988-2006. Diabetes Care 2010;33:562–568 340 DIABETES CARE, VOLUME 35, FEBRUARY 2012 340 care.diabetesjournals.org care.diabetesjournals.org Morrison, Shubina, and Turchin 12. American Diabetes Association. Standards of medical care in diabetesd2010. Di- abetes Care 2010;33(Suppl. 1):S11–S61 20. Blumenthal JA, Babyak MA, Hinderliter A, et al. Effects of the DASH diet alone and in combination with exercise and weight loss on blood pressure and cardio- vascular biomarkers in men and women with high blood pressure: the ENCORE study. Arch Intern Med 2010;170:126–135 treatment intensiﬁcation in patients with diabetes mellitus. Circulation 2008;117: 623–628 32. Lin DY. Cox regression analysis of multi- variate failure time data: the marginal ap- proach. Stat Med 1994;13:2233–2247 13. Boden G, Sargrad K, Homko C, Mozzoli M, Stein TP. Effect of a low-carbohydrate diet on appetite, blood glucose levels, and insulin resistance in obese patients with type 2 diabetes. Ann Intern Med 2005; 142:403–411 33. Deyo RA, Cherkin DC, Ciol MA. Adapting a clinical comorbidity index for use with ICD-9-CM administrative data- bases. J Clin Epidemiol 1992;45:613–619 21. He J, Whelton PK, Appel LJ, Charleston J, Klag MJ. Long-term effects of weight loss and dietary sodium reduction on incidence of hypertension. Hypertension 2000;35: 544–549 14. Anderson JW, Kendall CW, Jenkins DJ. Importance of weight management in type 2 diabetes: review with meta-analysis of clinical studies. J Am Coll Nutr 2003; 22:331–339 34. Turner RC, Cull CA, Frighi V, Holman RR; UK Prospective Diabetes Study (UKPDS) Group. Glycemic control with diet, sulfo- nylurea, metformin, or insulin in patients with type 2 diabetes mellitus: progressive requirement for multiple therapies (UKPDS 49). JAMA 1999;281:2005–2012 22. van de Laar FA, Akkermans RP, van Binsbergen JJ. Limited evidence for effects of diet for type 2 diabetes from systematic reviews. Eur J Clin Nutr 2007;61:929–937 15. Hughes TA, Gwynne JT, Switzer BR, Herbst C, White G. Effects of caloric re- striction and weight loss on glycemic control, insulin release and resistance, and atherosclerotic risk in obese patients with type II diabetes mellitus. Am J Med 1984; 77:7–17 23. Sacks FM, Campos H. Dietary therapy in hypertension. N Engl J Med 2010;362: 2102–2112 35. Kirk A, Mutrie N, MacIntyre P, Fisher M. References Effects of a 12-month physical activity counselling intervention on glycaemic control and on the status of cardiovascular risk factors in people with Type 2 di- abetes. Diabetologia 2004;47:821–832 24. Nield L, Moore HJ, Hooper L, et al. Dietary advice for treatment of type 2 diabetes mellitus in adults. Cochrane Database Syst Rev 2007;3:CD004097 16. Uusitupa M, Laitinen J, Siitonen O, Vanninen E, Pyörälä K. The maintenance of improved metabolic control after in- tensiﬁed diet therapy in recent type 2 di- abetes. Diabetes Res Clin Pract 1993;19: 227–238 25. West KM. Diet therapy of diabetes: an analysis of failure. Ann Intern Med 1973; 79:425–434 36. Huang J, Yu H, Marin E, Brock S, Carden D, Davis T. Physicians’ weight loss coun- seling in two public hospital primary care clinics. Acad Med 2004;79:156–161 26. Sawyer L, Gale EA. Diet, delusion and diabetes. Diabetologia 2009;52:1–7 37. Galuska DA, Will JC, Serdula MK, Ford ES. Are health care professionals advising obese patients to lose weight? JAMA 1999; 282:1576–1578 17. Appel LJ, Champagne CM, Harsha DW, et al.; Writing Group of the PREMIER Collaborative Research Group. Effects of comprehensive lifestyle modiﬁcation on blood pressure control: main results of the PREMIER clinical trial. JAMA 2003;289: 2083–2093 27. Hooper L, Bartlett C, Davey SG, Ebrahim S. Advice to reduce dietary salt for prevention of cardiovascular disease. Cochrane Data- base Syst Rev 2004;1:CD003656 38. Lopez-Quintero C, Berry EM, Neumark Y. Limited English proﬁciency is a barrier to receipt of advice about physical activity and diet among Hispanics with chronic diseases in the United States. J Am Diet Assoc 2010;110(Suppl.):S62–S67 28. American Diabetes Association. Standards of medical care for patients with diabetes mellitus. Diabetes Care 2000;23(Suppl. 1):S32–S42 18. The Trials of Hypertension Prevention Collaborative Research Group. Effects of weight loss and sodium reduction in- tervention on blood pressure and hyper- tension incidence in overweight people with high-normal blood pressure. The Trials of Hypertension Prevention, phase II. Arch Intern Med 1997;157:657–667 29. The sixth report of the Joint National Committee on Prevention, Detection, Evalu- ation,andTreatmentofHighBloodPressure. Arch Intern Med 1997;157:2413–2446 pp 39. Morrato EH, Hill JO, Wyatt HR, Ghushchyan V, Sullivan PW. Are health care professionals advising patients with diabetes or at risk for developing diabetes to exercise more? Diabetes Care 2006;29: 543–548 30. Turchin A, Goldberg SI, Breydo E, Shubina M, Einbinder JS. Copy/paste documentation of lifestyle counseling and glycemic control in patients with diabetes: true to form? DIABETES CARE, VOLUME 35, FEBRUARY 2012 341 References Arch Intern Med 2011;171:1393–1394 19. Sacks FM, Svetkey LP, Vollmer WM, et al.; DASH-Sodium Collaborative Research Group. Effects on blood pressure of re- duced dietary sodium and the Dietary Ap- proaches to Stop Hypertension (DASH) diet. N Engl J Med 2001;344:3–10 40. Hazlehurst B, Sittig DF, Stevens VJ, et al. Natural language processing in the elec- tronic medical record: assessing clinician adherence to tobacco treatment guide- lines. Am J Prev Med 2005;29:434–439 31. Turchin A, Shubina M, Chodos AH, Einbinder JS, Pendergrass ML. Effect of board certiﬁcation on antihypertensive DIABETES CARE, VOLUME 35, FEBRUARY 2012 341 341 care.diabetesjournals.org
https://openalex.org/W2150332528	https://www.research-collection.ethz.ch/bitstream/20.500.11850/290928/1/acp-10-7739-2010.pdf	English	null	What can be learned about carbon cycle climate feedbacks from the CO&lt;sub&gt;2&lt;/sub&gt; airborne fraction?	Atmospheric chemistry and physics	2,010	cc-by	12,155	ETH Library What can be learned about carbon cycle climate feedbacks from the CO2 airborne fraction? Abstract. The ratio of CO2 accumulating in the atmosphere to the CO2 ﬂux into the atmosphere due to human activ- ity, the airborne fraction AF, is central to predict changes in earth’s surface temperature due to greenhouse gas induced warming. This ratio has remained remarkably constant in the past ﬁve decades, but recent studies have reported an appar- ent increasing trend and interpreted it as an indication for a decrease in the efﬁciency of the combined sinks by the ocean and terrestrial biosphere. We investigate here whether this interpretation is correct by analyzing the processes that con- trol long-term trends and decadal-scale variations in the AF. To this end, we use simpliﬁed linear models for describing the time evolution of an atmospheric CO2 perturbation. We ﬁnd ﬁrstly that the spin-up time of the system for the AF to converge to a constant value is on the order of 200–300 years and differs depending on whether exponentially increasing fossil fuel emissions only or the sum of fossil fuel and land use emissions are used. We ﬁnd secondly that the primary control on the decadal time-scale variations of the AF is vari- ations in the relative growth rate of the total anthropogenic CO2 emissions. Changes in sink efﬁciencies tend to leave a smaller imprint. Therefore, before interpreting trends in the AF as an indication of weakening carbon sink efﬁciency, it is necessary to account for trends and variations in AF stem- ming from anthropogenic emissions and other extrinsic forc- ing events, such as volcanic eruptions. Using atmospheric CO2 data and emission estimates for the period 1959 through 2006, and our simple predictive models for the AF, we ﬁnd that likely omissions in the reported emissions from land use change and extrinsic forcing events are sufﬁcient to explain the observed long-term trend in AF. Therefore, claims for a decreasing long-term trend in the carbon sink efﬁciency over the last few decades are currently not supported by atmo- spheric CO2 data and anthropogenic emissions estimates. the observed long-term trend in AF. Therefore, claims for a decreasing long-term trend in the carbon sink efﬁciency over the last few decades are currently not supported by atmo- spheric CO2 data and anthropogenic emissions estimates. Journal Article Author(s): Gloor, Manuel; Sarmiento, Jorge L.; Gruber, Nicolas This page was generated automatically upon download from the ETH Zurich Research Collection. For more information, please consult the Terms of use. This page was generated automatically upon download from the ETH Zurich Research Collection. For more information, please consult the Terms of use. Atmos. Chem. Phys., 10, 7739–7751, 2010 www.atmos-chem-phys.net/10/7739/2010/ doi:10.5194/acp-10-7739-2010 © Author(s) 2010. CC Attribution 3.0 License. Atmospheric Chemistry and Physics What can be learned about carbon cycle climate feedbacks from CO2 airborne fraction? M. Gloor1, J. L. Sarmiento2, and N. Gruber3 1The School of Geography, University of Leeds, Leeds, LS2 9JT, UK 2Atmospheric and Oceanic Sciences Department, Princeton University, 300 Forrestal Road, Sayre Hall, Princeton, NJ 08544, USA 3Institute of Biogeochemistry and Pollutant Dynamics, ETH Z¨urich, Universit¨atsstr. 16, 8092 Z¨urich, Switzerland Received: 22 March 2010 – Published in Atmos. Chem. Phys. Discuss.: 8 April 2010 Revised: 18 June 2010 – Accepted: 21 July 2010 – Published: 20 August 2010 1 Introduction AFFF ≡ dC(t) dt FF(t), or AFFF+LU ≡ dC(t) dt FF(t)+LU(t) AFFF ≡ dC(t) dt FF(t), or AFFF+LU ≡ dC(t) dt FF(t)+LU(t) (Canadell et al., 2007; Raupach et al., 2008; Le Qu´er´e et al., 2009; Knorr, 2009). We analyze here the time-evolution of the AF as deﬁned by these recent studies. (Canadell et al., 2007; Raupach et al., 2008; Le Qu´er´e et al., 2009; Knorr, 2009). We analyze here the time-evolution of the AF as deﬁned by these recent studies. y We brieﬂy outline the organization of our paper. We start in Sect. 2 with a characterization of the time course of an- thropogenic CO2 emissions and carbon sinks, thereby high- lighting that there have been strong variations in the relative growth rate of fossil fuel emissions over the last century. In Sect. 3 we introduce a simple linear model of the evolution of an atmospheric CO2 perturbation, thereby also clarifying the meaning of “sink efﬁciency”. In Sect. 4 we explore how the time course of the anthropogenic emissions controls vari- ations in the AF, using a predictive equation implied by our simple model. We demonstrate that (i) for an atmospheric CO2 perturbation which is not following an exact exponen- tial function, there is an adjustment time for the AF to con- verge to its constant asymptotic value, which is on the or- der of centuries, and that (ii) variations in the relative growth rate of the anthropogenic emissions are a major control on variations of the AF. Therefore, in order to unravel trends in the AF caused by trends in carbon sink efﬁciency or extrin- sic non-anthropogenic events, like volcanic eruptions, sig- natures due to incomplete “spin-up” and fossil fuel growth rate variations, need ﬁrst to be removed from the observed AF. We can achieve this using our predictive equation for the AF (Sect. 5). We then examine the remaining signal for trends not explained by known extrinsic non-anthropogenic forcings or omissions in anthropogenic ﬂuxes, to conclude whether there is indeed evidence for trends in the carbon sink efﬁciency trends in the observed AF record (Sect. 6). This terminates our main analysis. Section 7 in addition ex- plores the signal to noise ratio of AF trends caused by sink efﬁciency trends, and ﬁnally we discuss and conclude. Before proceeding, it is important to recognize that deﬁni- tions of the AF in the literature vary. 1 Introduction Central for predicting future temperatures of the Earth’s sur- face is how much and for how long carbon dioxide from fossil fuel emissions and land use change stays in the atmo- sphere, and how much gets removed by the carbon sinks on land and in the ocean (e.g. Solomon et al., 2009). A straight- forward measure of this redistribution is the ratio between the increase rate in atmospheric CO2 and the CO2 emit- ted to the atmosphere by human activity (fossil fuel burn- ing and land use change). Keeling (1973) termed this quan- tity the “airborne fraction” (AF) and it was investigated in many subsequent studies (e.g. Bacastow and Keeling, 1979; Oeschger and Heimann, 1983; Enting, 1986). Because of the large uncertainties in land ﬂuxes, these early studies could estimate the value of AF only to within a wide range from 0.38 to 0.78 (Oeschger and Heimann, 1983). Recently sev- eral studies have extended the estimation of AF over the last two decades, with a suggestion of a positive trend in AF (Canadell et al., 2007; Raupach et al., 2008; Le Qu´er´e et al., 2009). Moreover, this positive trend has been interpreted as evidence for a decreasing trend in the efﬁciency of the ocean and land carbon sinks. Given the model-based projection of a substantial reduction in the sink strength of the ocean and land in the future (e.g. by a large-scale dieback of the Ama- zon old-growth forest, Cox et al., 2000), the notion that the sinks have already begun to deviate from a linear response Correspondence to: M. Gloor (eugloor@googlemail.com) Correspondence to: M. Gloor (eugloor@googlemail.com) Published by Copernicus Publications on behalf of the European Geosciences Union. opernicus Publications on behalf of the European Geosciences Union. 7740 M. Gloor et al.: Airborne fraction trends and carbon sink efﬁciency to the atmospheric CO2 perturbation is a source of substan- tial concern. While there remains discussion about whether this trend in the AF is actually statistically signiﬁcant (Knorr, 2009), we focus our discussion here on whether the inferred conclusion is defensible, i.e. whether an increasing trend in the AF implies a decreasing efﬁciency of the carbon sinks. (Oeschger and Heimann, 1983) where C is atmospheric car- bon dioxide, ti,tf are the beginning and the end time of the period considered, FF is fossil fuel emissions and LU is the ﬂux to the atmosphere due to land use change. 1 Introduction Studies from the 1970s and 1980s deﬁned airborne fraction from cumulative carbon inventory changes as 1 Introduction The more re- cent studies deﬁne airborne fraction from annual or monthly inventory changes as either Determinants of the AF are the magnitude and time course of the human induced emissions of CO2 into the atmosphere and the removal of this anthropogenic carbon by the ocean and land biosphere. It has been known since the early 1970’s, possibly earlier, that the AF will eventually asymptote to a constant value if (i) the CO2 uptake by the oceans and land ecosystems is linear and (ii) if CO2 emissions to the atmo- sphere follow exactly an exponential function (Bacastow and Keeling, 1979). Thus, given that fossil fuel emissions have risen approximately exponentially over the last 250 years, and that natural systems tend to respond linearly to small per- turbations, it is natural to inquire whether time trends in AF may inform us about changes in the linear behavior of car- bon uptake by the oceans and land ecosystems (Canadell et al., 2007; Raupach et al., 2008; Le Qu´er´e et al., 2009; Rafel- ski et al., 2009). However, closer examination shows that the relative growth rate RGR≡1 FF dFF dt of fossil fuel emissions FF has varied by more than a factor of two in the last 100 years (e.g. Raupach et al., 2008). In addition, emissions from land use change exhibited an even more varied time course, so that the total emissions only very approximately followed a single exponential. Furthermore, trends in AF may be an ar- ticulation of an incomplete spin-up of the system, so that the AF is still changing along its way toward reaching its asymp- totic value. We examine here the impact of these deviations and controls on the AF, and what the consequences are for the interpretation of the AF as an indicator for changes in the efﬁciency of carbon sinks, and in turn the state of the global carbon cycle. Our study builds on the seminal work of Ba- castow and Keeling (1979) who had stated already 30 years ago that “The global average airborne fraction will probably not remain near 56% in the future ... because fossil fuel re- sources are ﬁnite...”, i.e. that one important control of the AF is the growth rate of fossil fuel emissions. 2 Anthropogenic carbon emissions and carbon sinks 0.2 0.4 0.6 0.8 1 AF (−) modeled AF (FF only) modeled AF (FF+LU) observed AF (FF only) observed AF (FF+LU) e 2000 1750 1800 1850 1900 1950 −0.02 0 0.02 0.04 0.06 0.08 RGR = τf -1 (yr-1) 2000 RGR fossil fuel & land use (FF+LU) RGR fossil fuel (FF) d 0.5 1 1.5 2 LU (PgC yr-1) c 2000 land-use change (LU) 0 40 80 120 160 τf=(df/dt/f)-1 (yr) 2000 b τ of fossil fuel (FF) WW1 WW2 oil crisis end of USSR 1 FF (PgC yr-1) a 0.1 10 2000 fossil fuel (FF) Fig. 1. (a) Fossil fuel emissions estimated by Marland (2006), (b) time scale τf of relative rate of change of FF, (c) c atmosphere due to land use change estimated by Houghton et al. (2007), (d) relative growth rate of f =FF and f =FF+LU Fig. 1. (a) Fossil fuel emissions estimated by Marland (2006), (b) time scale τf of relative rate of change of FF, (c) carbon ﬂux to the atmosphere due to land use change estimated by Houghton et al. (2007), (d) relative growth rate of f =FF and f =FF+LU respectively, and (e) model predicted and observed AFFF and AFFF+LU . The time-scale τf of relative change of an anthropogenic ﬂux f to the atmosphere is deﬁned as the inverse of its loga- rithmic derivative: ( 1 FF 1FF 1t )−1 of ∼20 years (equivalent to a relative growth rate of 5% per year, Fig. 1d). The WW1, post WW1 and great de- pression period saw less growth, with both positive and neg- ative time-scales resulting in a substantially longer mean τFF. After WW2 (starting around 1948) there is again fast growth, paralleling the recovery of industrial countries’ economies, until the early 1970s with τFF of ∼20 years. From the early 1970s until approximately 1999 τFF increased again to ∼80 years (relative growth rate of ∼1.3% yr−1). Growth returned close to the 1830 to 1910 and post WW2 values starting around 2001. τf ≡( 1 f df dt )−1 ≃( 1 f 1f 1t )−1 τf ≡( 1 f df dt )−1 ≃( 1 f 1f 1t )−1 with 1f ≡f (t +1t) −f (t) and 1t = 1 year. The time- course of τf permits to identify periods with different fossil fuel emissions growth rates particularly well. 2 Anthropogenic carbon emissions and carbon sinks AFcum FF ≡C(tf)−C(ti) R tf ti FF(t)dt The main driver of the rapid increase in atmospheric CO2 is fossil fuel emissions, which are estimated from national energy statistics with an uncertainty of 6%–10% (90% con- ﬁdence interval) (Marland, 2006, updated by Boden et al., 2009; Marland, 2008). A logarithmic representation (Fig. 1a) reveals that fossil fuel emissions have increased roughly ex- ponentially, with the time-scale of relative change, the in- verse of the relative growth rate, varying roughly between ∼20 and 150 years (Fig. 1b). (Keeling, 1973; Bacastow and Keeling, 1979; Enting, 1986) or alternatively as (Keeling, 1973; Bacastow and Keeling, 1979; Enting, 1986) or alternatively as AFcum FF+LU ≡ C(tf)−C(ti) R tf ti FF(t)+LU(t)dt Atmos. Chem. Phys., 10, 7739–7751, 2010 Atmos. Chem. Phys., 10, 7739–7751, 2010 Atmos. Chem. Phys., 10, 7739–7751, 2010 www.atmos-chem-phys.net/10/7739/2010/ M. Gloor et al.: Airborne fraction trends and carbon sink efﬁciency 7741 M. Gloor et al.: Airborne fraction trends and carbon sink efﬁciency 7741 0.2 0.4 0.6 0.8 1 AF (−) modeled AF (FF only) modeled AF (FF+LU) observed AF (FF only) observed AF (FF+LU) e 2000 1750 1800 1850 1900 1950 −0.02 0 0.02 0.04 0.06 0.08 RGR = τf -1 (yr-1) 2000 RGR fossil fuel & land use (FF+LU) RGR fossil fuel (FF) d 0.5 1 1.5 2 LU (PgC yr-1) c 2000 land-use change (LU) 0 40 80 120 160 τf=(df/dt/f)-1 (yr) 2000 b τ of fossil fuel (FF) WW1 WW2 oil crisis end of USSR 1 FF (PgC yr-1) a 0.1 10 2000 fossil fuel (FF) Fig. 1. (a) Fossil fuel emissions estimated by Marland (2006), (b) time scale τf of relative rate of change of FF, (c) carbon ﬂux to the atmosphere due to land use change estimated by Houghton et al. (2007), (d) relative growth rate of f =FF and f =FF+LU respectively, and (e) model predicted and observed AFFF and AFFF+LU . M. Gloor et al.: Airborne fraction trends and carbon sink efﬁciency M. Gloor et al.: Airborne fraction trends and carbon sink efﬁciency With the ﬂux parameterization given by Eq. (1), the time evolution for 1C implied by the atmospheric CO2 mass bal- ance is determined by The second cause of the rise in atmospheric carbon, and at the same time the least well constrained positive component of the atmospheric carbon budget, is carbon ﬂuxes released from land to the atmosphere due to land use change (for ex- ample rainforest to pasture conversion in the tropics, or peat burning during 1997/98 in Indonesia due to conversion of swamp forests to rice paddies at a large spatial scale, Page et al., 2002). Estimates of Houghton et al. (2007) indicate that this term has also risen in time, but at a considerably smaller rate than fossil fuel emissions (Fig. 1c). The uncertainty in ﬂuxes associated with land use change is large, on the order of 40–100%, as revealed by the range of published estimates (e.g. Grainger, 2008; Houghton et al., 2007; DeFries et al., 2002; Achard et al., 2002). d1C dt = f (t)−Fat→oc −Fat→ld = f (t)−( 1 τoc + 1 τld )1C = f (t)−1C τs . (2) (2) Here t is time, the subscript s stands for “system”, 1 τs ≡1 τoc + 1 τld 1 τs ≡1 τoc + 1 τld is the proportionality constant between the atmospheric CO2 perturbation and the total C ﬂux out of the atmosphere, and f (t) is the anthropogenic CO2 ﬂux into the atmosphere, which we can view as the forcing of the system. For our problem f is mostly FF+LU although we will also consider the case of f =FF alone. The atmospheric CO2 accumulation rate is well con- strained by atmospheric concentration records (Keeling, 1960; Etheridge et al., 1996). Estimates of ocean uptake of anthropogenic carbon based on various methods have also converged over recent years to 2.2±0.2 PgC yr−1 for a nom- inal period of ∼1995–2000 (Sabine et al., 2004; Sweeney et al., 2007; Sarmiento et al., 2010; Gruber et al., 2009; Khati- wala et al., 2009). The net land sink, the sum of the land sink and the CO2 ﬂux to the atmosphere due to land use change, can then be calculated as the difference between fossil fuel emissions, the atmospheric CO2 accumulation rate and ocean uptake. M. Gloor et al.: Airborne fraction trends and carbon sink efﬁciency The implied net land sink stayed roughly constant with a mean value of nearly zero from the 1930s to 1990 and then increased to a magnitude of approximately 1 PgC yr−1 for the 1990s and early 2000s (e.g., Sarmiento et al., 2010). It is necessary to consider to what extent the assumption of a linear relationship between the ﬂux out of the atmosphere and the anthropogenic atmospheric CO2 perturbation is jus- tiﬁed based on our understanding of the dominant processes. In the case of ocean carbon uptake, this assumption is well underpinned, because the driving force for the uptake is the air-sea CO2 disequilibrium. In addition, the rate-limiting step of the oceanic uptake, the transport of the anthropogenic CO2 into the ocean’s interior, is a linear process (e.g. Sarmiento et al., 1992; Maier-Reimer and Hasselmann, 1987). The linear scaling of the ocean uptake with the perturbation in atmo- spheric CO2 is supported by 3-D ocean model simulations (e.g., Sarmiento and Le Qu´er´e, 1996), although such simu- lations also show a strong deviation from linearity once at- mospheric CO2 has risen to values where the surface ocean buffer factor begins to change rapidly (Sarmiento and Le Qu´er´e, 1996). Using the model-based scaling used by Gloor et al. (2003) and Mikaloff-Fletcher et al. (2006) and the an- thropogenic ocean carbon inventory estimated from oceans surveys for 1995 (Sabine et al., 2004), we obtain an estimate of τoc ≃81.4 yr (Appendix C). 2 Anthropogenic carbon emissions and carbon sinks Variations in the time-scale of relative change of fossil fuel emissions, τf, are mainly due to economic cycles and wars. Thus there was an approximately 80 year period from around 1830 to 1910 (approximately the start date of World War one (WW1)) with a roughly constant τFF ≡( 1 FF dFF dt )−1 ≃ Atmos. Chem. Phys., 10, 7739–7751, 2010 www.atmos-chem-phys.net/10/7739/2010/ 7742 4 Airborne fraction for idealized cases (3) To get a general sense of the implications of our simple model (Eq. 2) for the time-course of AF, we have calculated AF for three idealized forcing functions f (t): (i) exponential forcing f (t) = f et/τf with a single characteristic time-scale τf (or equivalently relative growth rate 1/τf); the subscript f refers to “forcing”, (ii) the sum of an exponential func- tion and a constant, and (iii) the sum of several exponential functions with different characteristic time-scales or relative growth rates (Appendix A and Fig. 2). The ﬁrst case is an idealization of forcing the atmosphere with fossil fuel burn- ing CO2 alone, while the latter two cases mimic forcing of the atmosphere with the sum of fossil fuel emissions and land use change emissions. derived in Appendix B. In order to interpret this equation it is helpful to notice that it is quite similar to Eq. (2). The analogue of 1C is AF, the analogue of the “forcing” ﬂux to the atmosphere f (t) is 1 f df dt + 1 τs dτs dt , and the analogue of the exponential damping term −1C τs is −( 1 τs + 1 f df dt + 1 τs dτs dt )AF. Growth of AF is thus largely dictated by the relative growth rate RGR= 1 f df dt of f (instead of f itself; 1 f df dt ≫1 τs dτs dt un- less there is a very strong feedback), and AF is damped to- wards zero at a rate 1 τs + 1 f df dt + 1 τs dτs dt (instead of 1 τs ). s f s s To predict the variations in AF according to our simple model, we integrate the equation numerically assuming a constant sink efﬁciency, i.e. τs = const. We choose a value for τs such that the mean observed and predicted AF are equal over the period 1959–2006 using least squares, which results in τs=42 years for AFFF and τs=37.5 years for AFFF+LU. Be- sides using the requirement for agreement of the mean AF over the period from 1959 to 2006 to estimate τs, we may also determine τs from the mass conservation requirement that predicted and observed increase in atmospheric CO2 agree. The two estimates agree well. 4 Airborne fraction for idealized cases The predicted variations in AF based on the fossil fuel time-series estimated by Marland (2006) alone, as well as the sum of the fossil fuel and land use time-series, used as forcing, are shown in Figs. 1e and 3a. As shown previously (e.g. Bacastow and Keeling, 1979), AF is constant for a purely exponential forcing function (Ap- pendix A and Fig. 2). For the forcing functions differing from an exact exponential function, AF converges to an asymptotic value after some spin up time. The asymptotic value of AF is the same for the three forcing functions and is given by AF(∞) = 1 1+ τf τs . It is thus controlled by the ratio between the forcing time- scale and the system response time-scale. If the forcing is not exactly exponential, then the time-scale for convergence is roughly on the order of 200–300 years (Fig. 2), depending on the exact functional form of the forcing. For the case of forcing by the sum of several exponentials it is the τf of the fastest growing exponential function that determines the asymptotic value of AF (see last equation in Appendix A). In order to assess the importance of restricting ourselves to a single ocean and land pool description for our results, we repeated this calculation using a more generalized form of the predictive equation for AF. The generalized form is based on a linear multi-pool representation of the ocean, or equivalently a sum of impulse response functions (Greens functions) with characteristic time-scales of τ0 = ∞,τ1 = 433.3 yr, τ2 = 83.9 yr, τ3 = 11.2 yr, and τ4 = 0.8 yr (see Ap- pendices B and D). The predicted AF is nearly the same as AF predicted by the simple single pool model, conﬁrming that the simple model sufﬁces to analyse the controls on the AF during the 1959–2006 period. The reason is that ocean carbon uptake during this period is primarily governed by one Green’s function, the one associated with τ2 = 83.9 yr (which is close to τoc). An intuitive explanation for the existence of a spin-up pe- riod is as follows. The constancy of AF for a purely exponen- tial forcing reﬂects the balance between two exponential pro- cesses, exponential damping of the atmospheric perturbation via carbon sinks and exponential forcing (Appendix B). 3 A simple carbon cycle model This is because fossil fuel emis- sions rise approximately exponentially but land use change emissions rise more slowly and thus their sum will not equal an exact exponential function (Sect. 2 and Fig. 1a and c). ﬁrst step the land uptake as being linearly related to the atmo- spheric perturbation with a single time-scale, with the inten- tion to generalize the description if the data were to contain sufﬁcient information. One could question the realism of our simple model on the grounds that the model treats both the oceans and the land vegetation as just one integral pool, while for both, sev- eral pools with different characteristic exchange time scales is more realistic. We tested this for the ocean and it turns out that inclusion of multiple ocean pools does not alter our conclusions for the reason explained in Sect. 5. 5 Predicted and observed airborne fraction Instead of idealized cases we now predict the time-course of AF using the observed FF and LU emissions. For this purpose we use the differential equation for AF implied by our model Eq. (2) dAF dt = ( 1 f df dt + 1 τs dτs dt )−( 1 τs + 1 f df dt + 1 τs dτs dt )AF (3) 3 A simple carbon cycle model We investigate the processes controlling time-variations of the airborne fraction with simple linear models of the global perturbation of the carbon cycle. This is justiﬁed on two grounds. First any claim of a possible non-linear behaviour of the system must be shown to differ from the prediction of such linear models. Secondly, the concept of an efﬁciency, like the efﬁciency of a heat engine, is inherently linear. This is because an efﬁciency is deﬁned as the ratio between the magnitude of an effect and the magnitude of its cause. In our case the cause is the increase in atmospheric CO2 due to human activity and the effect is the carbon ﬂux from the at- mosphere to the ocean and land carbon pools. If we treat the ocean and land each as a single pool of carbon with a con- stant sink efﬁciency, then the ﬂuxes from the atmosphere to the oceans and to the land Fat→oc and Fat→ld, are given by For uptake by the land vegetation it is less clear whether the linearity concept applies. This is because uptake by land, unlike the oceans, is tied to processes such as productivity and the status of the land vegetation, some of which may be related to the atmospheric CO2 perturbation (speciﬁcally CO2 uptake during photosynthesis), while others like nutri- ent and micronutrient availability, plant and soil respiration, vegetation population dynamics, and land use change are not. Even if there were a productivity increase due to CO2 “fertilization”, it would likely be a linear response only dur- ing a limited period of time until land vegetation reaches a new steady state balance between growth and mortality. The linear response assumption of the land vegetation thus con- founds many processes and time-scales (e.g., Lloyd, 1999). Despite these obvious caveats, we nevertheless described in a Fat→oc = 1C τoc , Fat→ld = 1C τld . (1) (1) Here 1C ≡C(t) −C(1765) is the anthropogenic perturba- tion of atmospheric carbon dioxide, and τoc and τld are con- stants. In this context a weakening/strengthening of the sinks means that τoc and/or, τld are increasing/decreasing in time. Atmos. Chem. Phys., 10, 7739–7751, 2010 www.atmos-chem-phys.net/10/7739/2010/ 7743 M. Gloor et al.: Airborne fraction trends and carbon sink efﬁciency AFFF from lower values. 4 Airborne fraction for idealized cases If the forcing deviates from a pure exponential function, there will be a spin up period until the exponential component of the forcing dominates over other slower growing components of the forcing. An implication of the existence of a spin-up pe- riod is that we expect observed AFFF+LU to converge towards Atmos. Chem. Phys., 10, 7739–7751, 2010 www.atmos-chem-phys.net/10/7739/2010/ 7744 M. Gloor et al.: Airborne fraction trends and carbon sink efﬁciency −200 −150 −100 −50 0 50 100 150 200 250 300 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 Year Airborne fraction (−) f(t)=fet/τf f=FF0et/τf+LU0et/τLU, τLU=110 τf/τs=20/45=0.444... τf /τs=40/45=0.888... τf /τs=20/4.5=4.444... f(t)=fet/τf+0.1 Fig. 2. Predicted air-borne fraction for a range of forcings including purely exponential forcing f (t) = f et/τf (blue), mixed exponential and constant forcing f (t) = f et/τf +f0 with f0 = 0.1 (black), and forcing by the Fig. 2. Predicted air-borne fraction for a range of forcings including purely exponential forcing f (t) = f et/τf (blue), mixed exponential and constant forcing f (t) = f et/τf +f0 with f0 = 0.1 (black), and forcing by the sum of two exponential functions, f (t) = f et/τf +fLUet/τLU (red). −200 −150 −100 −50 0 50 100 150 200 250 300 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 Year Airborne fraction (−) f(t)=fet/τf f=FF0et/τf+LU0et/τLU, τLU=110 τf/τs=20/45=0.444... τf /τs=40/45=0.888... τf /τs=20/4.5=4.444... f(t)=fet/τf+0.1 Airborne fraction (−) Fig. 2. Predicted air-borne fraction for a range of forcings including purely exponential forcing f (t) = f et/τf (blue), mixed exponential and constant forcing f (t) = f et/τf +f0 with f0 = 0.1 (black), and forcing by the Fig. 2. Predicted air-borne fraction for a range of forcings including purely exponential forcing f (t) = f et/τf (blue), mixed exponential and constant forcing f (t) = f et/τf +f0 with f0 = 0.1 (black), and forcing by the sum of two exponential functions, f (t) = f et/τf +fLUet/τLU (red). sum of two exponential functions, f (t) = f e f +f If the forcing is not exactly exponential, then th 160 of 200-300 years (Fig. 4 Airborne fraction for idealized cases 2), depending on the exa An intuitive explanation for the existence of for a purely exponential forcing reﬂects the bala damping of the atmospheric perturbation via ca the forcing deviates from a pure exponential fu 165 nential component of the forcing dominates ov An implication of the existence of a spin-up p verge towards AFFF from lower values. This exponentially but land use change emissions ri The model computed AFFF and AFFF+LU agree re- markably well with the observed ones, calculated from atmospheric concentration data (from Mauna Loa) and anthropogenic emissions. The numerator of the observed AFFF and AFFF+LU were calculated using the atmospheric rate of change, dC/dt, taken from the monthly mean records from NOAA ESRL (co2 mm mlo.2009.txt obtained in November 2009 from ftp://ftp.cmdl.noaa.gov/ccg/co2/ trends/, Tans, 2009a). From these monthly data we ﬁrst cal- culated annual means centered on 31 December/1 January, from which we estimated the time derivative by differencing. We estimated the dC/dt from annual means, because the an- thropogenic emissions estimates are annually resolved. Our conclusions are not sensitive to this choice. e (red). me-scale for convergence is roughly on the order unctional form of the forcing. in-up period is as follows. The constancy of AF between two exponential processes, exponential n sinks and exponential forcing (appendix B). If on, there will be a spin up period until the expo- ther slower growing components of the forcing. d is that we expect observed AFFF+LU to con- b f il f l i i i i l The forcing during the period 1959–2006 has three dis- tinctly different phases: 1958–1973 fast growth, small τf ∼20 yr; 1973–1999: slow growth, large τf ∼30–150 years; 2000–2006: fast growth, small τf ∼25 years, (Fig. 1b). We thus expect the predicted AF to decrease during the 1973– 1999 period and then to increase again with some lag. This is indeed what we ﬁnd (Figs. 1e, 3a). The same signature seems to be present in the observations as well, although it tends to be masked by the larger variability. Furthermore for this model there is indeed a tight relation between AF and the relative growth rate RGR of anthropogenic emissions (com- pare Fig. 1d and e). 4 Airborne fraction for idealized cases because fossil fuel emissions rise approximately more slowly and thus their sum will not equal an course of AF using the observed FF and LU As mentioned earlier on, because the forcing used to cal- culate AFFF+LU is approximately the sum of an exponential function, FF, and a less strongly increasing function, LU, we expect AFFF+LU (red dashed line) to be lower than AFFF (blue solid line) and to slope more upwards than AFFF, even- tually converging towards AFFF. This is indeed what is ob- served and predicted (Fig. 1e). exact exponential function (section 2). 170 5 Predicted and observed airborne fraction Instead of idealized cases we now predict the t emissions. For this purpose we use the differen Observed AFFF and AFFF+LU records (Figs. 1e, 3a) ex- hibit large inter-annual variability, which is missing in the AFFF and AFFF+LU predicted by the linear model. This is because our model is forced solely by carbon ﬂuxes from fossil fuel and land use change, thus variations due to non- anthropogenic forcings, like volcanic eruptions or climate os- cillations, are not captured. The large inter-annual variations in observed air-borne fraction are largely due to inter-annual variability in the rate of change of atmospheric CO2, dC dt , an observation known since the 1970s to be associated with El Ni˜no/La Ni˜na and post volcanic periods (Agung, El Chichon, Pinatubo; Bacastow, 1976). M. Gloor et al.: Airborne fraction trends and carbon sink efﬁciency 0.2 0.4 0.6 0.8 AF (−) −0.5 0 0.5 AFobs - AFpred −2 −1 0 1 2 ∆f + ∆s (PgC yr-1) −0.02 0 0.02 0.04 0.06 0.08 −1 0 1 2 Cpred - Cobs (ppm) Agung Pinatubo Indonesia Peat burning RGR = τf -1 (yr-1) 2010 2010 2010 2010 1955 1960 1965 1970 1975 1980 1985 1990 1995 2000 2005 2010 0.0 −2 observed AF modeled AF (FF + LU) modeled AF (FF + LU + ( ∆ f + ∆s)) RGR fossil fuel & land use (FF+LU) RGR fossil fuel (FF) 1963-65 1991-93 2002-03 1998 ? residual AF (FF + LU) residual AF (FF + LU + ( ∆ f + ∆s)) e d c b a Fig. 3. Time-series from 1955 through 2010: (a) Model predicted and observed AFFF+LU. Also shown is the model- predicted AF after the addition of the corrections shown in panel c, (b) Relative growth rate of f = FF and f = FF +LU, respectively. Shown are the annual values (symbols) and after ﬁltering it with a low-pass ﬁlter (11 year running mean); (c) Correction to land use and fossil fuel emissions 1f +1s calculated Fig. 3. Time-series from 1955 through 2010: (a) model predicted and observed AFFF+LU. Also shown is the model-predicted AF after the addition of the “corrections” shown in (c), (b) relative growth rate of f =FF and f =FF+LU, respectively. Shown are the annual values (symbols) and after ﬁltering it with a low-pass ﬁlter (11 year running mean); (c) correction to land use and fossil fuel emissions 1f +1s calculated by minimizing the least square difference between predicted and observed AFFF+LU. Periods with known extrinsic forcings are indicated with arrows. (d) Difference between observed and model predicted AFFF+LU and AFFF+LU+1(f +s), and (e) difference between predicted and observed atmospheric CO2. extrinsic forcings are indicated with arrows. (d) Differ and AFFF+LU+1f , and (e) difference between predi The forcing during the period 1959-2006 ha growth, small τf ∼20 yr; 1973-1999 slow growt assumption of a linear response were to ﬁt the data well, then we would not need to invoke a trend in sink efﬁciency (i.e. a trend in τs). The difference (residuals) between observed and predicted AF can thus give us an indication of potential non- linearities or possibly incompleteness of the linear model to describe the evolution of the anthropogenic atmospheric car- bon perturbation. equation for AF implied by our model equation 6 Causes for trends in observed airborne fraction Given the variation in AFFF and AFFF+LU due to variations in forcing (Figs. 1e, 3a), particularly in fossil fuel emis- sions, and the considerable time it takes for AF to converge to its asymptotic value, is there nonetheless a possibility to test whether there are trends in sink efﬁciency from the time course in AF? If our differential equation for AF based on the Atmos. Chem. Phys., 10, 7739–7751, 2010 www.atmos-chem-phys.net/10/7739/2010/ M. Gloor et al.: Airborne fraction trends and carbon sink efﬁciency M. Gloor et al.: Airborne fraction trends and carbon sink efﬁciency 7745 M. Gloor et al.: Airborne fraction trends and carbon sink efﬁciency 7745 0.2 0.4 0.6 0.8 AF (−) −0.5 0 0.5 AFobs - AFpred −2 −1 0 1 2 ∆f + ∆s (PgC yr-1) −0.02 0 0.02 0.04 0.06 0.08 −1 0 1 2 Cpred - Cobs (ppm) Agung Pinatubo Indonesia Peat burning RGR = τf -1 (yr-1) 2010 2010 2010 2010 1955 1960 1965 1970 1975 1980 1985 1990 1995 2000 2005 2010 0.0 −2 observed AF modeled AF (FF + LU) modeled AF (FF + LU + ( ∆ f + ∆s)) RGR fossil fuel & land use (FF+LU) RGR fossil fuel (FF) 1963-65 1991-93 2002-03 1998 ? residual AF (FF + LU) residual AF (FF + LU + ( ∆ f + ∆s)) e d c b a Fig. 3. Time-series from 1955 through 2010: (a) Model predicted and observed AFFF+LU. Also shown is the model- predicted AF after the addition of the corrections shown in panel c, (b) Relative growth rate of f = FF and f = FF +LU, respectively. Shown are the annual values (symbols) and after ﬁltering it with a low-pass ﬁlter (11 year running mean); (c) Correction to land use and fossil fuel emissions 1f +1s calculated b i i i i th l t diff b t di t d d b d AF P i d ith k Fig. 3. Time-series from 1955 through 2010: (a) model predicted and observed AFFF+LU. Also shown is the model-predicted AF after the addition of the “corrections” shown in (c), (b) relative growth rate of f =FF and f =FF+LU, respectively. Shown are the annual values (symbols) and after ﬁltering it with a low-pass ﬁlter (11 year running mean); (c) correction to land use and fossil fuel emissions 1f +1s calculated by minimizing the least square difference between predicted and observed AFFF+LU. Periods with known extrinsic forcings are indicated with arrows. (d) Difference between observed and model predicted AFFF+LU and AFFF+LU+1(f +s), and (e) difference between predicted and observed atmospheric CO2. M. Gloor et al.: Airborne fraction trends and carbon sink efﬁciency ce between observed and model predicted AFFF+LU d and observed atmospheric CO2. hree distinctly different phases (1958-1973 fast due to the omission of forcings caused by land use change, or associated with indirect, non-anthropogenic mechanisms such as volcanic eruptions, (ii) the response time scale (or equivalently sink efﬁciency) is changing over time indicating a non-linear behaviour, or (iii) the model is all too simplistic. arge τf ∼30-150 years; 2000-2006 fast growth, We investigate the ﬁrst explanation for the trend in the residuals by inquiring what corrections 1(f +s) to FF+LU would be needed to obtain a better ﬁt between observed and predicted AF. If we can attribute these ﬂux correc- tions 1(f + s) to sources or sinks 1s caused by extrinsic non-anthropogenic forcings, or omissions in land use and The trend of the residuals (the difference between ob- served and predicted AFFF+LU) is positive (Fig. 3d), indicat- ing that something is indeed at odds. There are three possible causes for the trend in the residuals: (i) incomplete forcing, Atmos. Chem. Phys., 10, 7739–7751, 2010 www.atmos-chem-phys.net/10/7739/2010/ M. Gloor et al.: Airborne fraction trends and carbon sink efﬁciency 7746 fossil fuel ﬂuxes 1f , then there is no need to invoke trends in sink efﬁciency (i.e. nonlinearities) and vice versa. To es- timate the ﬂux corrections 1(f +s), we minimize the cost function paralleled by compensating anomalies in respiration. Thus overall, with the possible exception of the 2002/2003 event, the four events in the residuals can be attributed to extrinsic forcings and omissions in land use change ﬂuxes. We may ﬁnally test whether there is a declining trend in sink efﬁciency by investigating the slope of 1(f + s) but with the post-Agung, post-Pinatubo, Indonesian peat pulse and 2002/2003 events excluded, as indicated by the red dashed line in Fig. 3c. The result of a t-test indicates that the chance for this slope to be signiﬁcantly differently from zero is very small (p < 0.01). Thus, after removing the four events there is no evidence for a sink efﬁciency trend in the AF. J(1(f +s)(1959),...,1(f +s)(2006)) = 2006 X yr=1959 (AFobs FF+LU(yr)−AFpred FF+LU(yr))2+ +((Cpred 2006 −Cpred 1959)−(Cobs 2006 −Cobs 1959))2 with respect to 1(f +s) (1959),...,1(f +s) (2006) using simulated annealing. The second term of the right hand side ensures that mass is conserved. M. Gloor et al.: Airborne fraction trends and carbon sink efﬁciency Because the weighting of the data is uniform, there should not be a signiﬁcant trend in the residuals after including the ﬂux corrections. To be sure, we used the standard t-test (e.g. Robinson, 1981, Appendix E) and found indeed no signiﬁcant slope. Our analysis is ambiguous regarding the possible sudden “positive feedback event” in 2002/2003. If the event is in- deed due to Siberian forest ﬁres then it may not necessarily be a sign of an nonlinear, irreversible event but rather part of a natural cycle of boreal forest population dynamics (birth, aging, death, caused e.g. by ﬁre) and thus carbon uptake and release (Wirth et al., 2002; Mollicone et al., 2002). There- fore measurements of future net carbon ﬂuxes in this region are necessary to determine to what extent these ﬂuxes could indeed reﬂect a positive feedback. The estimation procedure identiﬁes four events (Fig. 3c): increased sinks for atmospheric carbon in the aftermaths of the 1963 Agung and 1991 Pinatubo eruptions and carbon ﬂux pulses to the atmosphere in 1997/98 and similarly in 2002/03. A dip in the increase rate of atmospheric carbon is well known to occur after major volcanic eruptions, es- pecially those that inject material into the stratosphere (e.g., R¨odenbeck et al., 2003). The decrease in atmospheric CO2 is generally attributed to a land sink in the aftermath of the eruption. The mechanism may possibly be an increase in the ratio between diffuse and direct radiation, enhancing photo- synthesis (Roderick et al., 2001) and/or reduced soil respira- tion due to temporary cooling of the earth surface (Jones and Cox, 2001). The onset of increased land uptake in the early 1990’s is actually before the Pinatubo eruption as noticed by Keeling et al. (1995). To our knowledge the mechanism for this early onset remains unclear. The 1997/98 carbon ﬂux pulse to the atmosphere is also well studied, and largely at- tributed to peat burning in Indonesia in 1997/1998 (Page et al., 2002). This carbon ﬂux to the atmosphere seems to be missing from the Houghton et al. (2007) land use change ﬂux estimate, although it is the result of land use change (Page et al., 2002). Finally there are indications from several stud- ies of what the causes of the 2002 and 2003 ﬂux pulses to the atmosphere could be (Yurganov et al., 2005; Balzter et al., 2005; Jones and Cox, 2005). Speciﬁcally Yurganov et al. M. Gloor et al.: Airborne fraction trends and carbon sink efﬁciency (2005) documented air-column CO anomalies on the order of 50% at northern hemisphere mid-to high latitude stations, with anomalies occurring during the second half of the year 2002 and 2003. They associated these signatures with boreal forest ﬁres in Siberia, consistent with results from remote sensing ﬁre spot data, and results based on more reﬁned re- mote sensing methods (Balzter et al., 2005). Besides boreal forest ﬁres, the 2002/2003 events may also be related to the drought in Europe in summer 2003, which reduced net pri- mary production of the land vegetation (Ciais et al., 2005), although decreases in primary production are likely to be 8 Discussion A key motivation to undertake this study has been the re- cent claims by Canadell et al. (2007) and Raupach et al. (2008) that they have detected a long-term increasing trend in the AF and that this trend is due to positive feedbacks in the coupled carbon-climate system. Knorr (2009) already challenged these authors with regard to the detection of the trends, arguing that given the noise in the data, the trend is not detectable. Here we challenged the second claim of Canadell et al. (2007) and Raupach et al. (2008) that a posi- tive trend is indicative of a positive feedback between climate and the global carbon cycle. Our analyses suggest that this assumption cannot be made because trends in AF are not only caused by trends in sink efﬁciency but also by (i) variations in the relative growth rate of the emissions, (ii) incomplete spin-up, and (iii) omissions in the anthropogenic emissions. An alternative method to investigate carbon-climate feed- backs on the basis of the atmospheric CO2 record was re- cently proposed by Rafelski et al. (2009). They analysed a quantity that they termed the “constant airborne fraction anomaly”. This quantity is deﬁned as the difference between the atmospheric CO2 record and a ﬁxed fraction (57%) of the cumulated (time-integrated) fossil fuel emissions. While re- lated, this quantity differs fundamentally from our AF in two ways. First it uses the time-integrated emissions, whereas we use the instantaneous emissions. Secondly, it is expressed in terms of an absolute anomaly, i.e. the amount of anomalous CO2 in the atmosphere, whereas our AF is expressed relative to the magnitude of the emissions. One may argue that an analysis in terms of absolute anomalies is preferable, as the magnitude of the anomaly does not depend on the magnitude of the emissions. This dependency is actually a disadvan- tage of the AF analysis, since the same ﬂux anomaly leads to a larger deviation early in time when the emissions are small, and to a much smaller deviation in the latter part of the record, when emissions are large. A potential downside of the analysis of the “constant airborne fraction anomaly” is that because of its cumulative nature, it tends to suppress shorter-term variations. We focused our analysis here on the AF itself, primarily because our primary target was to inves- tigate the robustness of the conclusions of the analyses by Canadell et al. 7 Detecting trends in the efﬁciency of sinks Although our analysis suggests that the decadal-scale ob- served variations and trends in the AF primarily reﬂect changes in the relative growth rate of the total anthropogenic CO2 and incomplete spin-up, it is still interesting to analyze the relation between trends in sink efﬁciency and trends in AF within the framework of our simple model. For this pur- pose we investigate the hypothetical case where we impose a 50% decrease in the sink efﬁciency by the year 2008 com- pared to 1959. We achieve this strong feedback by setting τs(t) = 42 yr for t < 1959 and τs(t) = 42 yr+ϵ ∗(t −1959) with ϵ = 0.5 for t ≥1959. We then integrate Eq. (3) forward in time, starting from 1765 and compare the result with the record for the AF calculated for a constant τs. Such a weak- ening trend since 1959 would induce a difference in the trend of AF of δ 1AF 1t = 1AFϵ=0.5 1t −1AFϵ=0.0 1t δ 1AF 1t = 1AFϵ=0.5 1t −1AFϵ=0.0 1t ∼0.1(50 yr)−1 where 1AF 1t ≡AF(t = 2008)−AF(1959) 2008−1959 . where This shows ﬁrstly that a fairly strong positive feedback, oper- ating over a period of 50 years, causes a trend that is roughly of similar magnitude as variations caused by relative growth rate variations in fossil fuel emissions over the 1959–2009 This shows ﬁrstly that a fairly strong positive feedback, oper- ating over a period of 50 years, causes a trend that is roughly of similar magnitude as variations caused by relative growth rate variations in fossil fuel emissions over the 1959–2009 Atmos. Chem. Phys., 10, 7739–7751, 2010 www.atmos-chem-phys.net/10/7739/2010/ 7747 M. Gloor et al.: Airborne fraction trends and carbon sink efﬁciency Despite the fundamental differences in the approach, it is nevertheless of interest to compare our conclusions with those of Rafelski et al. (2009). Two main conclusions of their study are (i) that they expected a decrease in the airborne fraction anomaly after the early 1970s due to the decrease in the fossil fuel growth rates, and (ii) that the absence of this decrease in the observed anomaly is caused by enhanced land emissions due to a warming trend that began around the same time. Regarding their ﬁrst conclusion, it seems as if variations in the growth of the fossil fuel emissions matter irrespective of whether the AF is expressed instantaneously or cumulatively. 8 Discussion (2007) and Raupach et al. (2008). 7 Detecting trends in the efﬁciency of sinks This is likely a consequence of the fact that the integral of an exponential function is an exponen- tial with the same exponent, i.e. time-scale. Thus changes in the time-scale τf affect both deﬁnitions of the AF. In their second conclusion, their statement is equivalent to invoking the detection of a positive feedback between the carbon cy- cle and climate, i.e. they essentially support the conclusions of Canadell et al. (2007) and Raupach et al. (2008). This second conclusion of Rafelski et al. (2009) is based on a slightly better ﬁt of their model predicted time-evolution of the airborne fraction anomaly if they included a temperature dependent model of the land (and ocean). However, the ﬁt of this temperature-dependent model was only marginally bet- ter, and inclusion in the forcing of the additional processes we have identiﬁed in land-use change and variability could have equally led to an improvement over the temperature- independent model. Given our previous ﬁnding that rela- tively small omissions in the emissions from fossil fuel burn- ing and land-use change can alter the ﬁt (and trend) substan- tially, it may well be the case that once these omissions are added, the temperature-independent model may produce an equally good ﬁt. We thus conclude that the evidence for the detection of a carbon-cycle climate feedback is weak and not robust. A critical element to advance is the availability of much more accurate emission data, as this would permit to distinguish between alternative explanations. period. Secondly, the signal would be difﬁcult to detect. Us- ing a standard t-test, such a 50% sink efﬁciency decrease over a period of 50 years is detectable only at the 90% signiﬁcance level, but not at the 95% signiﬁcance level. This is because the’natural’ variation in AF of the order of 0.15 (Fig. 3a) will tend to mask any trend. In conclusion, variations in emis- sions and “noise” due to extrinsic non-anthropogenic forc- ings make the AF not a very suitable diagnostic for detecting trends in carbon sink efﬁciency. 9 Conclusions We have investigated the question of what controls trends and decadal scale variations in CO2 airborne fraction (AF) using simple linear models describing the evolution of an at- mospheric perturbation in CO2. Our analysis suggests ﬁrstly that variations of the relative growth rate of anthropogenic CO2 emissions are a major control of variations in AF. Sec- ondly, it suggests that there is a long spin-up time for AF to converge to its asymptotic value if the forcing is not exactly exponential. If the forcing is not exactly an exponential func- tion, as it is the case for the sum of fossil fuel burning and land use change emissions, this time-scale is of the order of 200–300 years. A ﬁrst consequence is that there is no one- to-one association between positive trends in AFFF+LU and negative trends in sink efﬁciency. A second consequence is Atmos. Chem. Phys., 10, 7739–7751, 2010 www.atmos-chem-phys.net/10/7739/2010/ M Gl t l Ai b f ti t d d b i k fﬁi 7748 M. Gloor et al.: Airborne fraction trends and carbon sink efﬁciency M. Gloor et al.: Airborne fraction trends and carbon sink efﬁciency The second identity holds because dC dt = d(C(t)−C(−∞)) dt = d1C dt . The second identity holds because dC dt = d(C(t)−C(−∞)) dt = d1C dt . that in order to detect trends in sink efﬁciencies from the time course of AFFF+LU, it is necessary to disentangle the spin-up time and fossil fuel growth rate variation signatures in the AF from signatures due to other causes. Our differential equation for AF permits us to do so by predicting the time course of AF due solely to these two factors. The remaining trends and variations in the residuals can then be explained by varia- tions in extrinsic forcings like volcanic eruptions and climate variations, omissions in the anthropogenic ﬂuxes to the at- mosphere, trends in sink efﬁciencies, or inadequacies in our model. We do indeed ﬁnd a positive trend in the residuals, but argue that this trend is not statistically signiﬁcant after cor- recting for known events such as the temporal distribution of the extrinsic forcings and likely omissions in the emissions (particularly from land-use change). We thus do not need to invoke a trend in carbon sink efﬁciencies to explain the trend in the AF. Appendix A AF= dC dt f = d1C dt f =1−1 τs 1C f =1−1 τs R t −∞G(t,t′)f (t′)dt′ f 9 Conclusions Our analysis also suggests that trends in AF are not a very good diagnostic to detect changes in carbon sink efﬁciency because variations in the signal are complex and the signal-to-noise ratio is small. dt For a forcing of the form f et/τf +f0 where f0 is constant, we may integrate the equation similarly to obtain AF = 1 1+ τf τs × e t τf e t τf +(f0/f ) . Finally for the case of a sum of exponential forcings with different time-scales, i.e. Pn i=1fiet/τi, we ﬁnd in a similar way AF = Pn i=1 1 1+ τi τs fiet/τi Pn i=1fiet/τi . Derivation of the differential equation for the time evolution of AF Although the surprisingly linear behaviour of the global carbon cycle for the past 50 years may suggest otherwise, it would be a mistake to assume that it will continue to operate in such a linear fashion into the future. For one thing, the continuous acidiﬁcation of the ocean will inevitably lead to a decrease in the oceanic uptake capacity for anthropogenic carbon (Sarmiento and Le Qu´er´e, 1996). Our analysis does not dispute a future reduction in sinks of anthropogenic car- bon compared to a linear system response. Rather, we argue that atmospheric concentration data if analysed adequately do not yet reveal a statistically signiﬁcantly signal. The basis for the derivation of the differential equation for the AF is the general solution of the Eq. (2) for an arbitrary forc- ing function f (t), which is again obtained with the method of “variation of constant”: 1C = Z t −∞ G(t,t′)f (t′)dt′ with G(t,t′) = e− R t t′ dt′′ τs(t′′) . (B1) (B1) G(t,t′) is called the Greens function of the problem. The in- terpretation of this expression is as follows. The atmospheric perturbation at time t is given by the sum of “ﬂux pulses” to the atmosphere, each of them damped exponentially in time by G(t,t′) from the moment they have been emitted into the atmosphere. From the deﬁnition of AF we then ﬁnd G(t,t′) is called the Greens function of the problem. The in- terpretation of this expression is as follows. The atmospheric perturbation at time t is given by the sum of “ﬂux pulses” to the atmosphere, each of them damped exponentially in time by G(t,t′) from the moment they have been emitted into the atmosphere. From the deﬁnition of AF we then ﬁnd Appendix E Appendix E Appendix C Thus Estimation of atmosphere ocean and atmosphere land exchange time constants τ oc and τ ld − Z t −∞ e−t−t′ τld (A0 τld + 4 X j=1 Aj( 1 τld + 1 τj )e −t−t′ τj )f (t′)dt′ Coupled carbon cycle ocean general circulation models show that there is an approximately linear relationship between the atmospheric perturbation of CO2 and ocean carbon uptake (e.g. Sarmiento and Le Qu´er´e, 1996). Furthermore we know ocean anthropogenic carbon inventories from ocean surveys (Sabine et al., 2004; Gruber et al., 2009). Based on the ap- proximate linearity = f (t)−I(t) using P4 j=0Aj = 1 and with I(t) ≡ Z t −∞ e−t−t′ τld (A0 τld + 4 X j=1 Aj( 1 τld + 1 τj )e −t−t′ τj )f (t′)dt′ . Fat→oc(t) = Fat→oc(tref) pCOat 2 (t)−pCOat 2 (1765) pCOat 2 (tref)−pCOat 2 (1765) and thus Therefore τoc = pCOat 2 (tref)−pCOat 2 (1765) F(tref) = 81.4 yr. dAF dt = d dt ( d1C dt f ) = 1 f df dt I(t) f (t) −1 f dI dt = 1 f df dt (1−AF) −1 f dI dt Here tref = 1995 and F(1995) = 2.2 PgC yr−1 is from Gru- ber et al. (2009), pCO2(1765)=276.7 ppm (Etheridge et al., 1996), pCO2(1995)=360.9 ppm, and 1 ppm CO2=2.1276 Pg C for the earth’s atmosphere (e.g., Sarmiento et al., 2010). Given τs = 37.5 years (from the main text) and using the relation with dI dt = (A0 τld + 4 X j=1 Aj( 1 τld + 1 τj ))f (t)+ − Z t −∞ e−t−t′ τld (A0 τ 2 ld + 4 X j=1 Aj( 1 τld + 1 τj )2e −t−t′ τj )f (t′)dt′. 1 τs = 1 τld + 1 τoc 1 τs = 1 τld + 1 τoc from Eq. (2) from the main text we furthermore ﬁnd τld ≃ 69.5 yr. 1 τs = 1 τld + 1 τoc from Eq. (2) from the main text we furthermore ﬁnd τld ≃ 69.5 yr. Solutions of the differential equation for 1C for idealized cases Therefore dAF dt = ( 1 f df dt + 1 τs dτs dt )(1−AF)−1 τs AF = ( 1 f df dt + 1 τs dτs dt )−( 1 τs + 1 f df dt + 1 τs dτs dt )AF. d dt Z t 0 G(t,t′)f (t′)dt′=G(t,t)f (t)dt dt + Z t −∞ dG(t,t′) dt f (t′)dt′ = f (t)−1 τs Z t −∞ G(t,t′)f (t′)dt′. Th f d dt Z t 0 G(t,t′)f (t′)dt′=G(t,t)f (t)dt dt dt Z 0 dt + Z t −∞ dG(t,t′) dt f (t′)dt′ = f (t)−1 τs Z t −∞ G(t,t′)f (t′)dt′. h f Goc(t,t′) = A0 +PN j=1Aje −t−t′ τj ,PN j=0Aj = 1. The Greens ′ + Z t −∞ dG(t,t′) dt f (t′)dt′ = f (t)−1 τs Z t −∞ G(t,t′)f (t′)dt′. oc( , ) 0 +P j=1 j ,P j=0 j function Goc(t,t′) for the oceans is available from Sarmiento et al. (1992) and Maier-Reimer and Hasselmann (1987), cal- culated using coupled ocean circulation carbon cycle models. Therefore Therefore dAF dt = ( 1 f df dt + 1 τs dτs dt )(1−AF)−1 τs AF = ( 1 f df dt + 1 τs dτs dt )−( 1 τs + 1 f df dt + 1 τs dτs dt )AF. The perturbation of atmospheric carbon 1C due to anthro- pogenic emissions is then given by 1C = Z t −∞ e−t−t′ τld (A0 + 4 X j=1 Aje −t−t′ τj )f (t′)dt′ . Thus d1C dt = 4 X j=0 Ajf (t)+ − Z t −∞ e−t−t′ τld (A0 τld + 4 X j=1 Aj( 1 τld + 1 τj )e −t−t′ τj )f (t′)dt′ 1C = Z t −∞ e−t−t′ τld (A0 + 4 X j=1 Aje −t−t′ τj )f (t′)dt′ . Thus d1C dt = 4 X j=0 Ajf (t)+ − Z t −∞ e−t−t′ τld (A0 τld + 4 X j=1 Aj( 1 τld + 1 τj )e −t−t′ τj )f (t′)dt′ = f (t)−I(t) using P4 j=0Aj = 1 and with I(t) ≡ Z t e−t−t′ τld (A0 + 4 X Aj( 1 + 1 )e −t−t′ τj )f (t′)dt′ 1C = Z t −∞ e−t−t′ τld (A0 + 4 X j=1 Aje −t−t′ τj )f (t′)dt′ . Solutions of the differential equation for 1C for idealized cases or equivalently In order to calculate the AF for idealized cases we integrate the differential equation (1−AF) = 1 τs R t −∞G(t,t′)f (t′)dt′ f . d1C dt = −1C τs +f (t) The time-derivative of AF is thus dAF dt = 1 f df dt 1 τs R t −∞G(t,t′)f (t′)dt′ f with initial condition 1C(−∞) = 0 (since 1C is the pertur- bation of atmospheric carbon). For purely exponential forc- ing f (t) = f e t τf , τf constant, we ﬁnd by the method of “vari- ation of constant” − d 1 τs dt R t −∞G(t,t′)f (t′)dt′ f −1 τs d dt R t −∞G(t,t′)f (t′)dt′ f . 1C(t) = f 1 τs + 1 τf e t τf 1C(t) = f 1 τs + 1 τf e t τf Applying Leibniz’s rule d dt Z h(t) g(t) m(t,s)ds and thus and thus AF ≡ dC dt f e t τf = d1C dt f e t τf = 1 1+ τf τs = constant. = m(t,h(t))dh dt −m(t,g(t))dg dt + Z h(t) g(t) dm(t,s) dt ds Atmos. Chem. Phys., 10, 7739–7751, 2010 Atmos. Chem. Phys., 10, 7739–7751, 2010 Atmos. Chem. Phys., 10, 7739–7751, 2010 www.atmos-chem-phys.net/10/7739/2010/ M. Gloor et al.: Airborne fraction trends and carbon sink efﬁciency 7749 to the third term on the right gives to the third term on the right gives of of a system of ordinary differential equations is similar to the solution 1C given in Eq. (B1), Appendix B, for Eq. (2) but with Greens function G(t,t′) = Gld(t,t′)Goc(t,t′) and Goc(t,t′) = A0 +PN j=1Aje −t−t′ τj ,PN j=0Aj = 1. The Greens function Goc(t,t′) for the oceans is available from Sarmiento et al. (1992) and Maier-Reimer and Hasselmann (1987), cal- culated using coupled ocean circulation carbon cycle models. The perturbation of atmospheric carbon 1C due to anthro- pogenic emissions is then given by of a system of ordinary differential equations is similar to the solution 1C given in Eq. (B1), Appendix B, for Eq. (2) but with Greens function G(t,t′) = Gld(t,t′)Goc(t,t′) and ′ g g d dt Z t 0 G(t,t′)f (t′)dt′=G(t,t)f (t)dt dt + Z t −∞ dG(t,t′) dt f (t′)dt′ = f (t)−1 τs Z t −∞ G(t,t′)f (t′)dt′. References Houghton, R. A.: Balancing the global carbon budget, Ann. Rev. Earth Plan. Sc., 35, 313–347, 2007. Achard, F., Eva, H. D., Stibig, H.-J., Mayaux, P., Gallego, J., Richards, T., and Malingreau, J.-P.: Determination of deforesta- tion rates of the World in humid tropical forests, Science, 297, 999–1002, 2002. Jones, C. and Cox, P.: Modelling the volcanic signal in the at- mospheric CO2 record, Global Biogeochem. Cy., 15, 453–465, 2001. Jones, C. and Cox, P.: On the signiﬁcance of atmospheric CO2 growth rate anomalies in 2002/2003, Geophys. Res. Lett., 32, L14816, doi:10.1029/2005GL023027, 2005. Bacastow, R. B.: Modulation of atmospheric carbon dioxide by the Southern Oscillation, Nature, 261, 116–118, 1976. Bacastow, R. B. and Keeling, C. D.: Models to predict future at- mospheric CO2 concentrations, Workshop on the Global Effects of Carbon Dioxide from Fossil Fuels, edited by: Elliott W. P. and Machta L., United States Department of Energy, Washing- ton DC, 20545, 72–90, 1979. Knorr, W.: Is the airborne fraction of anthropogenic CO2 emissions increasing?, Geophys. Res. Lett., 36, L21710, doi:10.1029/2009GL040613, 2009. Keeling, C. D.: The Concentration and Isotopic Abundances of Car- bon Dioxide in the Atmosphere, Tellus, 12, 200–203, 1960. Balzter, H., Gerard, F. F., George, C. T., Rowland, C. S., Jupp, T. E., McCallum, I., Shvidenko, A., Nilsson, S., Sukhinin, A., Onuchin, A., and Schmullius, C.: Impact of the Arctic Oscillation pattern on interannual forest ﬁre vari- ability in Central Siberia, Geophys. Res. Lett., 32, L14709, doi:10.1029/2005GL022526, 2005. Keeling, C. D.: The carbon dioxide cycle: Reservoir models to de- pict the exchange of atmospheric carbon dioxide with the oceans and landplants, Chemistry of the Lower Atmosphere, Rasool, I. S., Plenum, New York, 251–329, 1973. Keeling, C. D., Whorf, T. P., Wahlen, M., and Van Der Plicht, J.: Interannual extremes in the rate of rise of atmospheric carbon dioxide since 1980, Nature, 375, 666–670, 1995. Boden, T. A., Marland, G., and Andres, R. J.: Global, regional, and national CO2 emissions, Carbon Dioxide Information Analysis Center, Oak Ridge National Laboratory, Oak Ridge, 2009. Khatiwala, S., Primeau, F. and Hall, T.: Reconstruction of the his- tory of anthropogenic CO2 concentrations in the ocean, Nature, 462, 346–349, doi:10.1038/nature08526, 2009. Canadell, J. G., Le Qu´er´e, C., Raupach, M. R., Field, C. B., Buiten- huis, E. T., Ciais, P., Conway, T. J., Gillett, N. P., Houghton, R. t-test for signiﬁcance of a trend For completeness we give here the test statistic for the signif- icance of the slope b of a regression line y = bx +a to data (xi,yi), i = 1,...,N: Instead of one differential equation for the evolution of at- mospheric 1C we consider a system of ordinary differen- tial equations describing carbon exchange between differ- ent volumes of the ocean. In this case the sink efﬁcien- cies are given by the inverse of the exchange time con- stants between the different ocean volumes. The solution t = b−β √( s2 Ns2x ) Atmos. Chem. Phys., 10, 7739–7751, 2010 www.atmos-chem-phys.net/10/7739/2010/ M. Gloor et al.: Airborne fraction trends and carbon sink efﬁciency M. Gloor et al.: Airborne fraction trends and carbon sink efﬁciency M. Gloor et al.: Airborne fraction trends and carbon sink efﬁciency 7750 with DeFries, R. S., Houghton, R. A., Hansen, M. C., Field, C. B., Skole, D., and Townshend, J.: Carbon emissions from tropical deforestation and regrowth based on satellite observations for the 1980s and 1990s, P. Natl. Acad. Sci. USA, 99, 14256–14261, 2002. s2 = 1 N −2 N X i=1 (yi −a −bxi)2 Enting, I. G. and Pearman, G. I.: The use of observations in cali- brating and validating carbon cycle models. A Global Analysis, J. Trabalka and D. Reichle, Springer Verlag, 425–458, 1986. and and s2 x = 1 N N X i=1 (xi −x)2 . s2 x = 1 N N X i=1 (xi −x)2 . Etheridge, D. M., Steele, L. P., Langenfelds, R. L., Francey, R. J., Barnola, J.-M., and Morgan, V. I.: Natural and anthropogenic changes in atmospheric CO2 over the last 1000 years from air in Antarctic ice and ﬁrn, J. Geophys. Res., 101, 4115–4128, 1996. The t statistic is distributed as a t-distribution with N-2 de- grees of freedom. Because we want to test whether b differs signiﬁcantly from 0, we use the statistic for β = 0. Grainger, A.: Difﬁculties in tracking the long-term global trend in tropical forest area, P. Natl. Acad. Sci. USA, 105, 818–823, 2008. Gloor M., Gruber, N., Sarmiento, J. L., Sabine, C., Feely, D., and R¨odenbeck, C.: A ﬁrst estimate of present and preindus- trial air-sea CO2 ﬂux patterns based on ocean interior carbon measurements and models, Geophys. Res. Lett., 30(1), 1010, doi:10.1029/2002GL015594, 2003. Acknowledgements. t-test for signiﬁcance of a trend We thank Pieter Tans and Tom Conway for permitting us to use the NOAA ESRL CO2 data, and Stephen Sitch, Oliver Phillips, Jon Lloyd, Simon Lewis and two anonymous reviewers for comments and suggestions. This work was supported by the United Kingdom National Environmental Research Council (NERC) grant NE/F005806/1. Gruber, N., Gloor, M., Mikaloff Fletcher, S. E., Doney, S. C., Dutkiewicz, S., Follows, M. J. Gerber, M., Jacobson, A. R., Joos, F., Lindsay, K., Menemenlis, D., Mouchet, A., M¨uller, S. A., Sarmiento, J. L., and Takahashi, T.: Oceanic sources, sinks, and transport of atmospheric CO2, Global Biogeochem. Cy., 23, GB1005, doi:10.1029/2008GB003349, 2009. Edited by: M. Heimann M. Gloor et al.: Airborne fraction trends and carbon sink efﬁciency M. Gloor et al.: Airborne fraction trends and carbon sink efﬁciency 7751 model, Clim. Dynam., 2, 63–90, 1987. Sarmiento, J. L., Orr, J. C., and Siegenthaler, U.: A perturbation simulation of CO2 uptake in an ocean general circulation model, J. Geophys. Res., 97, 3621–3645, 1992. Marland, G., Boden, T. A., and Andres, R. J.: Global, regional, and national CO2 emissions. Trends: A Compendium of Data on Global Change. Carbon Dioxide Information Analysis Center, Oak Ridge National Laboratory, US Department of Energy, Oak Ridge, Tenn., USA, 2006. Sarmiento, J. L. and Le Qu´er´e, C.: Oceanic CO2 uptake in a model of century-scale global warming, Science, 274, 1346– 1350, 1996. Sarmiento, J. L., Gloor, M., Gruber, N., Beaulieu, C., Jacobson, A. R., Mikaloff Fletcher, S. E., Pacala, S., and Rodgers, K.: Trends and regional distributions of land and ocean carbon sinks, Bio- geosciences, 7, 2351–2367, doi:10.5194/bg-7-2351-2010, 2010. Marland, G.: Uncertainties in Accounting for CO2 from Fos- sil Fuels, J. Ind. Ecol., 12(2), 136–139, doi:10.1111/j.1530- 9290.2008.00014.x, 2008. Mikaloff Fletcher, S. E., Gruber, N., Jacobson, A. R., Doney, S. C., Dutkiewicz, S., Gerber, M., Follows, M., Joos, F., Lindsay, K., Menemenlis, D., Mouchet, A., M¨uller, S. A., and Sarmiento J. L.: Inverse estimates of anthropogenic CO2 uptake, transport, and storage by the ocean, Global Biogeochem. Cy., 20, GB2002, doi:10.1029/2005GB002530, 2006. Sabine, C. L., Feely, R. A., Gruber, N., Key, R. M., Lee, K., Bullister, J. L., Wannikhof, R., Wong, C. S., Wallace, D. W. R., Tilbrook, B., Millero, F. J., Peng, T.-H., Kozyr, A., Ono, T., and Rios, A. F.: The oceanic sink for anthropogenic CO2, Science, 305, 367–371, 2004. Sweeney C., Gloor, E., Jacobson, A. R., Key, R. M., McKin- ley, G., Sarmiento, J. L., and Wanninkhof, R.: Constrain- ing global air-sea gas exchange for CO2 with recent bomb 14C measurements, Glob. Biogeochem. Cy., 21, GB2015, doi:10.1029/2006GB002784, 2007. Mollicone, D., Achard, F., Belelli-Marchesini, L., Federici, S., Wirth, C., Leipold, M., Rosellini, S., Schulze, E.-D. and Valen- tini R.: A remote sensing based approach to determine forest ﬁre cycle: case study of the Yenisei Ridge dark taiga, Tellus B, 54, 688–695, doi:610.1034/j.1600-0889.2002.01338.x, 2002. Solomon, S., Plattner, G.-K., Knutti, R., and Friedlingstein, P.: Irre- versible climate change due to carbon dioxide emissions, P. Natl. Acad. Sci., 106, 1704–1709, 2009. Oeschger, H. and Heimann, M.: Uncertainties of Predictions of Future Atmospheric CO2 Concentrations, J. Geophys. Res., 88, 1258–1262, 1983. Page S. E., Siegert, F., Rieley, J. References A., and Marland G.: Contributions to accelerating atmospheric CO2 growth from economic activity, carbon intensity, and efﬁ- ciency of natural sinks, P. Natl. Acad. USA, 104(47), 18866– 18870, doi:10.1073/pnas.0702737104, 2007. Le Qu´er´e, C., Raupach, M. R., Canadell, J. G., Marland, G. et al.: Trends in the sources and sinks of carbon dioxide, Nature Geosci., 2, 831–836, doi:10.1038/ngeo689, 2009. Lloyd, J.: The CO2 dependence of photosynthesis, plant growth re- sponses to elevated CO2 concentrations and their interaction with soil nutrient status, II. Temperate and boreal forest productivity and the combined effects of increasing CO2 concentrations and increased nitrogen deposition at a global scale, Funct. Ecol., 13, 439–459, 1999. Ciais, P., Reichstein, M., Viovy, N., et al.: Europe-wide reduction in primary productivity caused by the heat and drought in 2003, Nature, 437, 529–533, 2005. Cox, P. M., Betts, R. A., Jones, C. D., Spall, S. A., and Totter- dell, I. J.: Acceleration of global warming due to carbon-cycle feedbacks in a coupled climate model, Nature, 408, 184–187, doi:10.1038/35041539, 2000. Mainer-Reimer, E. and Hasselmann, K.: Transport and storage of CO2 in the ocean – an inorganic ocean-circulation carbon cycle Atmos. Chem. Phys., 10, 7739–7751, 2010 www.atmos-chem-phys.net/10/7739/2010/ M. Gloor et al.: Airborne fraction trends and carbon sink efﬁciency O., Boehm, H. V. Jaya, A., and Limin, S.: The amount of carbon released from peat and forest ﬁres in Indonesia during 1997, Nature, 420, 61–65, 2002. Tans. P. and Conway, T.: NOAA ESRL CO2 data available online at: http://www.esrl.noaa.gov/gmd/ccgg/trends/, ftp://ftp. cmdl.noaa.gov/ccg/co2/trends/, 2009. Yurganov, L. N., Duchatelet, P., Dzhola, A. V., Edwards, D. P., Hase, F., Kramer, I., Mahieu, E., Mellqvist, J., Notholt, J., Nov- elli, P. C., Rockmann, A., Scheel, H. E., Schneider, M., Schulz, A., Strandberg, A., Sussmann, R., Tanimoto, H., Velazco, V., Drummond, J. R., and Gille, J. C.: Increased Northern Hemi- spheric carbon monoxide burden in the troposphere in 2002 and 2003 detected from the ground and from space, Atmos. Chem. Phys., 5, 563–573, doi:10.5194/acp-5-563-2005, 2005. Rafelski, L. E., Piper, S., and Keeling, R. F.: Climate effects on atmospheric carbon dioxide over the last century, Tellus, 61B, 718–731, 2009. Raupach, M. R., Canadell, J. G., and Le Qu´er´e, C.: Anthro- pogenic and biophysical contributions to increasing atmospheric CO2 growth rate and airborne fraction, Biogeosciences, 5, 1601– 1613, doi:10.5194/bg-5-1601-2008, 2008. Robinson E.: Least squares regression analysis in terms of linear algebra, Internat. Human Resources Development Corporation, Boston, 497 pp., 1981. Wirth C., Schulze E. D., Kusznetova V., Milyukova I., Hardes G., Siry M., Schulze B., and Vygodskaya, N. N.: Comparing the inﬂuence of site quality, stand age, ﬁre and climate on above- ground production in Siberian Scots pine forests, Tree Phys., 22, 537–552, 2002. Roderick, M. L., Farquhar, G. D., Berry, S. L., and Noble, I. R.: On the direct effect of clouds and atmospheric particles on the productivity and structure of vegetation, Oecologia, 129(25), 21– 30, 2001. R¨odenbeck, C., Houweling, S., Gloor, M., and Heimann, M.: CO2 ﬂux history 1982–2001 inferred from atmospheric data using a global inversion of atmospheric transport, Atmos. Chem. Phys., 3, 1919–1964, doi:10.5194/acp-3-1919-2003, 2003. Atmos. Chem. Phys., 10, 7739–7751, 2010 www.atmos-chem-phys.net/10/7739/2010/ www.atmos-chem-phys.net/10/7739/2010/
https://openalex.org/W4298679996	https://periodicos.unespar.edu.br/index.php/nupem/article/download/5522/3550	Portuguese	null	Expediente e Sumário	Revista NUPEM	2,017	cc-by	1,815	ISSN 2176-7912 Online ISSN 2176-7912 Online Volume 9 – Número 16 – 2017 ISSN 2176-7912 Online Volume 9 – Número 16 – 2017 ISSN 2176-7912 Online Editor Frank Antonio Mezzomo Editor Frank Antonio Mezzomo Conselho Editorial Alessandra Augusta Pereira da Silva Cristina Satiê de Oliveira Pátaro Marcos Clair Bovo Conselho Consultivo Artur Cesar Isaia, Universidade Federal de Santa Catarina, Brasil Anete Abramowicz, Universidade Federal de São Carlos, Brasil Eduardo Portanova Barros, Universidade do Vale do Rio dos Sinos, Brasil Elpídio Serra, Universidade Estadual de Maringá, Brasil Francisco Imbernon Muñoz, Universidade de Barcelona, Espanha Héctor Fabio Ospina, Universidad de Manizales, Colômbia Hélio Sochodolak, Universidade Estadual do Centro-Oeste do Paraná, Brasil Joaquim Dolz, Université de Geneve, Suíça José Machado Pais, Universidade de Lisboa, Portugal Marcos Aurélio Saquet, Universidade Estadual Paulista Júlio de Mesquita Filho, Brasil Mariana Rodrigues de Almeida, Universidade de São Paulo, Brasil Marília Pinto de Carvalho, Universidade de São Paulo, Brasil Michel Maffesoli, Universidade de Sorbonne, França Renilson José Menegassi, Universidade Estadual de Maringá, Brasil Rosalice Pinto, Universidade de Lisboa, Portugal Sergio Grez Toso, Universidade do Chile, Chile Tarcísio Vanderlinde, Universidade Estadual do Oeste do Paraná, Brasil Trinidad Donoso-Vazquez, Universidad de Barcelona, Espanha Ursula Prutsch, Universität München, Alemanha Conselho Consultivo Artur Cesar Isaia, Universidade Federal de Santa Catarina, Brasil Anete Abramowicz, Universidade Federal de São Carlos, Brasil Eduardo Portanova Barros, Universidade do Vale do Rio dos Sinos, Brasil Elpídio Serra, Universidade Estadual de Maringá, Brasil Francisco Imbernon Muñoz, Universidade de Barcelona, Espanha Héctor Fabio Ospina, Universidad de Manizales, Colômbia Hélio Sochodolak, Universidade Estadual do Centro-Oeste do Paraná, Brasil Joaquim Dolz, Université de Geneve, Suíça José Machado Pais, Universidade de Lisboa, Portugal Marcos Aurélio Saquet, Universidade Estadual Paulista Júlio de Mesquita Filho, Brasil Mariana Rodrigues de Almeida, Universidade de São Paulo, Brasil Marília Pinto de Carvalho, Universidade de São Paulo, Brasil Michel Maffesoli, Universidade de Sorbonne, França Renilson José Menegassi, Universidade Estadual de Maringá, Brasil Rosalice Pinto, Universidade de Lisboa, Portugal Sergio Grez Toso, Universidade do Chile, Chile Tarcísio Vanderlinde, Universidade Estadual do Oeste do Paraná, Brasil Trinidad Donoso-Vazquez, Universidad de Barcelona, Espanha Ursula Prutsch, Universität München, Alemanha Secretária Executiva Lara Grigoletto Bonini ISSN 2176-7912 Online Revista NUPEM, Campo Mourão, v. 9, n. 16, jan./abr. 2017 Revista NUPEM, Campo Mourão, v. 9, n. 16, jan./abr. 2017 Revista NUPEM, Campo Mourão, v. 9, n. 16, jan./abr. 2017 Diagramação Lara Grigoletto Bonini Diagramação Lara Grigoletto Bonini Revisão Cristina Satiê de Oliveira Pátaro Frank Antonio Mezzomo 1 Revista NUPEM, Campo Mourão, v. 9, n. 16, jan./abr. 2017 Volume 9 – Número 16 – 2017 ISSN 2176-7912 Online SUMÁRIO Apresentação Dossiê 07 Dossiê: O Espaço urbano: da pequena cidade à metrópole El espacio urbano: de la pequeña ciudad a la metrópolis Urban space: from the small town to the metropolis Ângela Maria Endlich e Marcos Clair Bovo 09 Calidad de vida y desigualdad en el municipio de Santa Fe (Argentina): un análisis desde dimensiones socioeconómicas y ambientales Qualidade de vida e desigualdade no município de Santa Fé (Argentina): uma análise de dimensões socioeconômicas e ambientais Quality of life and inequality in the city of Santa Fe (Argentina): an analysis of socioeconomic and environmental dimensions Néstor Javier Gómez 26 Desarrollo humano a escala local: experiencias en el municipio Urbano Noris, Holguín/Cuba Desenvolvimento humano em escala local: experiências no município Urbano Noris, Holguín/Cuba Human Development at local scale: experiences in the municipality of Urbano Noris, Holguín/Cuba Arnoldo Higinio Santos Assan e Olga Alicia Gallardo Milanés 38 Da imobilidade à mobilidade urbana: o papel dos veículos no planejamento das cidades De la inmovilidad a la movilidad urbana: el papel de los vehículos en la planificación de ciudades From immobility to urban mobility: the role of vehicles in the city planning Marcos Ferreira de Oliveira Filho e Rodrigo Machado Vilani 54 Circulação urbana: um estudo sobre a mobilidade em Ituiutaba, MG El tráfico urbano: un estudio sobre la movilidad en Ituiutaba, MG Urban road movement: a study on urban mobility in Ituiutaba, MG Daniel de Araujo Silva e Carlos Roberto Loboda Revista NUPEM, Campo Mourão, v. 9, n. 16, jan./abr. 2017 Volume 9 – Número 16 – 2017 ISSN 2176-7912 Online Volume 9 – Número 16 – 2017 ISSN 2176-7912 Online Volume 9 – Número 16 – 2017 ISSN 2176-7912 Online SUMÁRIO 07 Dossiê: O Espaço urbano: da pequena cidade à metrópole El espacio urbano: de la pequeña ciudad a la metrópolis Urban space: from the small town to the metropolis Ângela Maria Endlich e Marcos Clair Bovo 09 Calidad de vida y desigualdad en el municipio de Santa Fe (Argentina): un análisis desde dimensiones socioeconómicas y ambientales Qualidade de vida e desigualdade no município de Santa Fé (Argentina): uma análise de dimensões socioeconômicas e ambientais Quality of life and inequality in the city of Santa Fe (Argentina): an analysis of socioeconomic and environmental dimensions Néstor Javier Gómez 26 Desarrollo humano a escala local: experiencias en el municipio Urbano Noris, Holguín/Cuba Desenvolvimento humano em escala local: experiências no município Urbano Noris, Holguín/Cuba Human Development at local scale: experiences in the municipality of Urbano Noris, Holguín/Cuba Arnoldo Higinio Santos Assan e Olga Alicia Gallardo Milanés 38 Da imobilidade à mobilidade urbana: o papel dos veículos no planejamento das cidades De la inmovilidad a la movilidad urbana: el papel de los vehículos en la planificación de ciudades From immobility to urban mobility: the role of vehicles in the city planning Marcos Ferreira de Oliveira Filho e Rodrigo Machado Vilani 54 Circulação urbana: um estudo sobre a mobilidade em Ituiutaba, MG El tráfico urbano: un estudio sobre la movilidad en Ituiutaba, MG Urban road movement: a study on urban mobility in Ituiutaba, MG Daniel de Araujo Silva e Carlos Roberto Loboda Revista NUPEM, Campo Mourão, v. 9, n. 16, jan./abr. 2017 2 Volume 9 – Número 16 – 2017 ISSN 2176-7912 Online Volume 9 – Número 16 – 2017 ISSN 2176-7912 Online Artigos Revista NUPEM Campo Mourão v 9 n 16 jan /abr 2017 3 71 Os obstáculos de aprendizagens e as intervenções do professor em uma sequência didática sobre o gênero resposta a carta do leitor Obstáculos para el aprendizaje y las intervenciones del maestro en un secuencia de enseñanza sobre el género respuesta a la carta del lector The learning obstacles and the teacher’s interventions in a didactic sequences about the genre reply to the reader’s letter Joaquim Dolz, Carla Silva-Hardmeyer e Catherine Tobola Couchepin 87 O episcopado de Dom Luciano Mendes de Almeida (1976-2006): interfaces entre uma formação tomista e a Teologia da Libertação El episcopado del obispo Luciano Mendes de Almeida (1976-2006): interfaces entre la formación tomista y la Teología de la Liberación The episcopate of Dom Luciano Mendes de Almeida (1976-2006): interfaces between a thomist formation and the Liberation Theology Virginia Albuquerque de Castro Buarque 105 O corpo na Idade Média: alguns apontamentos El cuerpo en la Edad Media: algunas notas The body in the Middle Ages: some notes Luiz Alberto Ruiz da Silva, Maria de Lourdes dos Santos e Marcia Maria de Medeiros 116 A universalização da educação infantil nas universidades federais: um estudo a partir dos editais de abertura de vagas nas unidades La universalización de la educación infantil en las universidades federales: un estudio a partir de las convocatorias de apertura de vacantes en las unidades Universalization of early childhood education in Federal universities: a study from public notes of vacancies in the units Andrea Braga Moruzzi e Bianca Neves Borges da Silva 132 Mitologia e genealogia: Barthes e Nietzsche sobre linguagem e ideologia Mitología y genealogía: Barthes y Nietszsche sobre lenguaje e ideología Mythology and genealogy: Barthes and Nietzsche on language and ideology Enrique Nuesch 140 A atividade ervateira e a construção da identidade regional na literatura de Hernâni Donato e Hélio Serejo La actividad del cultivo de yerba mate y la construcción de la identidad regional en la literatura de Hernani Donato y Hélio Serejo 71 Os obstáculos de aprendizagens e as intervenções do professor em uma sequência didática sobre o gênero resposta a carta do leitor Obstáculos para el aprendizaje y las intervenciones del maestro en un secuencia de enseñanza sobre el género respuesta a la carta del lector The learning obstacles and the teacher’s interventions in a didactic sequences about the genre reply to the reader’s letter Joaquim Dolz, Carla Silva-Hardmeyer e Catherine Tobola Couchepin 71 Os obstáculos de aprendizagens e as intervenções do professor em uma sequência didática sobre o gênero resposta a carta do leitor Obstáculos para el aprendizaje y las intervenciones del maestro en un secuencia de enseñanza sobre el género respuesta a la carta del lector The learning obstacles and the teacher’s interventions in a didactic sequences about the genre reply to the reader’s letter Joaquim Dolz, Carla Silva-Hardmeyer e Catherine Tobola Couchepin 87 O episcopado de Dom Luciano Mendes de Almeida (1976-2006): interfaces entre uma formação tomista e a Teologia da Libertação El episcopado del obispo Luciano Mendes de Almeida (1976-2006): interfaces entre la formación tomista y la Teología de la Liberación The episcopate of Dom Luciano Mendes de Almeida (1976-2006): interfaces between a thomist formation and the Liberation Theology Virginia Albuquerque de Castro Buarque 116 A universalização da educação infantil nas universidades federais: um estudo a partir dos editais de abertura de vagas nas unidades La universalización de la educación infantil en las universidades federales: un estudio a partir de las convocatorias de apertura de vacantes en las unidades Universalization of early childhood education in Federal universities: a study from public notes of vacancies in the units Andrea Braga Moruzzi e Bianca Neves Borges da Silva 132 Mitologia e genealogia: Barthes e Nietzsche sobre linguagem e ideologia Mitología y genealogía: Barthes y Nietszsche sobre lenguaje e ideología Mythology and genealogy: Barthes and Nietzsche on language and ideology Enrique Nuesch 140 A atividade ervateira e a construção da identidade regional na literatura de Hernâni Donato e Hélio Serejo La actividad del cultivo de yerba mate y la construcción de la identidad regional en la literatura de Hernani Donato y Hélio Serejo 140 A atividade ervateira e a construção da identidade regional na literatura de Hernâni Donato e Hélio Serejo La actividad del cultivo de yerba mate y la construcción de la identidad regional en la literatura de Hernani Donato y Hélio Serejo Revista NUPEM, Campo Mourão, v. Revista NUPEM, Campo Mourão, v. 9, n. 16, jan./abr. 2017 Artigos 9, n. 16, jan./abr. 2017 Revista NUPEM, Campo Mourão, v. 9, n. 16, jan./abr. 2017 3 Volume 9 – Número 16 – 2017 ISSN 2176-7912 Online Volume 9 – Número 16 – 2017 ISSN 2176-7912 Online The yerba mate’s industry activity and construction of regional identity in Hernani Donato and Hélio Serejo literature Leandro Baller e Luiz Fernando Picolo 158 Oficinas de fotografia na pesquisa-intervenção: construção de coletivos de trabalho Talleres de fotografía en la investigación-intervención: la construcción de colectivos de trabajo Photography workshops in the intervention research: building working collectives Lúcia Regina Ruduit Dias, Andréa Vieira Zanella e Jaqueline Tittoni 4
https://openalex.org/W3049616876	https://cuadernoslinguistica.colmex.mx/index.php/cl/article/download/179/210	Spanish; Castilian	null	Reseña a Bauer, Laurie. 2019. Rethinking morphology. Edinburgo: Edinburgh University Press. pp. 150.	Cuadernos de lingüística de el colegio de México	2,020	cc-by	4,082	Cómo citar: Zacarías Ponce de León, Ramón. 2020. Reseña a Bauer, Laurie. 2019. Rethinking morphology. Edinburgo: Edinburgh University Press. pp. 150. Cuadernos de Lingüística de El Colegio de México 7, e179. doi: 10.24201/clecm.v7i0.179. Cuadernos de Lingüística de El Colegio de México 7, 2020, e179. doi:10.24201/clecm.v7i0.179 Cuadernos de Lingüística de El Colegio de México 7, 2020, e179. doi:10.24201/clecm.v7i0.179 issn: 2007–736X Cuadernos de Lingüística de El Colegio de México 7, 2020, e179. Reseña Reseña a Bauer, Laurie. 2019. Rethinking morphology. Edinburgo: Edin- burgh University Press. pp. 150. Review to Bauer, Laurie. 2019. Rethinking morphology. Edinburgh: Edinburgh University Press. pp. 150. Reseña Reseña a Bauer, Laurie. 2019. Rethinking morphology. Edinburgo: Edin- burgh University Press. pp. 150. Review to Bauer, Laurie. 2019. Rethinking morphology. Edinburgh: Edinburgh University Press. pp. 150. Ramón Zacarías Ponce de León Instituto de Investigaciones Filológicas, Universidad Nacional Autónoma de México rzacaria@hotmail.com Ramón Zacarías Ponce de León Instituto de Investigaciones Filológicas, Universidad Nacional Autónoma de México rzacaria@hotmail.com Ramón Zacarías Ponce de León Instituto de Investigaciones Filológicas, Universidad Nacional Autónoma de México rzacaria@hotmail.com Universidad Nacional Autónoma de México rzacaria@hotmail.com Este libro trata de la manera como construimos teorías sobre la estruc- tura de las palabras. El hecho observable es que algunas palabras parecen contener piezas de forma que se relacionan con significados predeci- bles. Estas son palabras del autor que aparecen en la introducción de su libro y en las cuales explica el objetivo que le llevó a escribir la obra que ahora reseño. Efectivamente, este libro conforma un panorama accesible a la estructura de las palabras y a las teorías morfológicas que se han abocado a explicar sus relaciones y patrones productivos. Lau- rie Bauer –experto en la morfología tanto teórica como didáctica– es considerado una autoridad en este campo de la lingüística y después de muchos años de trabajo que han producido una obra considerable, nos guía por un camino que él mismo ha recorrido y que le permite hacer un juicio crítico de las teorías más conocidas; este libro es una invita- ción a la reflexión y al análisis de las palabras con una nueva mirada. También es la presentación de una novedosa manera de pensar acerca Zacarías Ponce de León. 2020. Reseña a Rethinking morphology 2 de la estructura de la palabra, lo que el autor llama Exemplars and reso- nances (que se puede traducir aproximadamente como “ejemplares y resonancias”) desde la cual el autor aboga por una visión cognitiva del sistema lingüístico. El libro consta de siete partes. Cuadernos de Lingüística de El Colegio de México 7, 2020, e179. Cuadernos de Lingüística de El Colegio de México 7, 2020, e179. doi:10.24201/clecm.v7i0.179 Después de la introducción o primer capítulo, los siguientes tres hacen una reseña crítica de las tres principa- les teorías sobre la estructura de la palabra: morfología basada en mor- femas, morfología basada en palabras y sintaxis de la palabra. El quinto capítulo versa sobre la intrínseca relación que existe entre la morfología y la fonología. El penúltimo capítulo explora las fronteras de la morfología, a partir de fenómenos como acrónimos, cruces, acortamientos, deriva- ciones regresivas y analogías. Finalmente, la última parte presenta la pro- puesta del autor de acercarse a la estructura de la palabra desde una visión cognitiva, Ejemplares y resonancias. Además, el libro contiene un glosa- rio de términos lingüísticos, lo que permite utilizar este volumen como una introducción a la morfología. Al final de cada capítulo se incluyen secciones de notas y comentarios donde se toca información relacionada con el tópico principal de dicho capítulo, pero que no se desarrolla con profundidad. Asimismo, se incluyen recomendaciones de lecturas para ampliar el panorama de lo tratado en el capítulo y, por último, un apar- tado de preguntas para continuar la discusión. En los siguientes párrafos discutiré cada uno de los capítulos y su estrecha relación con el trabajo morfológico que se realiza actualmente. En la introducción se tratan algunos conceptos básicos que permiten presentar los objetivos del libro. En primer lugar, la estructura inter- Cuadernos de Lingüística de El Colegio de México 7, 2020, e179. Zacarías Ponce de León. 2020. Reseña a Rethinking morphology 3 na de la palabra y la relación forma-significado. No podía faltar en una obra sobre morfología lingüística una discusión sobre el concepto de palabra y todas las derivas que pueden seguirse para definirla. El autor opta por apegarse a una definición operativa basada en la representación ortográfica. Posteriormente, se ocupa de la distinción entre lexema, for- ma de palabra y palabra morfosintáctica. En un segundo apartado de esta introducción, se toca por primera vez el tópico principal del libro, es decir, la relación entre descripción y teoría. De manera muy clara, el autor muestra que los hechos concretos de la lengua pueden ser inter- pretados a partir de diversas teorías, las cuales se hacen preguntas distin- tas de acuerdo con su punto de vista. Lo que no cambia en ningún caso son los hechos concretos del lenguaje humano. Cuadernos de Lingüística de El Colegio de México 7, 2020, e179. doi:10.24201/clecm.v7i0.179 Independientemente de la teoría analítica, la morfología se ocupa fundamentalmente de la com- plejidad que presentan la forma y el significado de las palabras. ¿Qué teoría es mejor? se pregunta Bauer. ¿La que refleja mejor la forma en la que los hablantes nativos de una lengua tratan las estructuras lingüísti- cas en su mente, o bien, la teoría más económica? Termina este primer capítulo afirmando el objetivo del libro: proponer una nueva forma de mirar la estructura morfológica. En el capítulo dos, Morpheme-based morphology (morfología basa- da en morfemas), el autor remarca la inexistencia de una visión úni- ca acerca de lo que es un morfema y, como consecuencia, la aparición de variantes de la teoría morfémica. Para explicar este tipo de morfolo- gía recurre a ejemplos de la lengua inglesa para discutir los conceptos de morfema, morfo y alomorfo; morfo libre, morfo ligado y distribu- ción complementaria, lexema, raíz y afijos. Posteriormente, hace una Cuadernos de Lingüística de El Colegio de México 7, 2020, e179. Zacarías Ponce de León. 2020. Reseña a Rethinking morphology 4 exposición de los principales problemas que se encuentran en la noción de morfema. De manera muy atinada, presenta el problema a partir de datos de diversas lenguas e inmediatamente discute la defensa que se ha hecho del modelo en cuanto a tal problema. Esta manera de proce- der deja muy claro el concepto de morfema, sus alcances y limitacio- nes. Algunos de los argumentos en favor de la noción de morfema son más afortunados que otros y, en algunos casos, son francamente débiles. Los problemas que trata Bauer son los siguientes: la semejanza de for- ma que deben mantener los alomorfos; la semejanza de significado que deben mantener los alomorfos; el sobreanálisis de la estructura morfé- mica; los morfos vacíos; los morfos cero; el tratamiento de los procesos como morfos; morfos acumulativos, la exponencia extendida, el mor- fo superfluo, los morfos homófonos y la inversión de significado. Estos problemas demuestran la dificultad que tiene aplicar la noción de mor- fema indiscriminadamente, cuando en realidad, muchos fenómenos de la estructura de la palabra en diversas lenguas se resisten al escruti- nio de la morfología basada en morfemas. En una evaluación general de este modelo, el autor nota que ninguno de los problemas descritos podría por sí solo constituirse como un argumento contra este mode- lo. Cuadernos de Lingüística de El Colegio de México 7, 2020, e179. Cuadernos de Lingüística de El Colegio de México 7, 2020, e179. Cuadernos de Lingüística de El Colegio de México 7, 2020, e179. doi:10.24201/clecm.v7i0.179 Sin embargo, la abundancia de problemas es lo que pone en cues- tión la noción de morfema. Hoy en día la noción de morfema no es tan estricta y muchos de los problemas se han relativizado gracias al adveni- miento de teorías cognitivas del lenguaje que consideran los usos reales de la lengua antes que las estructuras fijas de los modelos. Sin embargo, advierte el autor, esa misma tendencia a priorizar los fenómenos cogni- tivos puede llevar a prescindir del morfema. En la última parte del capí- Cuadernos de Lingüística de El Colegio de México 7, 2020, e179. 5 Zacarías Ponce de León. 2020. Reseña a Rethinking morphology tulo, el autor introduce la noción de “morfoma” como alternativa. En los modelos de este tipo se le da más peso a la forma que al significa- do. Se explican las estructuras morfológicas, por ejemplo, los paradig- mas verbales, a partir de las relaciones formales que establecen entre sí las formas de palabra de un lexema, buscando patrones formales prede- cibles independientemente del significado que se les pueda asignar. La noción de morfoma ha permitido encontrar regularidades en las conju- gaciones de las lenguas romance, por ejemplo. Para finalizar el capítulo, en la sección de notas se describen las particularidades del modelo mor- fémico más conocido, Item and arrangement (Hockett 1954). El capítulo tres, Word-based morphology, versa sobre la morfología basada en palabras. En este tipo de morfología no se afirma, por ejem- plo, que la palabra gatos tiene un significado de pluralidad porque con- tiene el morfema -s que significa ‘plural’; lo que se interpreta es que gatos es la forma plural del lexema gato. Conviene aclarar que, aunque el capítulo hace referencia de manera general a una morfología basada en palabras, el propio autor específica que describirá el modelo de Palabra y Paradigma (Word-and-Paradigm morphology, de ahora en adelante wp) en particular el desarrollado por Matthews (1974) que es considerado un modelo realizacional que analiza segmentos de palabras con rasgos mor- fosintácticos. Bauer deja de lado versiones más recientes del modelo wp, como los llamados implicacionales abstractivos (Blevins 2016). Al prin- cipio del capítulo se establece que la unidad fundamental en este tipo de modelos es la palabra morfosintáctica. Un lexema puede ser representa- do por distintas formas de palabra que conllevan una serie de caracterís- ticas morfosintácticas que se adecuan a las exigencias de la sintaxis. Cuadernos de Lingüística de El Colegio de México 7, 2020, e179. Cuadernos de Lingüística de El Colegio de México 7, 2020, e179. doi:10.24201/clecm.v7i0.179 De Zacarías Ponce de León. 2020. Reseña a Rethinking morphology 6 acuerdo con la exposición de Bauer, este modelo enfrenta como dificul- tad principal que solo fue concebido para analizar la morfología flexiva, donde formas de palabras representan ciertas características morfosin- tácticas, y no para analizar derivaciones donde no se reconocen formas de palabras, sino nuevos lexemas. El problema es que no siempre es sen- cillo trazar la distinción entre flexión y derivación. En la siguiente parte del capítulo se lleva a cabo una discusión sobre los criterios que se han planteado para distinguir estos procesos morfológicos y sobre la distin- ción entre flexión inherente y flexión contextual. Utiliza en su argumen- tación casos fronterizos entre la derivación y la flexión provenientes del inglés, concretamente, los sufijos -ly e -ing. Hacia el final del capítulo, se pregunta nuestro autor si realmente es relevante la distinción entre fle- xión y derivación y si es posible considerar la existencia de paradigmas en la derivación. El capítulo siguiente, Word syntax, trata del modelo sintáctico de la palabra y termina con la revisión crítica que se hace de los tres principa- les modelos morfológicos. En este acercamiento se establece un paralelo con los principios que gobiernan la sintaxis. Se ve a la morfología como una cadena jerárquica de morfemas que pueden representarse en árboles sintácticos. La propuesta de este modelo es que dichos árboles sintácti- cos no deben terminar al nivel de la palabra, sino que deben crecer para analizar la estructura interna de la palabra. Después de señalar lo ante- rior, Bauer presenta ejemplos de diversas lenguas en los cuales este tipo de análisis resulta adecuado. Al considerar que la morfología puede tra- tarse como parte de la sintaxis se han sacado a la luz diversas preocupa- ciones teóricas de las cuales, las dos más importantes son discutidas en Cuadernos de Lingüística de El Colegio de México 7, 2020, e179. 7 Zacarías Ponce de León. 2020. Reseña a Rethinking morphology el libro: el núcleo (head)1 y la morfotáctica. Con respecto al concepto de núcleo, puede decirse que es aquella parte de la palabra que la encabeza y es la más importante de todas. 1 El término en inglés es head y de este puede derivarse headedness que también utiliza el autor. Para esta reseña, utilizaremos el término núcleo que es el que normalmente se utili- za en español. Cuadernos de Lingüística de El Colegio de México 7, 2020, e179. doi:10.24201/clecm.v7i0.179 Señala el autor los distintos criterios que se toman en cuenta para identificar el núcleo de la palabra: a) determina la categoría gramatical de la palabra; b) es obligatorio; c) es el término superordinado de la palabra; d) tiene la misma distribución que la pala- bra; e) el núcleo puede determinar la forma morfológica de otros ele- mentos de la palabra. El autor pone a prueba cada uno de estos criterios con ejemplos del inglés y del alemán. Las palabras utilizadas son tanto derivaciones como flexiones y composiciones y encuentra que su aplica- ción en la derivación enfrenta numerosas dificultades, mientras que en la flexión, de plano, no es posible determinar con exactitud el núcleo. Por el contrario, la noción de núcleo parece funcionar extremadamente bien en la composición. Incluso, la versión extrema de núcleo, la regla llamada Right-Head Rule, la cual indica que el núcleo siempre está a la derecha, funciona bien en lenguas como el inglés y el alemán. El propio Bauer reconoce que, en otras lenguas como el vietnamita (y las lenguas romances, apunto), el núcleo puede aparecer a la izquierda mayormen- te. A pesar de esto, la noción de núcleo en la composición de palabras es valiosa. Habrá que considerar, sin embargo, que esta noción de núcleo no es la misma que se utiliza en sintaxis ya que los criterios son diferen- tes en ambos niveles. En cuanto a la morfotáctica, lo que se busca es establecer la forma en la cual se ordenan los morfemas. Por principio de 1 El término en inglés es head y de este puede derivarse headedness que también utiliza el autor. Para esta reseña, utilizaremos el término núcleo que es el que normalmente se utili- za en español. Cuadernos de Lingüística de El Colegio de México 7, 2020, e179. Cuadernos de Lingüística de El Colegio de México 7, 2020, e179. Zacarías Ponce de León. 2020. Reseña a Rethinking morphology 8 cuentas, en una derivación simple nos podemos preguntar si la base eli- ge el afijo o si el afijo elige a la base. Estas preguntas conducen al con- cepto de rivalidad entre afijos, la cual es ejemplificada con el caso de -ise vs -ify. Otra consideración tiene que ver con clases de afijos. En inglés, por ejemplo, se distinguen afijos nativos de afijos no-nativos (a veces lla- mados “latinos”). Cuadernos de Lingüística de El Colegio de México 7, 2020, e179. Cuadernos de Lingüística de El Colegio de México 7, 2020, e179. doi:10.24201/clecm.v7i0.179 Cuando hay confluencia de sufijos de ambos tipos, el orden más común implica que los sufijos no-nativos se encuentran más cerca de la base y los afijos nativos son más externos (personalness, don- de -al es un sufijo no-nativo y -ness es un sufijo nativo). Este orden, sin embargo, presenta muchas excepciones. El orden también puede verse con respecto a la semántica. Los sufijos aportan su propio significado a la base. Sin embargo, si la base es más compleja, entonces, el alcance del sufijo puede ser sobre la palabra completa o sobre uno de sus compo- nentes. El orden, entonces, puede estar determinado por el alcance que tenga cada sufijo. Lo que queda claro, afirma el autor, es que la morfotác- tica no puede determinarse de manera simple. Hacia el final del capítu lo, el autor reconoce que la identificación del núcleo y la morfotáctica resultan ser muy complejos. Es claro que la teoría de sintaxis de la pala- bra presenta diversas lagunas que aún esperan su resolución. El capítulo siguiente, The phonological correlates of concatenation (Los correlatos fonológicos de la concatenación), es el más breve porque, según afirma el autor, en un libro dedicado principalmente a la morfo- logía, no parece apropiado detenerse mucho en la discusión de teorías fonológicas. El objetivo entonces es discutir ciertos fenómenos en los que interactúan fonología y morfología. Como su nombre lo indica, los fenó- menos tratados en este capítulo pueden verse principalmente como un Cuadernos de Lingüística de El Colegio de México 7, 2020, e179. 9 Zacarías Ponce de León. 2020. Reseña a Rethinking morphology correlato al modelo basado en morfemas. Dichos fenómenos se dividen en tres tipos: eliminación, adición y modificación de material fonológi- co. En el primer caso, se discuten dos tipos de eliminación, la haplología y el truncamiento, que en ambos casos se ejemplifican con palabras del inglés. En cuanto a la adición de material, se discute únicamente el caso de inserción de elementos fonológicos o extensiones, ya sea en la base o bien en los afijos. Dichas inserciones son conocidas también como inter- fijos. Finalmente, en el caso de la modificación de material fonológico, se revisan casos de asimilación, de umlaut, simplificación de clúster y de alternancia vocálica. Después de analizar estos fenómenos, Bauer resal- ta la importancia de considerar dentro del estudio de la morfología las distintas influencias de la fonología en diversos contextos morfológicos. Cuadernos de Lingüística de El Colegio de México 7, 2020, e179. Cuadernos de Lingüística de El Colegio de México 7, 2020, e179. doi:10.24201/clecm.v7i0.179 El capítulo seis del libro, The border of morphology (Las fronteras de la morfología), toca un tema muy interesante y polémico. Se describen diversos casos de formaciones de palabras a partir de esquemas no pro- ductivos, o bien, que hasta cierto punto no se consideran procesos mor- fológicos. En los fenómenos discutidos se involucra la estructura interna de las palabras, sin embargo, su estatus morfológico es controversial. Entre estos fenómenos se encuentran los casos de segmentos formales sin significado que parecen formar series de palabras (plan, planet, plangent, plantain); acrónimos (UNICEF, United Nations International Children’s Emergency Found, pronunciado como una palabra) y siglas (UNHCR, United Nations High Commissioner for Refugees, pronunciado letra por letra); fonoestemas (glare, gleam, glimmer, glimpse, glow, etc., que son palabras en las que puede interpretarse un segmento gl- con el significado de ‘luz’); blends o cruces (guestimate, formado a partir de guess y estima- Cuadernos de Lingüística de El Colegio de México 7, 2020, e179. 10 10 Zacarías Ponce de León. 2020. Reseña a Rethinking morphology te, palabras que se traslapan en el segmento /es/); clippings o acortamien- tos (binocs, de binoculars; flu de influenza); formaciones regresivas (edit, derivado de editor; burgle derivado de burglar). Existen otros casos aún más complejos de palabras técnicas que han sido diseñadas con cuidado y no se forman a partir de la productividad inconsciente de los hablantes, como el caso de amoxycillin creada a partir de AMino + hydrOXYphenyl + peniCILLIN. Otros casos pueden clasificarse como creaciones por ana- logía, es decir, palabras que siguen un modelo identificable pero que no proceden de patrones morfológicos regulares, como en el caso de useta- bes (used to be), formado a partir de wannabes (want to be), donde se pue- de identificar una analogía con una palabra existente, pero no un patrón morfológico. Aquí la frontera empieza a ser difusa ya que, para diversos investigadores, los esquemas de formación de palabras son analogías, si bien, repetidas muchas veces. Bauer concluye el capítulo afirmando que la formación de palabras morfológica es solo una parte de la formación de palabras general. El séptimo capítulo, Exemplars and resonances (Ejemplares y resonan- cias), describe la propuesta central del libro que plantea una nueva pos- tura en la morfología lingüística. Cuadernos de Lingüística de El Colegio de México 7, 2020, e179. Cuadernos de Lingüística de El Colegio de México 7, 2020, e179. doi:10.24201/clecm.v7i0.179 De acuerdo con el autor, de la misma manera que en la fonología donde los fonemas no aparecen al princi- pio del proceso de adquisición, la morfología no se adquiere a partir de morfemas aislados, sino de palabras y frases. Los hablantes usan palabras complejas sin necesidad de reconocer su estructura morfológica, así que usar palabras de forma coherente no es indicativo de que procese o reco- nozca su estructura interna. Después de puntualizar lo anterior, Bauer presenta la Teoría ejemplar (Exemplar Theory), que es una teoría psico- Cuadernos de Lingüística de El Colegio de México 7, 2020, e179. Zacarías Ponce de León. 2020. Reseña a Rethinking morphology 11 lógica acerca de la percepción y la categorización del mundo. Según la teoría, cuando escuchamos una palabra, esta se retiene en la memoria donde se almacenan no solo su fonética, sino también detalles lingüís- ticos y sociales que la acompañan. Entre más veces se escucha la pala- bra, más precisa es la imagen que guardamos de ella. De la misma forma que almacenamos palabras, podemos almacenar frases y segmentos de palabras recurrentes. Por otro lado, agrupamos palabras que contienen segmentos similares y que aumentan su fuerza de asociación proporcio- nalmente a su frecuencia de uso. Cuando necesitamos utilizar una pala- bra, nos enfrentamos a un par de opciones: buscamos en la memoria para ver si alguna palabra adecuada está ya disponible, o bien, construi- mos una nueva. Es un modelo de ruta dual. Cuando necesitamos cons- truir una palabra, buscamos en los conjuntos de palabras una analogía adecuada para la nueva creación. Los morfemas no son otra cosa que patrones frecuentes y disponibles. Sin embargo, otro tipo de patrones están también disponibles, como aquellos que se discutieron en el capí- tulo 6. Sin lugar a duda, este modelo ejemplar guarda muchas similitudes con la Teoría de prototipos, algo que reconoce Bauer en los comentarios finales del capítulo. La segunda parte del modelo es la llamada Resonancias, basada en una de las últimas propuestas de Hockett (1987) en la cual hace una fuer- te crítica al modelo morfémico, a partir de lo que este conocido lin- güista llama “el fraude aglutinante”. Según él, existen muchos procesos de formación de palabras que no pueden ser representados a través de morfemas. Para Bauer, las resonancias de Hockett son totalmente com- patibles con la Teoría ejemplar. Cuadernos de Lingüística de El Colegio de México 7, 2020, e179. Cuadernos de Lingüística de El Colegio de México 7, 2020, e179. doi:10.24201/clecm.v7i0.179 Las resonancias afectan la manera como Cuadernos de Lingüística de El Colegio de México 7, 2020, e179. 12 Zacarías Ponce de León. 2020. Reseña a Rethinking morphology 12 las palabras son percibidas, analizadas y creadas. El morfema clásico es simplemente un tipo de resonancia. Al final, la conclusión del capítu- lo es que la Teoría ejemplar y la Teoría de resonancias permiten incluir muchos patrones, hasta hoy ignorados, en el estudio de la morfología. Es una visión más realista acerca de cómo opera la morfología en la men- te humana y a partir de esta desaparecen muchos de los problemas de la morfología clásica. Este libro tiene muchas maneras de leerse, independientemente de estar o no de acuerdo con los planteamientos del autor. Por un lado, es un panorama de los principales conceptos teóricos de la morfología y de los modelos que se ocupan de su estudio. Resalta la relectura que hace Bauer de los últimos escritos de Hockett. En este sentido, puede servir como un libro introductorio al campo. Por otro lado, este libro es una propuesta teórica muy importante que compromete a los estudiosos de la morfología a poner una mirada moderna en la disciplina. Con un estilo claro y sucinto, Bauer nos entrega un libro que refleja su conocimiento y su experiencia amplia en el estudio de la morfología. Cuadernos de Lingüística de El Colegio de México 7, 2020, e179. Cuadernos de Lingüística de El Colegio de México 7, 2020, e179. Referencias Blevins, James 2016. Word and paradigm morphology. Oxford: Oxford University Press. Hockett, Charles 1954. Two models of grammatical description en Joos, Martin (ed.), Readings in Linguistics I. Chicago: University of Chi- cago Press. Cuadernos de Lingüística de El Colegio de México 7, 2020, e179. 13 Zacarías Ponce de León. 2020. Reseña a Rethinking morphology 13 Zacarías Ponce de León. 2020. Reseña a Rethinking morphology Hockett, Charles 1987. Refurbishing our foundations. Amsterdam/Phila- delphia: John Benjamins. Matthews, Peter 1974. Morphology. Cambridge: Cambridge Universi- Hockett, Charles 1987. Refurbishing our foundations. Amsterdam/Phila- delphia: John Benjamins. Matthews, Peter 1974. Morphology. Cambridge: Cambridge Universi- ty Press. delphia: John Benjamins. Matthews, Peter 1974. Morphology. Cambridge: Cambridge Universi- ty Press.

Subsets and Splits

No community queries yet

The top public SQL queries from the community will appear here once available.